Photoluminescent Copper Nanoclusters in “Turn-Off/Turn-On” Sensing of Picric Acid/Hydrogen Peroxide

: In this paper, we illustrate the synthesis, characterization, and application of a Bovine Serum Albumin-stabilized copper nanocluster (BSA@CuNCs)-based photoluminescence (PL) bifunctional sensor for the selective and rapid sensing of picric acid (PA) and hydrogen peroxide (H 2 O 2 ). Blue-emitting copper nanoclusters were synthesized using one-pot synthesis at room temperature. The PL intensity of BSA@CuNCs was shown to be quenched (“Turn-off”) with an increase in the concentration of PA and intensified (“Turn-on”) with the addition of H 2 O 2 . The quenching of PL intensity of BSA@CuNCs was shown to be extremely selective and rapid towards PA. A linear decrease in the PL emission intensity of BSA@CuNCs was observed with a PA concentration in the range of 0–15 µ M. An extremely low detection limit of 60 nM (3 σ /k) was calculated. The as-prepared BSA@CuNCs also exhibited superior selectivity for PA detection in aqueous medium. The developed sensor was also utilized for the sensing of PA in natural water samples. The probe was found to be extremely sensitive towards the detection of H 2 O 2 . An increase in the PL intensity of BSA@CuNCs was seen with the addition of H 2 O 2 , with a detection limit of 0.11 µ M.


Introduction
Metal nanoclusters, which consist of a few to hundreds of atoms, have garnered significant attention in various fields of interest in academia as well as industry. This is due to their remarkable PL intensity, biocompatibility, large quantum yield, large Stokes shift, and excellent solubility in water. They have been used in chemical sensing, heterogeneous catalysis, and cell imaging applications [1][2][3][4]. For a number of different analytical applications, researchers have concentrated on developing Gold (Au) and Silver (Ag) nanoclusters as luminous probes during the past decades [5][6][7][8]. Copper nanoclusters (CuNCs) have garnered great interest because they are relatively inexpensive, are earth abundant, have excellent optophysical properties, and are readily available for commercial applications. However, their application is hampered due to the difficulty in obtaining smaller particles because of surface oxidation [9][10][11][12][13][14][15]. As a result, researchers are eager to find ways to synthesise highly stable, surface-oxidation-free CuNCs that are soluble in water. [10,[16][17][18] The luminescence of the NCs is influenced by several factors, such as the size and type of the NCs, the surface chemistry, and even the solvent. Hence, the fluorescence signals can be amplified effectively using metal enhanced fluorescence (MEF), which has been extensively researched. MEF occurs when a fluorophore is situated in an amplified electromagnetic field produced by far-field excitation of plasmonic metal nanoparticles and surfaces. In addition to the signal amplification, other significant spectral alterations, such as shortened lifetime, improved photo-stability, and longer distances for resonance energy transfer (RET) of fluorescence, have been documented in MEF [19]. It has also been observed that shape and surface

Synthesis
BSA-stabilized copper nanoclusters were synthesized at ambient temperature using a greener approach. In a typical synthesis, we mixed 2 mL of 0.1 M freshly prepared Cu(NO 3 ) 2 (aqueous) solution with 2 mL of 0.1 M aqueous solution of L-ascorbic acid. The reaction mixture was stirred continuously. Into the reaction mixture, 3 mL of BSA (15 mg/mL) was introduced, and the final volume of the reaction mixture was maintained at 15 mL by the addition of Millipore water. The reaction was constantly stirred at ambient temperature for 24 h in darkness to obtain the final BSA-capped CuNCs. In this synthesis, ascorbic acid acts as a reducing agent, which reduces Cu 2+ ions to CuNCs, and BSA is used as a capping agent to stabilize CuNCs. After the completion of reaction, the obtained solution was centrifuged to remove insoluble particles. The solution was then dialyzed to remove unbound BSA, ascorbic acid, and other impurities. Finally, the BSA-capped CuNCs solution was stored at 4 • C when not in use.

PA Detection
Eight nitroaromtic compounds were chosen to perform PL experiments. In a typical experiment, 5 µM solution of nitroaromtic compound was successively added to 50 µL of BSA@CuNCs solution. The PL experiment was performed. The same process was repeated for all the compounds. The samples were prepared at room temperature.

H 2 O 2 Detection
In a typical experiment, 3 µM of H 2 O 2 was gently added to 50 uL BSA@CuNCs solution to carry out the PL experiments. The measurements were taken at ambient temperature.

Results and Discussion
Scheme 1 displays the synthesis scheme for BSA@CuNCs. We utilized transmission electron microscopy using a JEM-2100 electron microscope system, operated at 200 kV, to investigate the obtained shape and size of as-synthesized BSA@CuNCs. As shown in the TEM image in Figure 1a, the average size of BSA@CuNCs was found to be 4 ± 1 nm of the NCs, which agrees with the results reported in the literature. Further, dynamic light scattering (DLS) measurement using a Malvern Zetasizer Nano S was also performed (Figure 1b), from which the hydrodynamic diameter was determined to be 5 nm.
Sustain. Chem. 2022, 3, FOR PEER REVIEW 3 temperature for 24 h in darkness to obtain the final BSA-capped CuNCs. In this synthesis, ascorbic acid acts as a reducing agent, which reduces Cu 2+ ions to CuNCs, and BSA is used as a capping agent to stabilize CuNCs. After the completion of reaction, the obtained solution was centrifuged to remove insoluble particles. The solution was then dialyzed to remove unbound BSA, ascorbic acid, and other impurities. Finally, the BSA-capped CuNCs solution was stored at 4 °C when not in use.

PA Detection
Eight nitroaromtic compounds were chosen to perform PL experiments. In a typical experiment, 5 μM solution of nitroaromtic compound was successively added to 50 μL of BSA@CuNCs solution. The PL experiment was performed. The same process was repeated for all the compounds. The samples were prepared at room temperature.

H2O2 Detection
In a typical experiment, 3 μM of H2O2 was gently added to 50 uL BSA@CuNCs solution to carry out the PL experiments. The measurements were taken at ambient temperature.

Results and Discussion
Scheme 1 displays the synthesis scheme for BSA@CuNCs. We utilized transmission electron microscopy using a JEM-2100 electron microscope system, operated at 200 kV, to investigate the obtained shape and size of as-synthesized BSA@CuNCs. As shown in the TEM image in Figure 1a, the average size of BSA@CuNCs was found to be 4 ± 1 nm of the NCs, which agrees with the results reported in the literature. Further, dynamic light scattering (DLS) measurement using a Malvern Zetasizer Nano S was also performed ( Figure  1b), from which the hydrodynamic diameter was determined to be 5 nm.  The UV-vis spectroscopy measurements were also performed on both BSA and BSA@CuNCs using a UV-170 Shimadzu spectrophotometer. Figure 2a,b show the recorded UV-Vis spectra for BSA and BSA@CuNCs, respectively. The BSA is characterized by the presence of an absorption peak at 280 nm, as shown in Figure 2a [52]. The UV-vis spectra of copper nanoparticles is reported to have a peak at 560 nm. As there is no peak in the UV-Vis spectra of BSA@CuNCs around 560 nm, this confirms the formation of smaller nanoclusters and not nanoparticles. The UV-vis spectroscopy measurements were also performed on both BSA and BSA@CuNCs using a UV-170 Shimadzu spectrophotometer. Figure 2a,b show the recorded UV-Vis spectra for BSA and BSA@CuNCs, respectively. The BSA is characterized by the presence of an absorption peak at 280 nm, as shown in Figure 2a [52]. The UV-vis spectra of copper nanoparticles is reported to have a peak at 560 nm. As there is no peak in the UV-Vis spectra of BSA@CuNCs around 560 nm, this confirms the formation of smaller nanoclusters and not nanoparticles. The UV-vis spectroscopy measurements were also performed on both BSA an BSA@CuNCs using a UV-170 Shimadzu spectrophotometer. Figure 2a,b show the re orded UV-Vis spectra for BSA and BSA@CuNCs, respectively. The BSA is characteriz by the presence of an absorption peak at 280 nm, as shown in Figure 2a [52]. The UV-v spectra of copper nanoparticles is reported to have a peak at 560 nm. As there is no pe in the UV-Vis spectra of BSA@CuNCs around 560 nm, this confirms the formation smaller nanoclusters and not nanoparticles.

Photoluminescence Study
In this study, we employed photoluminescence spectroscopy using an Agilent Cary spectrophotometer to evaluate the potential applicability of the as-synthesized BSA@CuNCs for selective sensing of PA. With the variation in the excitation wavelength from 300 to 350 nm, the emission peak was observed to be shifted from 400 to 430 nm. A single peak centred around 405 nm was experimentally observed in the fluorescence spectrum of BSA@CuNCs with the 320 nm excitation wavelength. As demonstrated in Figure 3a, the maximum intensity was recorded when the excitation wavelength was 320 nm and the emission peak was centred at 405 nm. We also performed the PL measurements of the synthesized sample at different times. As shown in Figure 3b, the PL intensity was noticed to be same for 48 h with 320 nm excitation wavelength. In fact, the sample seemed extremely stable. No relevant change in the PL was noticed even after a few days. We also investigated the pH dependence of the sample, and the PL intensity was found to be linearly increasing with respect to the increase in the pH of the buffer. The fluorescence intensity of our analytical system was shown to be decreasing with increasing PA solution content, as shown in Figure 4a. For the 75 µM concentration of PA solution, the fluorescence intensity of BSA@CuNCs was decreased up to 90%. This clearly indicates the extreme sensitivity of the as-developed probe towards the sensing of PA. When a similar amount of other nitro explosives were added to the BSA@CuNCs solution, almost no or very little change in the PL intensity was observed, as shown in Figure 4b.
We also performed fluorescence titration experiments to determine the sensitivity of the BSA@CuNCs probe for the sensing of PA. The emission intensity of the BSA@CuNCs at 405 nm was recorded for the identification of the sensitivity of the developed system towards the sensing of PA. With the increase in the PA concentration in the range of 0-75 µM, the emission intensity of the BSA@CuNCs was observed to be decreasing ( Figure 5a). The change in the emission intensity of BSA@CuNCs shows a good linear behaviour in the 0-15 µM concentration of PA ( Figure 5b). This can be linearly fitted with the equation (F0/F)-1 = −0.0036 × X + 0.0431 (R 2 = 0.99167), where X denotes the PA concentration. The detection limit was calculated as 60 nM using the equation 3σ/k, where σ is the standard deviation of the blank sample and k is slope of linear calibration plot. The present method thus exhibits a relatively better LOD for detecting PA in comparison to the other recently reported fluorescence methods. A comparison of the synthesized probe for the detection of PA with other fluorescence-based methods is listed in Table 2. We also performed fluorescence titration experiments to determine the sensitivity of the BSA@CuNCs probe for the sensing of PA. The emission intensity of the BSA@CuNCs at 405 nm was recorded for the identification of the sensitivity of the developed system towards the sensing of PA. With the increase in the PA concentration in the range of 0-75 μM, the emission intensity of the BSA@CuNCs was observed to be decreasing (Figure 5a). The change in the emission intensity of BSA@CuNCs shows a good linear behaviour in the 0-15 μM concentration of PA ( Figure 5b). This can be linearly fitted with the equation (F0/F)-1 = −0.0036 × X + 0.0431 (R 2 = 0.99167), where X denotes the PA concentration. The detection limit was calculated as 60 nM using the equation 3σ/k, where σ is the standard deviation of the blank sample and k is slope of linear calibration plot. The present method thus exhibits a relatively better LOD for detecting PA in comparison to the other recently reported fluorescence methods. A comparison of the synthesized probe for the detection of PA with other fluorescence-based methods is listed in Table 1.   We also performed fluorescence titration experiments to determine the sen the BSA@CuNCs probe for the sensing of PA. The emission intensity of the BSA at 405 nm was recorded for the identification of the sensitivity of the develope towards the sensing of PA. With the increase in the PA concentration in the rang μM, the emission intensity of the BSA@CuNCs was observed to be decreasing (F The change in the emission intensity of BSA@CuNCs shows a good linear beh the 0-15 μM concentration of PA (Figure 5b). This can be linearly fitted with the (F0/F)-1 = −0.0036 × X + 0.0431 (R 2 = 0.99167), where X denotes the PA concentra detection limit was calculated as 60 nM using the equation 3σ/k, where σ is the deviation of the blank sample and k is slope of linear calibration plot. The presen thus exhibits a relatively better LOD for detecting PA in comparison to the othe reported fluorescence methods. A comparison of the synthesized probe for the of PA with other fluorescence-based methods is listed in Table 1. In this work, the fluorescence intensity of BSA@CuNCs was found to decr the addition of PA, as shown in Figure 4a. A plausible mechanism for the sens by BSA@CuNCs is shown in Scheme 2. To understand the quenching mechanism ried out further investigations. The mechanism for quenching of fluorescence in the presence of an analyte can be one of the following: fluorescence resonan transfer (FRET), the inner filter effect (IFE), the formation of a donor-accepto In this work, the fluorescence intensity of BSA@CuNCs was found to decrease with the addition of PA, as shown in Figure 4a. A plausible mechanism for the sensing of PA by BSA@CuNCs is shown in Scheme 2. To understand the quenching mechanism, we carried out further investigations. The mechanism for quenching of fluorescence intensity in the presence of an analyte can be one of the following: fluorescence resonance energy transfer (FRET), the inner filter effect (IFE), the formation of a donor-acceptor charge-transfer complex, and static and dynamic quenching effects [53]. The fluorescence resonance energy transfer (FRET) mechanism is found to be operative in the quenching process if the absorption spectrum of the quencher overlaps with the emission spectrum of the fluorophore. In our experiments, the photoluminescence emission spectrum of BSA@CuNCs was found at 405 nm, whereas the UV-Vis spectrum of PA was observed at 355 nm. As can be seen in Figure 6a, there is a significant spectral overlap between the absorption spectrum of PA and the emission spectrum of BSA-CuNCs, which meets the criteria of FRET. In order to gain more insight into the mechanism behind the quenching of emission intensity of BSA@CuNCs in the presence of PA, time-resolved photoluminescence (TRPL) measurements were also carried out using a TRPL FLS920 spectrophotometer, Edinburgh, UK (Figure 6b). As is reported in the literature, fluorescence lifetime, which remains constant for static quenching and changes proportionally with the quencher concentration for dynamic quenching, can be utilised to differentiate between static and dynamic quenching [47][48][49]. We used a thrice exponential decay function to fit the decay in the fluorescence intensity. It was found that with the increase in the concentration of PA, fluorescence lifetime became shorter (Table 1). This reduction in the lifetime indicates that the fluorescence resonance electron transfer from BSA@CuNCs to PA is in good agreement with previous reports [29]. Thus, the FRET mechanism controls the excellent selectivity of BSA@CuNCs towards PA in comparison to other nitro compounds. ustain. Chem. 2022, 3, FOR PEER REVIEW transfer complex, and static and dynamic quenching effects [53]. The fluorescence nance energy transfer (FRET) mechanism is found to be operative in the quenching cess if the absorption spectrum of the quencher overlaps with the emission spectru the fluorophore. In our experiments, the photoluminescence emission spectru BSA@CuNCs was found at 405 nm, whereas the UV-Vis spectrum of PA was observ 355 nm. As can be seen in Figure 6a, there is a significant spectral overlap betwee absorption spectrum of PA and the emission spectrum of BSA-CuNCs, which mee criteria of FRET. In order to gain more insight into the mechanism behind the quenc of emission intensity of BSA@CuNCs in the presence of PA, time-resolved photolum cence (TRPL) measurements were also carried out using a TRPL FLS920 spectrophot ter, Edinburgh, UK ( Figure 6b). As is reported in the literature, fluorescence life which remains constant for static quenching and changes proportionally with quencher concentration for dynamic quenching, can be utilised to differentiate bet static and dynamic quenching [47][48][49]. We used a thrice exponential decay function the decay in the fluorescence intensity. It was found that with the increase in the co tration of PA, fluorescence lifetime became shorter ( Table 2). This reduction in the life indicates that the fluorescence resonance electron transfer from BSA@CuNCs to PA good agreement with previous reports [29]. Thus, the FRET mechanism controls th cellent selectivity of BSA@CuNCs towards PA in comparison to other nitro compoun

Detection of PA in Water Samples
We also investigated the application of BSA@CuNCs for the detection of PA in water samples. For this purpose, we performed a recovery study where a known amount of the PA was added to the water sample. A standard addition method was applied to determine the recovery of PA in water samples. The proposed method shows excellent recovery (in the range of 98-99%) and good reproducibility, as shown in Table 3. The results obtained confirm that the developed photoluminescence-based sensor has tremendous potential for detecting PA in natural water samples.

Turn-On-Based Sensing of H2O2
H2O2 is a very important molecule in the area of biological and chemical sciences. Developing new methods for its sensing and determination is of extreme importance. In this work, we also explored the possibility of H2O2 sensing using BSA@CuNCs under optimized conditions. Fluorescence experiments were carried out with the slow addition of H2O2 in the BSA@CuNCs solution. As presented in Figure 7a the fluorescence emission peak of BSA@CuNCs at 405 nm increased slowly with increasing H2O2 concentration. The plot of (1-F0/F) vs. the increasing concentration is reported in Figure 7b. The inset of Figure  7b exhibits the relative fluorescence intensity (1-F0/F), displaying linear response between the emission intensity and concentration of H2O2 in the range 0-36 μM. The detection limit obtained was 0.11 μM, which is comparable to the results reported in the literature. The relative standard deviation (RSD) was calculated to be 5.3% from the five repeated measurements of 10 μM H2O2, which confirms the extremely high reproducibility of the present system for H2O2. This increase in the PL intensity is due to the surface etching of CuNCs in the presence of H2O2.

Detection of PA in Water Samples
We also investigated the application of BSA@CuNCs for the detection of PA in water samples. For this purpose, we performed a recovery study where a known amount of the PA was added to the water sample. A standard addition method was applied to determine the recovery of PA in water samples. The proposed method shows excellent recovery (in the range of 98-99%) and good reproducibility, as shown in Table 3. The results obtained confirm that the developed photoluminescence-based sensor has tremendous potential for detecting PA in natural water samples.

Turn-On-Based Sensing of H2O2
H2O2 is a very important molecule in the area of biological and chemical sciences. Developing new methods for its sensing and determination is of extreme importance. In this work, we also explored the possibility of H2O2 sensing using BSA@CuNCs under optimized conditions. Fluorescence experiments were carried out with the slow addition of H2O2 in the BSA@CuNCs solution. As presented in Figure 7a the fluorescence emission peak of BSA@CuNCs at 405 nm increased slowly with increasing H2O2 concentration. The plot of (1-F0/F) vs. the increasing concentration is reported in Figure 7b. The inset of Figure  7b exhibits the relative fluorescence intensity (1-F0/F), displaying linear response between the emission intensity and concentration of H2O2 in the range 0-36 μM. The detection limit obtained was 0.11 μM, which is comparable to the results reported in the literature. The relative standard deviation (RSD) was calculated to be 5.3% from the five repeated measurements of 10 μM H2O2, which confirms the extremely high reproducibility of the present system for H2O2. This increase in the PL intensity is due to the surface etching of CuNCs in the presence of H2O2.

Detection of PA in Water Samples
We also investigated the application of BSA@CuNCs for the detection of PA samples. For this purpose, we performed a recovery study where a known amou PA was added to the water sample. A standard addition method was applied to d the recovery of PA in water samples. The proposed method shows excellent rec the range of 98-99%) and good reproducibility, as shown in Table 3. The results confirm that the developed photoluminescence-based sensor has tremendous for detecting PA in natural water samples.

Turn-On-Based Sensing of H2O2
H2O2 is a very important molecule in the area of biological and chemical Developing new methods for its sensing and determination is of extreme impo this work, we also explored the possibility of H2O2 sensing using BSA@CuNCs u timized conditions. Fluorescence experiments were carried out with the slow ad H2O2 in the BSA@CuNCs solution. As presented in Figure 7a the fluorescence peak of BSA@CuNCs at 405 nm increased slowly with increasing H2O2 concentra plot of (1-F0/F) vs. the increasing concentration is reported in Figure 7b. The inset 7b exhibits the relative fluorescence intensity (1-F0/F), displaying linear response the emission intensity and concentration of H2O2 in the range 0-36 μM. The detec obtained was 0.11 μM, which is comparable to the results reported in the litera relative standard deviation (RSD) was calculated to be 5.3% from the five repeat urements of 10 μM H2O2, which confirms the extremely high reproducibility of th system for H2O2. This increase in the PL intensity is due to the surface etching o in the presence of H2O2.

Detection of PA in Water Samples
We also investigated the application of BSA@CuNCs for the detection of PA in water samples. For this purpose, we performed a recovery study where a known amount of the PA was added to the water sample. A standard addition method was applied to determine the recovery of PA in water samples. The proposed method shows excellent recovery (in the range of 98-99%) and good reproducibility, as shown in Table 3. The results obtained confirm that the developed photoluminescence-based sensor has tremendous potential for detecting PA in natural water samples.  Figure 7a the fluorescence emission peak of BSA@CuNCs at 405 nm increased slowly with increasing H 2 O 2 concentration. The plot of (1-F 0 /F) vs. the increasing concentration is reported in Figure 7b. The inset of Figure 7b exhibits the relative fluorescence intensity (1-F 0 /F), displaying linear response between the emission intensity and concentration of H 2 O 2 in the range 0-36 µM. The detection limit obtained was 0.11 µM, which is comparable to the results reported in the literature. The relative standard deviation (RSD) was calculated to be 5.3% from the five repeated measurements of 10 µM H 2 O 2 , which confirms the extremely high reproducibility of the present system for H 2

Conclusions
To summarize, we reported on the development of a dual bifunctional fluorescen based sensor constructing copper nanoclusters with BSA as a stabilizer. The so-synt sized BSA@CuNCs, with an excitation wavelength at 320 nm, displayed a sharp photo minescence emission peak centred at 405 nm. The fluorescence intensity of BSA@CuN was observed to quench ("Turn-off") and enhance ("Turn-on") in the presence of PA a H2O2, respectively. The emission spectra of the BSA@CuNCs was found to be remarka decreased while adding PA, and it was observed to be extremely selective. The sugges fluorescence-based sensor revealed the detection of PA with a detection limit of 60 n The application of as-synthesized materials towards the detection of PA in water samp also produced satisfactory results, which confirms its practical applications for real sa ples. The as-developed material was also tested for H2O2 detection. It was found that H could increase the photoluminescence of BSA@CuNCs. A low value of 0.11 μM for detection limit was calculated. This simplistic methodology demonstrates the applicatio of BSA@CuNCs in the sensing of a broad range of pollutants and thus demonstrates th relevance in environmental applications.

Conclusions
To summarize, we reported on the development of a dual bifunctional fluorescencebased sensor constructing copper nanoclusters with BSA as a stabilizer. The so-synthesized BSA@CuNCs, with an excitation wavelength at 320 nm, displayed a sharp photoluminescence emission peak centred at 405 nm. The fluorescence intensity of BSA@CuNCs was observed to quench ("Turn-off ") and enhance ("Turn-on") in the presence of PA and H 2 O 2 , respectively. The emission spectra of the BSA@CuNCs was found to be remarkably decreased while adding PA, and it was observed to be extremely selective. The suggested fluorescence-based sensor revealed the detection of PA with a detection limit of 60 nM. The application of as-synthesized materials towards the detection of PA in water samples also produced satisfactory results, which confirms its practical applications for real samples. The as-developed material was also tested for H 2 O 2 detection. It was found that H 2 O 2 could increase the photoluminescence of BSA@CuNCs. A low value of 0.11 µM for the detection limit was calculated. This simplistic methodology demonstrates the applications of BSA@CuNCs in the sensing of a broad range of pollutants and thus demonstrates their relevance in environmental applications.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.