Aggregation induced emission based fluorenes as dual-channel fluorescent probes for rapid detection of cyanide: applications of smartphones and logic gates

Rational modification of molecular structure by incorporating electron donating groups can play a potential role for designing aggregation induced emission (AIE) active fluorescent probes. Based on this principle, fluorescent probes (1a–c) were synthesized, and they displayed excellent aggregation induced emission (AIE) behavior in a H2O/DMF (4 : 1, v/v) mixture due to restrictions in intramolecular charge transfer (ICT). As a comparison, probe 1d was synthesized by installing an electron withdrawing (–NO2) group that surprisingly quenched the aggregation behaviour. Additionally, AIE active probes 1a–c displayed a highly sensitive dual channel (fluorometric and colorimetric) response towards rapid detection of CN−, which is an active toxic material. Probes 1a–c showed selectively enhanced fluorescence emission behavior towards CN− with detection limits of 1.34 ppb, 1.38 ppb, and 1.54 ppb, respectively. The sensing mechanism involves Michael type adduct formation due to the nucleophilic addition reaction of cyanide with probes and was confirmed through 1H NMR titration experiments. In contrast, probe 1d containing an electron withdrawing moiety showed insensitivity towards CN−. Therefore, this study provides the efficient strategy to induce AIE character in fluorescent probes and expands the mechanistic approach toward the sensing of toxic CN−.


Introduction
The rational design and synthesis of aggregation-induced emission (AIE) active probes has acquired a lot of attention due to their broad range of applications such as uorescence sensors 1 photodynamic therapy, 2 data security, 3 and organic light-emitting diodes (OLEDs). 4 However, many conventional uorescent probes display high uorescence emission in dilute solution, whereas the planarity, p-p stacking interaction, or excimers in solid or aggregated state quench their uorescence emission. This phenomenon is known as aggregation-caused quenching (ACQ). 5 It markedly hampers the performance of uorescent probes in high-tech applications because uorescent probes are generally used in their solid/aggregated state for real-world applications. In contrast to ACQ, aggregation induced emission (AIE) active probes exhibit high uorescence emission in the aggregated state and less or no emission in solution forms, which enormously increases their practical applicability. 6 Considering the extensive applicability, a large variety of AIE active uorescent probes viz. tetraphenylethylene, 7 distyrylanthracene, 8 silole, 9 and carbazole derivatives 10 have been designed and synthesized depending upon different approaches. Previous studies proposed numerous mechanisms to decipher the AIE phenomenon such as restricted intramolecular rotation (RIR), 11 twisted intramolecular charge transfer (TICT), 12 intramolecular charge transfer (ICT), 13 and cis-trans isomerization. 14 One effective strategy to induce AIE character is the installation of strong electron donor (D) moieties in uorescent probes. In our recent approach aimed at construction of AIE active materials, it was intended to modify our previously reported uorescent probe by incorporating strong electron donating groups in simple uorene. 15 Interestingly, slight structural modications (introduction of electron donors) in the attached moieties induced completely newer and unique feature of aggregation induced emission (AIE) due to electronic push-pull effect (Fig. 1). More importantly, this study extends the mechanistic approach that how an exquisite change in the molecular structure of uorescent probe can modulate aggregation induced emission (AIE) properties. Further, it was noticed that the introduction of electron withdrawing group (-NO 2 ) in uorene core (probe 1d) quenched aggregation (ACQ) due to intersystem crossing. Therefore, this study brings a strategy to facilitate the development of efficient AIE active uorescent probes.
Keeping in mind the broad applications of AIE active uorescent probes, newly developed probes (1a-d) were efficiently used for the uorescent detection of cyanide ions (CN À ), which is an active hazardous material. Long-term skin exposure of cyanide ions can lead to numerous health hazards, for example, hypoxia, loss of consciousness, endocrine disorders, convulsions, respiratory damage, and even death. 16 Up to present, various traditional detection methods viz. chromatography, voltammetry, potentiometry, and electrochemical methods have been developed for the efficient detection of toxic CN À . [17][18][19][20] Yet, various disadvantages are associated with these traditional techniques i.e. complicated detection process, time-consuming, need bulky and expensive equipment that provides some room to exploit alternate appropriate detection methods. To compensate conventional methodologies, uorescent probes are employed for the recognition and determination of targeted anions. They hold some promising predominance over the previous methodologies including high sensitivity, cost effective, facile operation, and efficient biological diagnosis. 21 At present, CN À probes have been categorized into different classes including hydrogen bonding, 22 nucleophilic addition, 23 coordination, 24 anion-metal affinity, 25 and deprotonation 26 depending upon their interaction with cyanide. Deprotonation and H-bonding based cyanide probes have quick response time but the detection process can easily be disturbed by other competing anions such as phosphate and uoride. CN À probes based on coordination mechanism commonly involve two steps (viz. addition of metal ions and then adding cyanide) that make the recognition process quite inconvenient and tedious. On the other hand, nucleophilic addition reaction based CN À probes has anti-interference ability, sensitivity, and excellent selectivity due to the distinguished nucleophilic character of cyanide ions. In this context, olen based uorescent probes were designed and synthesized by Guo et al. for the detection of cyanide where CN À disturbed the conjugation system and thereby resulted in the interruption of intramolecular charge transfer (ICT) process. 27 Cheng et al. synthesized indole based carboxycoumarin probe that underwent C]N nucleophilic addition reaction upon the attack of CN À to the indole unit. 28 However, aggregation caused quenching (ACQ) effect, slow response time, high detection limit, complicated synthetic procedures, and inefficiency of prior reported reaction based CN À probes in aqueous medium limit their applications in real environment.
In continuation of our efforts to design uorescent probes, 6,15,[29][30][31][32][33][34][35][36][37][38] newly synthesized probes (1a-d) offered valuable insight into the mechanistic approach involved in the sensing of CN À . The introduction of -NO 2 substituted aromatic ring in probe 1d seems to play decisive role, because electron withdrawing -NO 2 group did not allow the probe to bind or react with CN À . Reduced reactivity of olen functionality toward CN À may occur due to the presence of electron withdrawing -NO 2 group and formation of less stable carbocation. In fact, electron withdrawing groups make olen moiety less favorable for nucleophilic addition reaction and vice versa. Sensing mechanism of nucleophilic addition reaction of probes and CN À was conrmed through 1 H NMR spectroscopy. The sensing ability of probes towards cyanide was further investigated through uorescence emission, UV-visible absorption, computational studies, dynamic light scattering (DLS), and Jobs plots. To prove practicability, probes were utilized to detect CN À in easy to make test strips and for the detection of endogenous CN À ions in actual samples including peach kernel, bitter almond, and cassava.
The same procedure was opted to synthesize compounds 1b, 1c, and 1d that resulted in the yellow, yellow, and grey colored crystals, respectively.
The uorescence experiments were performed at an excitation wavelength (l exc ) of 340 nm opting slit width of 2/2 nm.

Calculation of uorescence quantum yield
The uorescence quantum yield (F) of probes (1a-d) was determined by using uorescein as a standard compound (F ¼ 0.95) at an excitation wavelength (l exc ) of 340 nm and slit width of 2/2 nm on the spectrouorophotometer. The quantum yields (F) of probes was calculated through eqn (1), where F s and F r represent the relative quantum yield of the samples and reference, respectively. A s and A r are equivalent to the absorbance of samples and reference, respectively. D s is the area of emission of samples, while D r is the area of emission of reference compound. The lengths of absorption cells is represented by L s and L r , whereas N r and N s are the refractive indices of the reference and sample solutions.
Instruments and reagents used during this study are provided in the ESI (SI-1 †).

Statistical data analysis
Fluorescence sensing experiments were presented as mean and standard deviation with n ¼ 3. Statistical analysis of the obtained results was executed by employing GraphPad Prism with paired Student's t-test. Further, the linear t model was evaluated through analysis of variance (ANOVA) (Table S1 †); the value of p < 0.05 is statistically important. Similarly, normal data distribution (random error) and a condence interval of 95% were considered for statistical evaluation.

Effect of solvents
Since solvents' polarity may affect the ground and/or excited state energy levels and consequently shi the emission/absorbance of a probe to a lower/higher wavelength. 39 Hence, the uorescence spectral properties of probes 1a, 1b, 1c, and 1d were evaluated in different solvents including ethyl acetate, tetrahydrofuran (THF), ethanol, methanol, acetonitrile (CH 3 CN), dichloromethane (DCM), chloroform (CHCl 3 ), and toluene. Probes were insoluble in ethanol and methanol. Meanwhile, increasing solvents polarity from weakly polar toluene to strongly polar DMF, the uorescence emission of probes 1a, 1b, 1c, and 1d was progressively red-shied from 409, 410, 407, and 408 nm to 418, 414, 410, and 412 nm, respectively (Fig. S1 †). The redshi in uorescence emission was also associated with a considerable decrease in emission intensity. Conversely, the UV-visible absorption of probes 1a, 1b, 1c, and 1d at 370, 352, 353, and 300 nm displayed insignicant changes on varying the polarity of solvents (Fig. S1 †). These spectral changes show that increase in solvents' polarity perturbs the intramolecular charge transfer (ICT) process, thereby affecting the uorescence emission.

Aggregation induced emission (AIE) effect
Organic compounds are readily soluble in organic solvents but are commonly insoluble in water. Water insoluble compounds tend to agglomerate upon the addition of water that may subsequently alter the uorescence emission. The possible changes in uorescence emission of probes were investigated by using binary solvent system with varying water fractions from 0 to 90%. As shown in Fig. 2 and S2, † probes 1a (20 mM), 1b (50 mM), and 1c (10 mM) were almost non-emissive in DMF. The uorescence emission of probes was retained constant with a marginal decrease in emission along with a signicant redshi until the water fraction reaches 30%. This is probably due to effective involvement of ICT process. Upon further addition of water fractions (f w ) from 30% to 80%, signicant enhancement in uorescence emission of probes 1a, 1b, and 1c was noticed. It is due to the aggregate formation of probe molecules due to the addition of polar water that led to the restrictions in intramolecular rotations (IRs) and partially conned intramolecular charge transfer (ICT). It activates radiative decay process that ultimately results in enhanced uorescence emission. 40 Meanwhile, when the water fraction increased from 80% to 90%, the uorescence emission of probes slightly reduced that indicates the formation of amorphous aggregates at higher water contents ( Fig. 2 and S2 †). In contrary to this, the addition of water fractions (0-90%) considerably attenuated the uorescence emission of probe 1d (20 mM) (Fig. S2 †). It shows that the introduction of nitro (-NO 2 ) group to 1d led to the uorescence quenching that is probably due to the internal conversion (IC) or intersystem crossing (ISC). 41 All these uorescence changes are associated with color changes of probes (1a-d) under UV radiations (365 nm) upon gradual increase in water fractions in DMF solution. As   Fig. 3, the emission of probes (1a-c) was gradually increased up to 80% water and noticeably diminished upon the addition of water from 80 to 90%. However, uorescence emission of probe 1d was continuously reduced upon changing the water fractions from 0 to 90%. Next, the uorescence quantum yield of probes (1a-d) was measured by using eqn (1) (given in experimental part) to further decipher the aggregation induced emission (AIE) character. The quantum yields (F) of probes 1a, 1b, 1c, and 1d were calculated as 0.23, 0.28, 0.35, and 0.59, respectively in DMF. The quantum yields (F) of probes 1a, 1b, and 1c were substantially increased to 0.67, 0.63, and 0.58 in H 2 O/DMF mixture with 80% water fractions (f w ). However, probe 1d observed a notable decrease in quantum yield to 0.25. These outcomes support the strong emissive behavior of probes 1a, 1b, and 1c in aggregated state.

evidenced in
Further, the uorescence emission of probes (1a-c) was investigated in ethylene glycol/DMF mixture with different ethylene glycol fractions (f w ). It was assumed that the uorescence emission of probes would be substantially enhanced upon the addition of more viscous solvent, ethylene glycol. It is demonstrated in Fig. S3 † that the emissions of probes 1a, 1b, and 1c showed distinct response in ethylene glycol/DMF mixture (ethylene glycol; 0-90%), suggesting the aggregate formation as a reason for their enhanced uorescence emissions. This splendid uorescence emission enhancement was quantied by establishing a linear t plot taking uorescence intensity as a function of ethylene glycol fractions (Fig. S3 †). Additionally, the aggregation induced emission (AIE) behavior was justied through the concentration dependent uorescence emission response of probes (1a-c) in H 2 O/DMF (4 : 1, v/v). Upon increasing the concentrations of probes from 0 to 50 mM, probes 1a, 1b, and 1c displayed very weak uorescence emissions with marginal better emission at 20 mM, 50 mM, and 10 mM (Fig. S4 †) concentrations. These results evidently favor that uorescence emission of probes is enhanced due to aggregate formation (AIE) and not because of concentration changes.
Sensing studies pH effect. Occasionally, small changes in pH inuence the uorescence response of probes towards anions. To check the applicability of probes, uorescence spectra of probes were recorded in H 2 O/DMF (4 : 1, v/v) system before and aer the addition of CN À (500 nM) over a wide pH range (2.0-12.0). Fig. S5 † implies the enhanced uorescence response of probes towards CN À in the pH range of 7.5-8.5. However, probes displayed insignicant uorescence response in the acidic pH (0-6.5 pH) which shows that probes didn't undergo a nucleophilic addition reaction with CN À in an acidic medium. Furthermore, study of pH effect articulates that probes are ideal to detect CN À in aqueous medium with a rapid response time since the addition of excess amount of NaCN in water changes its pH from 7.0 to 8.3-8.5 that relates the optimized pH of probes.
characteristic band at 370, 352, 353, and 300 nm, respectively. Upon the addition of CN À , the intensity of (1a, 1b, and 1c) characteristic peaks was increased and a completely new sharp peak appeared at 505, 590, and 425 nm (Fig. S6 †). However, other anions were unable to perturb the absorption of probes. Interestingly, probe 1d remained insensitive towards all targeted anions. These experimental ndings clearly indicate that probes are highly selective towards the detection of CN À except probe 1d that remained insensitive towards targeted anions. Selective detection of CN À is probably ascribed to its greater nucleophilicity, strong basicity, weak hydration enthalpy (À295 kJ mol À1 ) in semi-aqueous medium as compared to other anions. 42 However, the inability of probe 1d to sense CN À is probably due to its electron withdrawing group (-NO 2 ) that didn't favor the nucleophilic reaction to occur. Next, the sensitivity of probes 1a (20 mM), 1b (50 mM), 1c (10 mM) towards CN À was elaborated through UV-visible titration of probes by adding incremental concentration of CN À . As the concentration of cyanide was increased from 0 to 500 nM, gradual hyperchromic shi in the characteristic UV-visible absorption band of probes was noticed along with an appearance of a completely new band at 505, 590, and 425 nm that denotes the formation of new species (Fig. 4). Besides, obvious color change of probes 1a, 1b, and 1c from yellow, greenish, and light yellow to dark red, brown, and bright yellow during the titration experiment also favors the formation of new species ( Fig. 4; insets).
Following the absorption experiments, a linear calibration curve was plotted for each of the probes 1a, 1b, and 1c to exclusively quantify the direct relation between the gradual increases in the CN À ions concentration (0-500 nM) and elevation in the absorption intensity (Fig. S7 †).

Fluorescence emission study
To evaluate the sensing potential of probes toward CN À , the uorescence emission studies of probes 1a (20 mM), 1b (50 mM), 1c (10 mM), and 1d (20 mM) were carried out in H 2 O/DMF (4 : 1, v/v) without and with the addition of targeted anions viz. CN À , F À , Cl À , Br À , I À , ClO 4 À , N 3 À , NO 3 À , H 2 PO 4 À , NO 2 À , and CH 3 -COO À . It is evident in Fig. S8 † that the concomitant addition of different anions, except CN À , didn't change the uorescence emission of probes 1a, 1b, and 1c, respectively. With the addition of CN À , the uorescence emission intensity of probes 1a, 1b, and 1c was abruptly increased with an observable redshi of 13, 8, and 5 nm, respectively. The selective uorometric response of probes toward CN À is probably due to the disruption in p conjugation of probes upon the addition of CN À ions. 43 As expected, the uorescence emission of probe 1d at 412 nm was retained unaltered upon the addition of targeted anions (Fig. S8 †). These results substantiated the excellent selectivity of probes 1a, 1b, and 1c towards CN À . In addition, effect of potential interferences was also investigated by simultaneous addition of targeted anions (20 equiv.) to the H 2 O/DMF (4 : 1, v/ v) solution of probes containing CN À (500 nM). As evidenced from the results presented in Fig. S9, † uorescence enhancement efficiencies of probes 1a (34 folds), 1b (27 folds), and 1c (21 folds) against CN À ions were not affected in the presence of other interferences, signifying favored selectivity of probes toward CN À even in complex environment.
To elaborate the uorescence sensing potential of probes, the uorescence titration experiments were performed by the progressive addition of CN À (0-500 nM) to H 2 O/DMF (4 : 1, v/v) solution of probes 1a (20 mM), 1b (50 mM), and 1c (10 mM). As a result, the uorescence emission of probes centered at 418, 414, and 410 nm was gradually increased with a concomitant redshi to 431, 422, and 415 nm, respectively (Fig. 5). A linear correlation was observed between relative uorescence intensity (F/F 0 ) and CN À concentrations in the range of 0-500 nM. This response was justied through linear calibration curves, correlation coefficient along with regression equation, provided in these curves (Fig. S10 †). The precision in the uorescence determination of the CN À ions was evaluated through relative standard deviation (RSD) (eqn (2)). It was calculated aer three replicate determinations (0-500 nM) of the CN À ions and was calculated to be 1.44%.
(2) where s p represents the standard deviation, and x p denotes the average of a given analytical standard (n ¼ 3). Further, these spectral changes were accompanied by the appearance of bright emission of probes under UV radiations (365 nm) upon the addition of CN À (1 mM) ( Fig. 5; insets). The binding constant (K a ) for the reaction of probes with CN À was calculated through uorescence titration experiments using the Benesi-Hildebrand equation. Plots of relative increase in uorescence emission of probes with increasing concentrations of CN À displayed excellent linear behavior and estimated binding constant values were determined to be 2.23 Â 10 6 M À1 (1a), 1.0 Â 10 6 M À1 (2a), and 3.42 Â 10 5 M À1 (3a) (Fig. S11 †). The high magnitude of the binding (association) constants (K a ) implied a strong association between probes and CN À . Moreover, the binding stoichiometry between probes and CN À was studied through the continuous variance method (Job's plot). For this purpose, equimolar solutions (50 mM) of probes and CN À were prepared and Job's plots were made between relative uorescence emission and increasing mole fractions of CN À . The resulting plots given in Fig. S12 † show the highest intensity at the composition of 0.5 : 0.5 of probes-CN À complex conrming the 1 : 1 binding stoichiometry and excellent sensitivity of probes with CN À . Distinguished sensitivity of probes for the sensing of CN À was further estimated through their limits of detection (LOD). The LODs of probes were determined through the IUPAC recommended protocols, using formula LOD ¼ 3s/S, where s is the standard deviation and S corresponds to the slope of the plot between uorescence intensity of probes versus cyanide concentration. The calculated detection limits of probes 1a, 1b, and 1c for the detection of CN À were found out as 1.34 ppb, 1.38 ppb, and 1.54 ppb, respectively. These values are sufficiently lower than the WHO limit of CN À concentration in aqueous medium (1.9 mM). 44 Calculated LOD values are based on the standard deviation from the blank signal intensity of the probes in triplicate experiments.
Sensing mechanism 1 H NMR titration study. 1 H NMR titration study of probe 1a without and with the addition of cyanide (in the form of NaCN) was carried out in DMSO-d 6 to delineate the sensing mechanism for the detection of CN À . As shown in Fig. 6, alone probe 1a displayed three signals appeared at d 7.80, 7.38-7.32, and 7.87-7.85 ppm corresponding to vinyl proton (H a ), two aromatic protons present in uorene core (H b ), and two aromatic protons present in attached phenyl unit (H c ). Upon successive addition of CN À (0-1.0 equiv.), vinyl proton peak was disappeared and all aromatic protons were shied completely upeld. All these spectral changes are evidence of covalent interaction between probe and CN À that resulted in the formation of a new Michael type adduct. However, to obtain the covalent interaction, the probe-CN À mixture was kept for 1 hour aer mixing the each equiv. (0.5 and 1.0) of CN À to probe.
The sp 2 -hybridized vinyl carbon of probe 1a (1b, 1c, and 1d) renders alkene carbon slightly electrophilic/carbocation. Electrophilic carbon (partially carbocation) is stabilized by conjugation with electron donating groups (in 1a, 1b, and 1c). As a result, stabilized vinyl carbon undergoes nucleophilic addition reaction with a strong nucleophile, CN À that consequently generates Michael type adduct as illustrated in Fig. 6. On the other side, electron withdrawing group (-NO 2 ) attached to probe 1d makes its vinyl carbon less stable and less favorable for nucleophilic addition reaction initiated by CN À . Fluorescence results clearly show that probes 1a, 1b, and 1c favorably react with CN À but probe 1d remained completely insensitive towards CN À .
Density functional theory (DFT) study. The sensing CN À was further substantiated through FMOs analysis via density functional theory (DFT) calculations by using Gaussian 09 soware, 45 at B3LYP/6-31G (d) method. 46,47 FMOs analysis is very useful for analyzing the change in electronic properties before and aer the complexation of probes. [48][49][50] FMOs plots of probes and their corresponding CN À complexes are given in Fig. 7 and S13. † As evident from Fig. 7 and S13, † the highest occupied molecular orbitals (HOMOs), in isolated probes, are mainly located on the attached aromatic moieties and vinyl position and the lowest unoccupied molecular orbitals (LUMO) are distributed over uorene units. Such dispersion of molecular orbitals (MOs) accounts for the intramolecular charge transfer (ICT) process. However, HOMO/LUMOs distribution differs aer sensing of CN À at vinyl position, which demonstrates the disturbance in ICT transition and is responsible for changes in photophysical properties. Moreover, reduction in the HOMO-LUMO (H-L gap) energy gap of probes 1a (4.32 eV), 1b (4.27 eV), and 1c (4.11 eV) to 1.22 eV (1a-CN À ), 1.42 eV (1b-CN À ), and 1.57 eV (1c-CN À ) proves the formation of very stable probe-CN À adduct.
Stability test and response time study. Upon concomitant addition of CN À (500 nM), particle size of probes was signicantly reduced to 9.1 nm (1a-CN À , PDI: 0.331) and 164 nm (1b-CN À , PDI: 0.334). Reduction in particle size is clear evidence of disturbance in ICT transitions and new de-aggregated adduct formation that consequently resulted in the enhanced uorescence emission. 51 Practical applicability of a probe can be evaluated through its photostability and quick response time. To estimate extent of photostability, probes (1a, 1b, and 1c) and probe-CN À adducts were subjected to high energy excited radiations and their uorescence emission spectra were recorded aer regular intervals of time (10-100 s). Results given in Fig. S15 † disclose that uorescence emission of probes and probe-CN À adducts was not affected by their continuous exposure to high energy radiations that substantiate their photostability. Additionally, emission enhancement response of probes was studied in H 2 O/ DMF (4 : 1, v/v) as a function of time (0-60 s) aer the addition of CN À (500 nM).
It was noticed that probes 1a, 1b, and 1c displayed signicant uorescence enhancement behavior aer 20, 25, and 35 s of cyanide addition that ensures their quick response time towards sensing of CN À (Fig. S16 †).

Practical applications
Solid-supported detection of CN À . During the past few years, solid-state emitting materials have attracted a lot of attention in the domain of uorescence sensors and organic light emitting diodes (OLEDs). 52 An enhanced emission in solid-state is a prerequisite for the fabrication of devices to be used in organic electronics applications (i.e. OLEDs). Strong uorescence enhancement response of probes 1a, 1b, and 1c to CN À in solution stimulated us to develop an effective solid-supported emitters for the useful detection of CN À . Moreover, development of solid-supported detection tool greatly enhances the portability of sensing process and effectively reduces the detection cost. In this context, cyanide responsive test strips were prepared by immersing thin layer chromatographic (TLC) plates (300 mm) into THF solution of probes 1a (20 mM), 1b (50 mM), and 1c (10 mM) and air dried. Probe coated test strips were dipped in THF solution of CN À and other targeted anions (500 nM) and dried in the oven at 30 C for 20 min. Out of these test strips, the one dipped in CN À solution alone displayed strong uorescence emission under UV radiations (365 nm) and all other test strips were non-responsive to other treated anions as shown in Fig. 8 and S17. † These observations clearly indicate that newly developed probes' coated test strips can potentially be applied for instant colorimetric identication of CN À ions. The similar probes' coated test strips were used to detect CN À ions aer regular intervals of 30 and 60 minutes (Fig. S18 †). The results were comparable to previous observations. This demonstrates the detection accuracy and storage stability of the probes on test strips.
Detection of CN À in industrial waste. Cyanide liberates from pharmaceutical waste materials and form hydrogen cyanide (HCN) in air that is known to cause cyanide toxicity in nearby areas. Prolonged inhalation in HCN saturated area can cause nerve damage that may lead to the human death. 53 Hence, it is highly dispensable to sense cyanide in industrial wastewater in order to avoid cyanide toxicity. Accordingly, probe 1a was applied to recognize CN À in pharmaceutical wastewater (collected from Islamabad Pharma Abbottabad, Pakistan). For this purpose, collected wastewater samples were ltered to remove inessential particles followed by spiking different concentrations of CN À (200 and 500 nM). Prepared water samples were added to H 2 O/DMF (4 : 1, v/v) solution of probe 1a (20 mM) and uorescence emission spectra were recorded under already optimized parameters. Obtained results revealed that  addition of cyanide containing waste water resulted in signicant uorescence emission enhancement (Fig. S19 †). Fluorescence enhancement results were compared with uorescence response of probe towards laboratory sample to quantify CN À ions. Experimental outcomes given in Table 1 provided cyanide recovery more than 97% with <2.1 RSD that ensure the practical applicability of probes presented in this study. These experiments further justify the accuracy and stability of the method.
Detection of endogenous CN À in food samples. The accuracy of the method was validated by determining the CN À ions concentration in food samples. It is well known that peach kernel and bitter almonds have high medicinal signicance. However, endogenous enzymes have tendency to break cyanoside into hydrogen cyanide (HCN), if these kernels are not properly processed. 54 Similarly, cassava is appetizing because high starch is found in its rhizome. However, in unfavorable temperature and humid environment, cyanogenic glycosides undergo enzymatic hydrolysis to release hydrocyanic acid (HCN). HCN, consequently, can cause cyanide poisoning in human and animals. 54 Hence, it is important to detect endogenous cyanide in these food materials. To attain this object, peach kernel, bitter almond, and cassava powder (20 g) were mixed with 1.0 g NaOH and mixtures were subjected to sonication for 30 min at 25 C followed by ltration to eradicate undesired suspended particles.
Upon addition of peach kernel, bitter almond, and cassava powder containing solution to the H 2 O/DMF (4 : 1, v/v) solution of probes 1a (20 mM), 1b (50 mM), and 1c (10 mM), uorescence emission of probes was signicantly enhanced as shown in Fig. 9 and S20. † Further, developed probes' coated test strips also displayed signicant uorescence emission under UV radiations (365 nm), when a single drop of these solutions was applied ( Fig. 9 and S20; † insets). These results revealed that probes can be used for recognition of endogenous cyanide in food materials. Further, to validate the accuracy of the adopted method, a range of CN À concentrations (100-500 nM) was spiked in cassava samples and uorescence spectra were recorded. Aerward, a calibration curve was plotted that represents the direct co-relation between CN À concentration and enhanced uorescence emission (Fig. S21 †). These results justify the accuracy and stability of the adopted method.
Construction of logic gate. In recent past, fabrication of logic devices based on variations in uorescence signals into chemically encrypted information has drawn attention in the eld of an unconventional computing system. 60 Therefore, an obvious redshi in uorescence emission of probe 1a (418-431 nm) upon the addition of CN À (500 nM) impetus us to design logic gates. In this regard, AND, OR, NOT, and XOR gates were utilized to construct a logic circuit in which probe 1a lone and 1a-CN À adduct were considered as input-1 (In-1) and input-2 (In-2), respectively. Fluorescence emission at 431 nm was used as an output (OUT) signal (Fig. 10). Fluorescence wavelength of 431 nm was arbitrarily selected as threshold limit and output (OUT, emission wavelength) lower than threshold limit (431 nm) is designated as "0'' or "OFF" state whereas equal or larger than the threshold limit is considered as "1" or "ON" state. As it is evident in Fig. 10, only when 1a-CN À adduct (In-2) is present (0,1)/(1,1), output (OUT) at 431 nm is on "1" or "ON" state of the   system. Other variations such as when both 1a lone (In-1) and 1a-CN À (In-2) or only 1a lone are absent, the output (OUT) signal at 418 nm is lower than threshold limit and exhibits "0'' or "OFF" state. These variations are summarized in truth table (Table 2).
Smartphone APP based detection of CN À . In the present era, usage and availability of smartphones have tremendously increased around the globe. Smartphones have revolutionized the world in terms of offering excellent computing power, inbuilt sensors, and high-resolution coverage. These features drawn our attention towards quick quantitative detection of CN À in laboratory and real samples. As discussed earlier, probe 1a displayed sharp color change from yellow to dark red upon the addition of CN À . Herein, smartphone APP was used for convenient detection of CN À based on RGB (Red, Green, and Blue) color scale. On RGB color scale, each color is indicated by a specic whole number in between 0 and 255. For example, the number scale [0,0,0] corresponds to absolute black while [255,255,255] is attributed to the true white. Using a smartphone (model: Apple iPhone 6s Plus), color assist (freely available APP) was used to record variations in RGB (Red, Green, and Blue) values when naked eye color change was noticed upon the progressive addition of CN À (0-500 nM) to the H 2 O/DMF (4 : 1, v/v) solution of probe 1a. Subsequently, a plot was produced between the ratio of red and green (R/G) and increasing concentrations of CN À that displayed excellent linear response (R 2 ¼ 0.97923) and limit of detection (LOD) was found out as 13.7 nM (Fig. 11).
To substantiate the validity of the method, pharmaceutical wastewater containing specic concentration of CN À was tested quantitatively through color assist APP and uorescence spectrophotometer independently. Aer comparison of obtained results with laboratory samples, the concentration of CN À spiked in wastewater was determined as 237 nM through smartphone APP. Besides, concentration determined through uorescence enhancement response was 250 nM (Fig. 11). It revealed that smartphone APP based determination of CN À in real samples provided excellent results with 95% accuracy. These results provide portable, low cost and convenient approach in terms of probes (presented in this study) for the qualitative and quantitative detection of CN À .  Fig. 11 (a) Smartphone-assisted RGB (Red, Green, and Blue) responses for the determination of CN À ; (b) fluorescence spectral response for the determination of CN À . Comparison study. The uorescence sensing potential of probes towards cyanide was compared with CN À probes reported in literature and summarized in Table 3. Summarized data reveal that probes reported in this study are much better, exhibiting distinguished sensitive response towards CN À in a wide pH range and with a very low detection limit. Facile synthesis, aggregation induced emission (AIE) character, broad range of practicability, and dual channel sensing (uorescent and colorimetric) in semi-aqueous medium provides them an additional advantage in comparison to prior reported CN À sensors.

Conclusions
In summary, four new simple uorene based uorescent probes were synthesized through a single step Knoevenagel condensation reaction that displayed strong aggregation induced emission (AIE) behavior. AIE character was accomplished due to the restriction in intramolecular charge transfer (ICT). Further, aggregation caused quenching (ACQ) character exhibited by probe 1d (with no ICT) favors the restriction in intramolecular charge transfer (ICT) as a key factor for AIE. Additionally, AIE active probes displayed uorescence emission enhancement response towards the recognition of cyanide that was ascribed to the Michael type adduct formation. Cyanide reacts at vinyl position of probes that results in the disturbance of intramolecular charge transfer process, thereby causing enhanced uorescence response. Nucleophilic addition reaction was conrmed through NMR titration experiments, theoretical and DLS studies. Besides, probes exhibited selective colorimetric detection of CN À that was readout through smartphone assisted application. Moreover, enhanced uorescence response of probes for CN À was exploited to design logic circuit. Advantageously, simple uorescent probes were able to sense CN À in food materials, industrial samples and through cyanide responsive test strips. Conclusively, this study facilitates to understand structure-property relationship and provides an extended mechanistic approach for the sensing of CN À .

Conflicts of interest
The authors declare no conict of interest.