Nanomolar detection of hypochlorite in ground water samples by a norbornene-based polymeric sensor via unusual fluorescence turn-on response

Abstract Hypochlorite anion has been widely used as a bleaching and disinfecting agent in daily life. Selective and sensitive identification ofOCl-ions from water is very important for researchers. For this purpose, monomeric (NPh), and polymeric (PNPh-Peg) novel fluorescent sensors have been established for the specific and excellently unique sensors that exhibit selective characteristics like excellent resistance to bleaching and a high fluorescence brightness. A multi-functional random polymer (PNPh-Peg) with ICT (intramolecular charge transfer) active para–amino phenol functionality and reactive oxygen species (ROS) responsive, PEG attached are readily prepared via ROMP (ring-opening metathesis polymerization).: An ICT-active random polymer (PNPh-Peg) exhibited an unexpectedly strong cyan blue emission in a water medium compared to that in other common organic solvents, which was dramatically increased by adding a trace amount of NaOCl.: Incorporating PEG moiety in the polymeric backbone increases the water solubility of a copolymer, and the ROS-responsive groups make the polymer a good ROS scavenger. Upon oxidation of the phenol group into carbonyl, both the monomeric (NPh) and polymeric (Norp-PEG oh) sensors showed a selective, noticeable, unusual fluorescence turn-on response towardsanalyteOCl-ions with a very fast response (within three minutes). The detection limit (59.14 nM) and (126.93 nM) were calculated for monomeric and polymeric sensors, respectively. This was a selective, specific oxidation reaction of the completely water-soluble random polymeric sensor (PNPh-Peg) for hypochlorite anions and can be applicable for quantitative measurement of aqueous OCl-. This ICT-active random polymeric molecule (PNPh-Peg) can also be used as a fluorescent sensor for unique OCl- detection from contaminated water by preparing a sensor-coated paper strip. Thus, these multi-functional monomeric (NPh) and polymeric (PNPh-Peg) sensors are anticipated to apply to the environment. Graphical Abstract


Introduction
Several biological functions and activities are essentially monitored by various reactive oxygen and reactive nitrogen species where hypochlorite (OCl -) is one of the most important reactive oxygen species (ROS) which plays a vital role in various biological processes [1][2][3][4][5][6][7] .Biologically, in activated neutrophils, the hypochlorite ions are synthesized by a catalyzed reaction of myeloperoxidase from hydrogen peroxide and chloride ions [1,[8][9][10][11][12][13] .There are many other reactive oxygen species (ROS) and reactive nitrogen species, but among them, hypochlorite (OCl -) and hypochlorous acid (HOCl) have contributed vastly in daily environmental life.Sodium hypochlorite is used as a disinfectant and environment-friendly "bleach" [14,15] .Hypochlorite is a strong nucleophilic non-radical oxidant which makes OCl -ions a microbicidal used for natural defense.The enzymes required for its catalytic detoxification; its efficacy lies in the fact that neither bacteria nor mammalian cells can neutralize the toxic effects they lack [16][17][18][19][20] .Under physiological conditions, hypochlorous (HOCl) is a biologically important ROS and weakly acidic, partially forming a hypochlorite ion ( -OCl).Besides microorganism inversion prevention, progressing result from researcher suggests that -OCl also incorporated its importance in various human diseases, like neuron degeneration, cardiovascular diseases, and osteoarthritis due to abnormal levels of MPO.Also OCl -is highly harmful and is strongly firmly a reason for many other diseases, such as inflammatory diseases, acute lung injuries, nephropathies, cystic fibrosis, neurodegenerative disorders, and cancer [12,14,[21][22][23][24][25] .
Due to the absence of sensitive and specific good watersoluble monomeric and polymeric sensors for detecting hypochlorite, the action mechanism of OCl -in these diseases is not much recorded compared to other ROS and RNS [13,[26][27][28] .As hypochlorous acid can react with biomolecules, including DNA, RNA, fatty acids, cholesterol, and proteins, the abnormal presence of OCl -can also damage the biological systems very severely, although OCl -is a strong anti-bacterial agent in nature [29][30][31] .
Absence of an excellently efficient water-soluble sensor for detecting hypochlorite, the mechanism of action of hypochlorite in these diseases is not clear compared to interfering ROS and RNS.Therefore, to investigate the various biological effects of -OCl, much effort has been given by researchers [32][33][34][35] .The physiologically relevant OCl -in a living organism is 5-25 mM; therefore, in a recent article, we established an excellently reliable method for the instant detection of biological OCl -in living organisms which become very important and interesting among researchers [36][37][38] .Fluorimetry has evolved as a widely used prominent system for detecting biological analytes for its easy use, rapid response, and noninvasive detection mechanism.In recent years, the enormous efforts of chemical researchers helped to develop a vast number of fluorescent sensors for OCl -, which established a novel kind of system for OCl -probes by researchers in science [39][40][41] .The development of probes by researchers had inherent defects, such as single-channel detection, long response time, and small Stokes shifts, making them highly susceptible to real-life environments, limiting their applications for the environment [42] .Therefore, developing novel probes remains a challenging part of research without the earlier defects.For the modification, the researcher tries to develop luminescent sensors for -OCl by taking the oxidation properties of OCl -.
Additionally, fluorophores for hypochlorite ions usually modified the emission maximum, and researchers could not discard interference by bleaching properties [43,44] .Our recent article develops a quick responsive, highly sensitive monomeric NPhand polymericPNPh-Pegfluorescent probe for NaOCl with excellent selectivity over other potentially competing species to address these challenges.The overall mechanism of NaOCl detection is composed of the intramolecular charge transfer (ICT) process [45] .Para-amino phenol molecule has been used as the signal reporter due to its outstanding photophysical properties after oxidation, and the mechanism is unique in the literature.Secondly, the norbornene group was adopted as the masking and fluorescence turn-on prompting agent toward NaOCl.Overall making the detection of NaOCl insusceptible from other ROS and active interfering molecules, our design of sensor molecule is exceptionally active and selectiveNaOCl sensing moiety.The polymeric sensor has more advantages over monomeric sensors, including low detection time and high Sensitivity (lower limit of detection) due to side-chain interaction [46,47] .Therefore, we randomly co-polymerize our monomeric sensor NPh with Nor-PEG to the polymeric sensor PNPh-Peg molecule using the ring-opening metathesis (ROMP) polymerization process.Additionally, the PEG chain (polyethylene glycol) was incorporated via a random polymerization process at the end of the targeted probe which makes the molecule excellently water soluble.The hygroscopic nature of the PEG fragment also makes the polymeric sensor molecule capable of keeping the moisture, which helps detect NaOCl gas with a probe-based test paper for in-field detection of analytes.All sensor molecules were designed to investigate its application property in real environments to investigate its application properties in natural environments, such as in contaminated water and air.The recent sensor systems detected NaOClanion selectively in water and its gas phase without interference from other anions and gases.Our investigation implies that NPh, and PNPh-Pegmay be the first unusual fluorescent turn-on probes for NaOCl detection with high selectivity and sensitivity [48][49][50] .

Experimental section
Monomeric sensor (NPh) and polymeric sensor (NPhand Nor-PEG-oh) were successfully synthesized as shown in Synthetic Scheme 1 and Synthetic Scheme 2 and were characterized by different analytical methods.All the experimental procedures and characterization are given in supporting information.We synthesized the norbornene-attached amino phenol molecule (Scheme S1) by refluxing para-amino phenol and cis-5-Norbornene-Exo-2,3-dicarboxylic anhydride at 120 � C using dry toluene as a solvent.After successful synthesis and characterization, random ROMP (ring-opening metathesis polymerization) co-polymerization of monomer NPh, PNPhand Nor-PEG-oh molecules were obtained by using the second-generation Grubbs' catalyst (10 mM %) at room temperature in dry (99% dry) dichloromethane Synthetic scheme 1. Synthetic scheme of monomeric sensor NPh.
(DCM) under inert atmosphere into a glove box.The 1 H NMR and APC analysis confirmed the polymerization.After 12 h, new peaks were observed at 5.3-5.5 ppm, and norbornene olefinic protons at 6.10-6.17ppm had disappeared.Advanced polymer chromatography (APC) measurement of PNPhandNor-PEG-oh was carried out in dimethylformamide (DMF) as an eluent.The observed Mn was 15256, PDI ¼ 1.02314of the polymer (Nor-PEG-oh), Mn was 15285, and PDI ¼ 1.016022of the polymer Norp-oh (Figures S1-S8).

Co-solvents and buffers
Co-solvents are often used by researchers such as N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile, and ethanol; as most fluorescent probes have insolubility issues in pure water medium.Biochemical studies are done mostly in an aqueous environment as a high fraction of organic solvents could destroy the usual activity of biomolecules for their biochemical investigation.For biological investigation, organic co-solvents fraction should be as low as possible because they could destroy living organisms inherently.N-2 hydroxyethyl piperazine-N 0 -2-ethane sulfonic acid (HEPES), phosphate-buffered saline (PBS), tris-(hydroxymethyl)aminomethane (Tris) are used in fluorescent chemo-sensor systems for the adjustment in physiological environments.However, asOCl -could oxidize HEPES, it is not a good choice for physiologicalOCl -detection systems.Therefore, we used PBS buffer and DMSO as co-solvent for our recent biological studies.

Solvent assay for both monomer and polymers
The solvent assay was done to explore the solubility of monomeric and polymeric sensor molecules at is best % of the DMSO: H 2 O solvent mixture.DMSO and water solvent mixture is the best choice to solve the biological purposes if the compound is soluble in that solvent.So, we have prepared various solvent mixtures for dissolving all the sensor molecules 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8 (DMSO: H 2 O), water (pure) in PBS buffer at pH-7.4 and for all fraction of solvent fluorescence spectra were recorded by exciting at 350 nm.The result shows that the monomeric sensor NPh shows maximum fluorescence intensity in a 2:8 DMSO: H 2 O solvent mixture.The polymeric sensor PNPh also shows the maximum fluorescence emission for a 2:8 DMSO: H 2 O solvent mixture.But surprisingly, the PEG-incorporated random polymeric sensor PNPh-Peg shows a different result, the 4:6 (DMSO: H 2 O) showing maximum fluorescence emission intensity.And also, it was observed that the sensor was soluble in 3:7, 2:8, and pure water solvent systems (Figures S11-S19).

Time-dependent study for monomer nor-oh and polymer PNPh-Peg
Kinetic studies were carried out by monitoring the fluorescence intensity and reaction time to investigate the Sensitivity of monomeric sensor NPhand PNPh-Peg toward -OCl.Adding 60 mM of -OClto the solution of sensors NPh (25 mM) and PNPh-Peg (100 ml from 1 mg/1 ml stock solution)instantaneously resulted in a significant change in the fluorescence intensity, which was maximized within seconds.Subsequently, time-dependent fluorescence spectra were measured upon adding NaOCl for monomeric NPhand polymeric sensors PNPh-Peg at the same condition.The absorption maximums of NPh were selected as excitation wavelengths for all sensors.Free NPhand PNPh-Pegwere substantially non-fluorescent, regardless of the excitation wavelength.However, a cyan-bluish emission band generated at 435 nm upon adding NaOCl gradually emerged and reached the intensity plateau within 10 min.In this case, for the polymeric sensor, PNPh-Peg maximum intensity plateau was reached within 5 min.An approximate 70.0-fold intensity enhancement at 435 nm can be observed for both sensors (NPhand PNPh-Peg) (Figures S12, S13).

Preparation of the test solution
A stock solution of NPh (10 −3 M) was prepared in (8:2) DMSO: H 2 O, PNPhandNor-PEG-oh (1 mg/1 ml) in 3:7 DMSO: H 2 O was prepared.The test solution of NPh (10 −5 M), PNPh, and Nor-PEG-oh (100 ml from a stock solution of 1 mg/1 ml, 3:7 DMSO: H 2 O solvent) in pH 7.4 PBS was prepared.Solutions of various testing species were prepared by adding the stock solution with PBS buffer solution.The resulting solutions were shaken well and incubated for 15 min at room temperature before recording the various electronic spectra.

Experimental details for preparation and analysis of real water samples
A stock solution of NPh (10 −3 M) in (8:2) DMSO: H 2 O, PNPhandNor-PEG-oh (1 mg/1 ml) in 3:7 DMSO: H 2 O was prepared.The test solution of NPh (10 −5 M), PNPh, and Nor-PEG-oh (100 ml from a stock solution of 1 mg/1 ml, 3:7 DMSO: H 2 O solvent) PBS buffer pH-7.40 was prepared.Then 5 mM, 10 mM, and 15 mMNaOCl solutions of the Ganga river water and lab tap water with both monomeric and polymeric sensor NPh, PNPh, and Nor-PEG-oh were prepared and incubated.Then the resulting solutions mixture of analyte and sensor molecules was shaken well and incubated for 15 min at room temperature before recording the various electronic spectra.

Computation details
Time-dependent density functional theory (TDDFT) calculations were performed at the CAMB3LYP level as implemented in Gaussian 09 using 6-31 þ G(d) basis set for all atoms.Imaginary frequencies were 0 for all structures as a result of TDDFT.

Electronic (Uv-vis and fluorescence) spectroscopic analysis for NPh
To test our design's feasibility, we examined NPh's sensing behavior toward HClO/ -OCl in a PBS buffer of pH-7.40.
NPh displays an absorption band at 350 nm and no fluorescence band.There was no naked eye color change in the sensor solution mixture upon the addition of the NaOClinto solution mixture.Upon addition of two equivalents of -OCl, the probe immediately displayed a dramatic unusual cyanblue fluorescence turn-on response.The time-course fluorescence spectra showed that the fluorescence promptly increased to a maximum within 30 s after adding -OCl.Upon addition of increasing amount of -OClto the solution mixture of NPh, the absorption band centered at 350 nm and increased gradually.A new emission band appeared at 435 nm, a cyan-blue fluorescence color.
Interestingly, the fluorescence intensity of NPh was enhanced about 70-fold after treatment with -OCl, which can be another alternative signal for real sample analysis.Therefore, an examination of the fluorescence response of the sensor NPh toward various relevant interfering species, including ROS and RNS, was done to evaluate the sensor's selectivity toward -OCl.Furthermore, the probe exhibited no fluorescence response toward the small reactive biomolecules and RNS at biologically relevant concentrations and conditions.

Selectivity and sensitivity for NaOCl with monomer
In the selectivity test, 2.4 Equiv.-OCl as well as other interfering species and common ions such as H 2 O 2 , Cl -, Br -, H 2 S, , HPO 4 2-, NO 3 -, t BuOOH, ClO -, KO 2 , CaO 2, HO -have no impact on the recent monomeric sensors NPh fluorescence detection signal which was generated by the addition of -OClselectively.As shown in (Figure S14).From the pictorial presentation of the selectivity study with the monomeric sensor NPh, a significant cyan-blue fluorescence color was observed in the vial containing NaOCl under a hand-held laboratory UV lamp (wavelength ¼ 365 nm).Naked eye, no color change was observed from the other different vials containing various interfering ROS and anions except -OCl anion-containing vial.Other investigations suggested that the monomeric sensor NPh was not UV active, and the sensor's response toward NaOCl detection was fluorimetric but not colorimetric.The response was fluorometric by NPhonly toward analyteNaOCl (Figure 1).Concentration-dependent fluorescence titration spectroscopic analysis was done with monomeric sensor NPh (25 mM) by gradually adding NaOCl (0 mM −60 mM).From the concentration-dependent emission spectroscopic analysis with the gradual addition of NaOCl into 8:2 DMSO: H 2 O solution, observed that the fluorescence intensity at 435 nm was increased continuously.After adding 40 mM of NaOCl, a nearly complete turn-on response of the fluorescence intensity was observed by NPh.Linear regression plot was achieved by the PL intensity at 435 nm with NPh when -OClconcentration function was recorded.The minimum amount of -OCl that can be detected is evaluated to be 126.93nM (limit of detection, LOD), (R 2 ¼ 0.95778) (S/N ¼ 3) which is comparable to that of the previously reported oxidation of phenol-based fluorescence turn-onICTactive monomeric sensor.Fluorescence spectroscopic analysis was done with the monomeric sensor NPh after the addition of different concentration of -OCl, oxidation of NPh happens which can disturb the conjugation and changes the emission properties.By the gradual addition of the -OCl, the -OH functional group of NPhwas converted to the corresponding aldehyde through an oxidation reaction.The spectroscopic emission studies confirmed that the oxidation reaction was speedy (giving maximum response within 15 s of NaOCl addition) and was completed within 15 min.The emission maxima of the sensor NPhin aqueous medium at 435 nm increased upon continuous addition of -OCl, indicating the efficient oxidation of sensor NPhin in an aqueous environment [51,52] .The fluorescence emission maxima were increased significantly when -OCl is titrated from 0 to 60 mM.As depicted in Figure 2.This can be ascribed to conjugation breakage caused by the oxidation of aniline units on the backbone of NPh.The detection study of -OCl, results show that the sensor NPh shows an efficient fluorescence response to -OCl in the physiological pH-7.4.For real-life sample analysis, the sensor molecules must exhibit a significantly different signal toward the target analyte.So, our sensor system is a perfectly efficient sensor that can be applied to real-life problem-solving systems.denoted by I (9.472 ppm) were for -OH proton, which vanished in spectra [b] in the 1 H NMR titration spectra.This might be due to the deprotonation of the -OH group from the aromatic ring.Moreover, the aromatic -CH proton experienced a massive up-field shift from (7.0114 ppm, 6.827 ppm)to (6.65 ppm, 6.4 ppm) (Dk�0.4ppm)with considerable broadening and finally became indistinguishable.This confirms the direct involvement of the para-aminophenol moiety in the NaOCl-assisted oxidation reaction.Protons present in the spectra Other peaks for aliphatic carbon in spectra [1] corresponded to norbornene moieties and did not show so many changes in 13 C NMR spectra [2] After adding two equivalents of NaOCl.This confirms that an oxidation reaction happens on the 4-amino phenol entity of the monomeric sensor NPh.No involvement of norbornene moieties was observed.From the above analysis and observation, it was confirmed that there was a unique interaction of NaOCl with both the monomeric sensor molecules NPhand polymeric sensor PNPh-Peg(via oxidation reaction process of -OH to -C¼O) (Figure 3).

DFT calculation
To rationalize the experimental results, we optimized the structure of both NPhand Nor-al using the B3LYP/6-31G � level of theory.NPh'soptimized structure showed a geometry that does not facilitate the ICT interaction.Still, feasible ICT became possible after the addition of NaOCl and the formation of Nor-C¼O into the structure.Efficient intramolecular charge transfer led to the formation of a charge-separated state.From geometrical analysis, it was observed that NPhnon-planner geometry has the lowest energy and most stable.But after the oxidation reaction of NPh with NaOClNor-C¼O formed, which most stable geometry is comparatively planner in nature.A distribution of orbitals similar to Nor-C¼O is impossible in NPh due to its nonplanar geometry, which essentially restricts electronic communication between the donor and acceptor moiety (Figure 4).So, molecule NPh was not fluorescent.Still, Nor-C¼O was a fluorescent molecule due to HOMO-LUMO charge separation, and it was also ICT active molecule in nature.
The energy difference between the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), was obtained by frontier molecular orbital analysis of both NPhand NPh þ NaOCl.The lowest-energy electronic transition was found to be 0.186 a.u. and 0.172 a.u.for NPhand Nor-C¼O respectively.This significant difference in the HOMO − LUMO gaps quantifies the turn-on fluorescence spectra seen experimentally.Also, it was observed that in the case of Nor-C¼O, the difference in HOMO and LUMO decreased drastically; hence emission was observed.From the calculation, it was observed that the difference between the positions of HOMO and LUMO wave functions caused the prevention of non-radiative relaxation pathways for Nor-C¼O.The result was based on the optimized molecular geometries and NPhand Nor-C¼O energy calculations.But in the case of NPh, both HOMO and LUMO wave functions were located on para-aminophenol and norbornene moieties (Figure 5).Therefore, from the theoretical analysis of NPh, it was observed that HOMO and LUMO become non-planners in nature, which is responsible for the non-radiative relaxation pathway, and hence molecule was non-fluorescent [53][54][55] .The observed unusual bluish fluorescence revealed the possibility of the interaction between the norbornene double bond and the NaOCl functionality.From the above observation, we can confirm that this is the first report on the NaOCl sensor through unusual bluish fluorescence turn-on by all sensor molecules that carry the para-amino phenol moieties.

The sensing mechanism of sensors NPhtowards NaOCl
UV-vis and fluorescence spectroscopic analysis confirmed that ICT (intermolecular charge transfer) processes "off to on" mode was responsible for the drastic turn-on of intense bluish fluorescence at 435 nm for both monomeric and polymeric sensor NPhand PNPh-Peg.Job's plot analysis and calculation confirm a monomeric sensor's 1:1 (1 NPh:1NaOCl) interaction ratio(Figure S15).Both monomeric and polymeric sensor response was similar to NaOCl.The Job's plot result is also applicable to the polymeric sensor PNPh-Peg.
An overall mechanism was, after adding NaOCl in the reaction mixture, an oxidation reaction happened by -OCl.NaOCl is an oxidizing agent, so it can take electrons from HOMO of NPhas electron-rich norbornene moieties have a chlorine atom with a vacant d-orbital in its valence shell responsible for the accommodation of extra electrons.The above observation also was confirmed by DFT calculation and HOMO-LUMO position in the sensor molecule of NPh.NPhand PNPh-Peg with NaOCl DFT (density functional theory) were evaluated to ensure the interaction mechanism of sensors.DFT data confirmed that the ICT process was  possible for Nor-C¼O due to the prominent lesser energy difference (0.172 a.u.) between HOMO and LUMO compared to the molecule NPh(0.186a.u.).
The oxidation reaction mechanism between NPhand NaOCl was investigated and explained by 1 H NMR and 13 C NMR titration spectra analysis to explore the unusual blue fluorescence turn-on.As by the oxidation process, the aromatic peaks become aliphatic, it was found that NPharomatic protons, responsible for amino phenol moiety, shifted to the up-field region upon adding NaOCl.After adding NaOCl, the peak corresponding to -OH hydrogen (i, 9.472 ppm) vanished due to the oxidation of -OH to -CHO.Besides, all the protons regarding aliphatic norbornene moiety did not show significant change after adding two equivalents of NaOCl.Instead, a characteristic carbon signal at 180.021 ppm was observed for the newly generated -C¼O bond after NaOCl addition.The shift in the signals for only carbon of norbornene-anhydride was attributed to the oxidation reaction and loss of aromaticity of amino phenol moiety.All the peak from Norborne anhydride aliphatic carbon remains unchanged after the oxidation reaction by NaOCl.However, the ESI-MS experiment did not support the proposal.To confirm the formation of new molecules, TLC analysis was performed.
Interestingly, a fluorescent spot with higher polarity was observed.We proposed that two molecules with the same mass but different polarities could be possible only in the case of an intramolecular rearrangement reaction, based on mass and TLC analyses.This prompted us to hypothesize that the NaOCl detection process involved the oxidation reaction to form Nor-C¼O.
An IR test provided further evidence to support the reaction mechanism.IR stretching frequency for -C¼O of norbornene anhydride shifted from (1666.14 cm −1 ) to (1634.38 cm −1 ) after adding 2 equivalents of NaOCl.The observation was due to the loss of aromaticity by oxidation of aminophenyl moiety and the formation of aliphatic double bonds.The NPhstretching frequency of the -C¼O group was significant in the sensor due to the additional -I effect inserted by a conjugated aromatic moiety of amino-phenol segment.After the oxidation reaction, the -I effect does not happen, and due to that, the polarity difference decreases for the -C¼O bond and the stretching frequency also decreases.So, due to the oxidation of the phenol segment by NaOCl, a low energy charge separation happens, which is responsible for the delocalization of charge via ICT (intermolecular charge transfer) and turn on of unusual intense cyan blue fluorescence color (Figure S16).
From results, as mentioned above, demonstrate that the sacrificial reaction between NPh and NaOCl is an essential protective mechanism for NaOCl detection, as schematically illustrated in (Figure 6).Moreover, unlike the monomeric sensor, its water-soluble polymer also shows the same type of response in the presence of NaOCl.So, it is confirmed that our work (NPhand PNPh-Peg) shows the oxidation of aminophenol moiety based on an unusual fluorescence turnon response in the presence of NaOCl.

Selectivity and sensitivity for polymeric sensor toward NaOCl
From the previously discussed experiments, NPh is highly selective and sensitive toward NaOCl sensing by ICT active fluorescence turn-on mode response.But as usual, being a small, fully organic molecule, NPh suffered from insolubility in pure water, making the sensor molecule a less efficient probe for NaOCl detection in all infield application purposes.To resolve this outcome of a small sensor molecule, we synthesized a random copolymer between NPhand Nor-PEG using the ROMP technique (incorporation of PEG in the polymer).This random polymer becomes significantly water-soluble, which can detect NaOCl very efficiently from the aqueous environment.The sensing efficiency dynamics were recorded for PNPh-Peg and NaOCl in an effective aqueous medium, and the response time turned out to be less than 5 min, which was quite fast.The absorbance spectrum of PNPh-Peg consists of a peak around 350 nm which was decreased after the addition of analyteNaOCl.No emission in fluorescence, spectroscopy was observed for the blank polymeric sensor PNPh-Peg itself.The "on" mode of emission peak at 435 nm was observed due to the ICT (intermolecular charge transfer) from the HOMO of electron-rich oxidized amino phenol moiety toward electrondeficient LUMO of the same oxidized version of amino phenol moiety after the addition of NaOCl into the sensor solution mixture.When NaOCl was added to sensor PNPh-Peg, a strong cyan blue emission peak around 435 nm was recorded upon excitation at 350 nm.Surprisingly, the polymeric sensor's response time was much lower than the monomeric sensor NPh.Through naked eye detection, there was no visible color change in the solution.In contrast, an intense cyan-blue fluorescence color was observed after adding NaOCl into the sensor (PNPh-Peg) solution under a handheld UV-lamp.Upon addition of various inferring species like ROS and common anionsH 2 O 2 , Cl -, Br -, H 2 S, -OCl, NO 2 -, HCO 3 -, HSO 4 -, SO 3 2-, CO 3 2-, HPO 4 2-, NO 3 -, t BuOOH, ClO -, KO 2 , CaO 2, HO -no noticeable fluctuation of fluorescence spectra was observed.As shown in (Figure S17).The above observation confirmed that the exciting spectroscopic change was due to the intermolecular charge transfer from HOMO to LUMO of the oxidized form of para-amino phenol moiety of polymeric sensor molecule PNPh-Peg, which generated changes that occurred in the sensors by the interaction with NaOCl.From the pictorial presentation of the selectivity study of the monomeric sensor, polymeric sensorPNPh-Peg, a cyan-blue fluorescence color was observed in the vial into which NaOCl was added under a handheld laboratory UV lamp (wavelength ¼ 365 nm).Naked eye, no color change was observed from the different vials containing various interfering ROS and anions.So, the water-soluble PEG-incorporated polymeric sensor was not UV active, and the sensor's response toward NaOCl detection was not colorimetric.Still, only the fluorometric response by PNPh-Peg toward analyteNaOCl.The above spectroscopic result indicated that the sensor PNPh-Peg was susceptible to NaOCl, and the detection can be utilized in real-life applications (Figure 7).Fluorescence spectroscopy was done to determine the Sensitivity and efficiency of the polymeric sensor PNPh-Peg with analyteNaOCla concentration-dependent titration.After the gradual increase of NaOCl concentration, fluorescence intensity was increased gradually.Using this titration plot, an excellent linear relationship (R 2 ¼ 0.96969)was obtained between the fluorescence intensity and concentration of NaOCl in the 1-60 mM.Results (S/N ¼ 3) indicated that the LOD of sensor PNPh-Peg for detecting NaOCl was 59.14 nM, relatively lower than monomeric sensor NPh (Figure 8).This is another significant advantage of the polymeric sensor system over the monomeric sensor system.Also, it was observed that with the continuous addition of NaOCl into the solution mixture of the polymeric sensor for the fluorescence titration experiment, a very intense cyan blue-colored fluorescence was generated gradually from the non-fluorescent solution mixture.Therefore, the polymeric sensor PNPh-Peg was susceptible to NaOCl qualitatively and quantitatively.Furthermore, all spectroscopic analysis and observation confirmed that, unlike the monomeric sensor NPh, all the sensing mechanisms and interaction ratios were also applied to the polymeric sensor PNPh-Peg.

Real-life application of NPhandPNPh-Peg toward various types of water
Water solubility is the fundamental condition for a fluorescent active sensor molecule to work efficiently in real-environmental applications like the detection of industrial toxicants or in vivo detection of toxic molecules.Both the monomeric and polymeric sensor moleculesNPhand Nor-PEG-oh were used to investigate the efficiency of recovery of NaOCl from different water samples of the Ganga river water and tap water.Three types of NaOCl concentrations (5, 10, and 15 mM) were selected for the measurement of fluorescence intensity changes for Ganga River water and tap water in a similar test solution medium.All measured recovered concentrations of -OCl was excellently matched with the respective spiked concentrations.The recovery rates were within the range of 85-100%, concluding that monomeric sensor NPhand polymeric sensor PNPh-Peg was accurate for detecting NaOCl in natural water samples and potentially employed in every environmental system.NaOCl can be quantified in the concentration of 0-100 nM from different actual water samples with excellent linear correlation coefficients(0.95778and 0.96969, respectively), indicating  that NPh and PNPh-Peg very much suitable as an infield detection tool for NaOCl from real environmental water samples.The maximum level of NaOCl in drinking water permitted by the World Health Organization is 15 mM (500 ppb), NaOCl detected from the spiked samples was down to 1.5 mM.Therefore, the high Sensitivity suggests that monomeric NPhand polymeric PNPh-Peg sensors could be applied sensitively and efficiently to practical environmental analysis.They are shown in Table 1 and Table 2.

Bleaching test analysis by NPhand PNPh-Peg
For the analysis of the bleaching capability of both monomeric and polymeric sensor NPh and PNPh-Peg we have collected NaOCl-containing branded bleaching powder from nearly super market.Then 100 mg of bleaching powder was dissolved with 20 ml of HPLC water and the whole solution mixture was centrifuged for 30 min after that residual formed was filtered out by syringe filtration.Then the fluorescence spectroscopy was done by both monomeric and polymeric sensors NPh and PNPh-Peg with the addition of 100 mlfiltrate containing NaOCl solution in PBS buffer at pH 7.4 2:8 DMSO: Water solvent system.Unknown concentration was calculated using the linear regression plot of NPh and PNPh-Peg with NaOCl.Recovered NaOCl concentration was obtained 2.6032 � 10 −5 M and 6.6802 � 10 −5 M respectively.The fluorescence spectrum was given in (Figure S18) for NPhand (Figure S19) for PNPh-Peg.

Paper strip application by PNPh-Peg
If the probe could work in the solid state it would be more convenient for the application part in practical detection cases, like the preparation of paper strips.Thus, the sensing performance on test papers was excellently performed by coating with polymeric sensor PNPh-Peg.The test paper was fabricated by dropping of THF solution of polymeric sensor PNPh-Peg upon the TLC paper and was dried in the air, then with the help of a high vacuum for 24 h.The sensing behavior of polymeric sensor-coated paper was dependent on the concentration of sensor solution and under the handheld UV light, also it was observed that the PNPh-Peg coated paper strip was colorless.
An excellent and instant 'turn-on' (cyan blue fluorescence) response was observed when only a few drops of NaOClsolution were added to the polymeric sensor grafted paper strip.It was also observed that keeping the paper strip in the solution of NaOCl also created a prominent fluorescence color change in the paper strip.Furthermore, there was no response of the PNPh-Pegcoated strip in the presence of the other interfering chlorinated compounds, ROS,  and RNS water vapor was observed.The above experiments and observations reveal the ultra-sensitivity of our recent polymeric sensor toward real-life NaOCl contamination detection (Figure 9).

Comparison data for reported references
Some previously reported fluorescent probes as a detection site for NaOCl detection as to NPh and PNPh-Peg, including the limit of detection, time for response, and solvent medium listed in supporting information.Most organic sensors have unique applications in real-world life.Still, in our work, the monomeric sensor NPhand polymeric sensor PNPh-Peg have advantages in easy synthesis, extended wavelength emission, and more significant efficiency.Paper strip preparation and the reaction time of 10 min realize fast detection, making it possible for NPhand PNPh-Peg would be applicable for instant detection of the edibles.In addition, NPhand PNPh-Peg can recognize NaOCl in the actual water samples, which illustrates that NPhand PNPh-Peg may become a system for investigating NaOCl amount in real environmental systems Table S1.

Conclusion
In summary, a novel highly selective fluorescent sensor NPh was efficiently designed, synthesized, and characterized to detect -OCl.We first created and synthesized an ICT-active fluorescent sensor NPh incorporating the p-aminophenol moieties for their recognition unit of -OCl.As expected, NPh exhibited significant fluorescence turn-on emission at 435 nm, an instant response time (< 15 min), and excellent activity for detecting -OCl, via a unique -OCl, incorporated intramolecular charge transfer mechanism.NPh exhibits preeminent selectivity toward -OCl, over RNS, thiols, and other ROS, including H 2 O 2 , Cl -, Br -, H 2 S, -OCl, NO 2 -, HCO 3 -, HSO 4 -, SO 3 2-, CO 3 2-, HPO 4 2-, NO 3 -, t BuOOH, ClO -, KO 2 , CaO 2, HO -which might be ascribed to the oxidation of phenol moiety.Moreover, NPh can accurately determine -OCl, at the nanomolar level and is the first fluorescent probe for the picomolar level analysis of -OCl.More importantly, this response mechanism would provide a new strategy for constructing specific -OCl contamination detection indicators.Our recent analysis refers to easy-to-synthesize unique fluorescent turn-on sensors for the determination of -OCl, from the picomolar level of contamination and is a perfect system for real-life lower concentrations of -OClquantification from living organisms.
To investigate PEG-incorporated molecules' water solubility, we developed two types of random polymer PNPh(without peg)and PNPh-Peg.The sensor molecule was highly selective and sensitive compared to typical ROS.Unlike chromophore-containing small-molecule fluorescent sensors, this unique kind of polymeric sensor operates through fluorescence turn-on caused by oxidation-induced polymers.For efficient response to -OCl, in solution, applying the sensing materials for detecting -OCl, the paper strip was also successfully demonstrated.This novel type of work shows the first example of the potential, but still unexplored, applicability of water-soluble polymeric sensors for the detection of ROS (specifically NaOCl).This charge transfer fluorescence active water-soluble random copolymer can be directly used as a NaOCl sensor for environmental water analysis.

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-, KO 2 , CaO 2, HO -were added respectively to a 25mMsolution of NPh.The experimental solution mixture was kept in parallel at room temperature for 15 min, and then the fluorescence spectra were recorded.Monomeric sensor NPh caused a robust fluorescence enhancement (about 70-fold) with only -OCl, while the others exhibited almost no change in fluorescence behavior.Selectively, NPh shows excellent selectivity for -OCl without any interference from �OH and other reactive oxygen species (ROS), including H 2 S, t-BuOOH, and H 2 O 2 .Other interfering ROS likeH 2 O 2 , Cl -, Br -, H 2 S, -OCl, NO 2
[1] denoted by a (6.798 ppm), b (2.81 ppm), c (1.401 ppm), and d (3.152 ppm) were for norbornene protons, which remained unchanged before and after the addition of NaOCl.The 13 C NMR spectrum of NPh showed an upfield shift of peak denoted from (177.73 ppm, 137.827 ppm, 138.29 ppm, 128.69 ppm, 121.42 ppm, 115.95 ppm, 47.87 ppm, 45.47 ppm, 43.14 ppm) to (179.02 ppm, 138.68 ppm, 128.52 ppm, 123.01 ppm, 118.08 ppm, 48.12 ppm, 45.88 ppm, 43.42 ppm) upon interaction with NaOCl, presumably due to oxidation reaction the aromatic ring loses its aromaticity and conjugation with carbon corresponds to e.In 13 C NMR, a peak for j vanishes in spectra [2] after the addition of 2 equivalent of NaOCl due to oxidation of -OH to -C¼O group, which is further confirmed by the generation of a new peak at (180.02 ppm) denoted as i.Due to the oxidation process and loss of aromaticity, the peak denoted as f in spectra [1] also got an upfield shift from (121.42 ppm) to (123.01 ppm) in spectra [2].

Figure 5 .
Figure 5. Calculated FMO energy diagram for the most stable structure of NPh and nor-C ¼ O by DFT calculation.

Figure 6 .
Figure 6.Pictorial presentation of the interaction mechanism of NaOCl with NPh.

Figure 9 .
Figure 9. Paper strip coated with PNPh-Peg(100 ml from 1 mg/1 ml stock solution)shows the response based on the various concentrations of NaOCl (determination of the efficiency of our sensing method).

Table 1 .
Real water sample analysis by NPh spiked with NaOCl.

Table 2 .
Real water sample analysis by PNPh-Peg spiked with NaOCl.