A Dual Fluorometric and Colorimetric Sulﬁde Sensor Based on Coordinating Self-Assembled Nanorods: Applicable for Monitoring Meat Spoilage

: Psychrotrophic bacteria, commonly called spoilage bacteria, can produce highly toxic hydrogen sulﬁde (H 2 S) in meat products. Thus, monitoring the presence of hydrogen sulﬁde in meat samples is crucial for food safety and storage. Here, we report a unique chemical sensor based on supramolecular nanorods synthesized via copper ion induced self-assembly of N , N -bis[aspartic potassium salt]-3,4,9,10-perylenetetracarboxylic diimide (APBI-K). The self-assembled nanorods can speciﬁcally detect sulﬁde with a detection limit of 0.181 µ M in solution. The nanorods suspended in pure water show a turn-on ﬂuorescence sensing behavior along with color change, acting as a dual ﬂuorometric and colorimetric sensor. Spectroscopic investigation conﬁrms the sensing mechanism due to copper ion displacement induced by the association with sulﬁde. Based on the high selectivity and sensitivity, supramolecular nanorod sensors were successfully employed to detect H 2 S in spoiled meat sample as well as dissolved H 2 S in water.


Introduction
Hydrogen sulfide (H 2 S), being nearly as toxic as carbon monoxide gas, can cause severe health damage and even death [1]. The endogenous H 2 S is primarily produced by enzyme-catalyzed reactions from various sulfur-containing amino acids which include cysteine, homocysteine, and methionine in the metabolic pathway [2]. Under standard conditions, 70% of H 2 S is generated from cysteine and the residual 30% is produced from homocysteine [3]. Highly water soluble H 2 S exists primarily in three different forms, namely: S 2− , HS − and H 2 S. The form of the sulfide is dependent on the pH of the aqueous medium [4,5]. Serious physiological problems, like Down syndrome [6], diabetes [7], liver cirrhosis [8], and Alzheimer's [9], can arise from excess sulfide exposure. In addition to the industrial sources of H 2 S, the illegal use of sulfide additives in food such as sulfites, rongalite, and sodium sulfide can cause harmful effects on human health [10]. Nevertheless, sulfide is overly formed in the process of food rot. Meat and meat-based foods are very susceptible to spoilage. The Gram-negative bacteria, Shewanella putrefaciens, and Citrobacter freundii present in meat can be active at low temperatures and can rapidly produce sulfides when growing [11,12]. Thus, sulfide can act as a marker of meat spoilage. According to the World Health Organization (WHO), food containing harmful bacteria, pathogens, viruses, parasites, or toxic chemical additives cause more than 200 different diseases in humans [13]. Detection of sulfides in food is still a challenging task because of the complexity of food compositions and the presence of interfering ingredients. Thus, a sensitive and effective sulfide detection method is in great demand to ensure food safety and human health.
Several traditional detection techniques such as chromatographic assays [14,15], electrochemical analysis [16,17], metal-oxide-semiconductor-based electronic nose [18,19], and visible light colorimetric technology [20,21] have been employed for detecting levels of sulfides. However, most detection techniques are time-consuming and need to be conducted in specialized laboratory settings by highly trained individuals. Spoilage of food, especially meat, is very time dependent. Because of this, on-site detection of sulfides by simple instrumentation is highly desirable for real-time monitoring of food substrates. In recent years, naked-eye detection of sulfides, using either colorimetric or fluorometric probes, got enormous attention from the scientific community due to its simplicity, high selectivity, sensitivity, and fast response time [22][23][24][25][26][27][28][29]. An ideal sensor should feature fluorescence turn-on performance, fast response time, high sensitivity, and selectivity over interfering components. A color change with target analyte exposure is also desired. Unfortunately, a large number of sensors work only in pure organic solvents or in mixed aqueous solvents [2,[30][31][32]. The use of toxic organic solvents restricts the materials' applications as useful sensors in the field, especially in a food industry setting. Therefore, the development of a simple sensing platform that can detect sulfide contamination on-site without using complex instrumentation is still necessary.
Herein, we report a new, dual fluorometric and colorimetric probe using nanorods made by the coordination induced self-assembly of Cu 2+ ion and N,N-bis[aspartic potassium salt]-3,4,9,10-perylenetetracarboxylic diimide (APBI-K) [33]. The potassium salt of tetracarboxylic acid increases the water solubility of the perylene diimide (PDI) core and helps nanorods assemble via rapid complex formation with Cu 2+ ion. These coordinating self-assembled nanorods (APBI-Cu) were made by mixing the aqueous solutions of Cu(NO 3 ) 2 and APBI-K. Spectroscopic investigation suggests that APBI-Cu nanorods were formed via intermolecular intrinsic π-π stacking driven by Cu 2+ -APBI-K complexation accompanied by fluorescence quenching. Sulfides have a strong binding affinity for Cu 2+ ions, consistent with the extremely low solubility product constant of CuS, 7.9 × 10 −37 [34]. Thus, Cu 2+ present in nanorods acts as an active metal center towards sulfides. The presence of sulfide ions triggers the competitive binding with the Cu 2+ ion, resulting in the disassembly of APBI-Cu nanorods. Consequently, in the presence of Na 2 S (commercially available H 2 S donor) [35], a turn-on fluorometric and colorimetric response from APBI-Cu nanorods was observed.

Materials and Instrumentations
N,N-bis[aspartic potassium salt]-3,4,9,10-perylenetetracarboxylic diimide (APBI-K) was synthesized by following a previously published procedure [33]. Reagent grade starting materials were used as received from the commercial suppliers. Ultrapure Milli-Q water (Millipore) was used during all experiments. Fresh chicken mince was acquired from a local market (Salt Lake City, UT, USA).
UV-Vis spectra were obtained by using an Agilent Cary 100 spectrophotometer. Emission spectra were recorded by using an Agilent Cary Eclipse fluorescence spectrophotometer. Attenuated total reflectance-infrared (ATR-IR) spectra were measured on a Nicolet iS50 FTIR Spectrometer at room temperature. Scanning electron microscopy (SEM) images were obtained on the FEI Nova NanoSEM™ scanning electron microscope. 1 H NMR spectrums were carried out on a Varian Mercury 400 MHz spectrometer.

Synthesis of APBI-Cu Nanorods
An aqueous solution of Cu(NO 3 ) 2 (2 mL, 10 mM) was slowly mixed with an aqueous solution of APBI-K (2 mL, 1 mM). The mixture was aged overnight to complete the selfassembly process. The nanorods were collected by centrifugation, washed thoroughly with water and dried in an air oven. Dried samples were used for experimentation.

Fluorescence Sensing Experiment in Water
5 mg APBI-Cu nanorods were dispersed in water by sonication. 50 µL of APBI-Cu nanorod suspension was diluted in 2 mL water. Fluorescence emission was monitored Chemosensors 2022, 10, 500 3 of 13 at 547 nm, using an excitation wavelength of 490 nm upon incremental addition of Na 2 S solution. Similar experiments were carried out by substituting Na 2 S with other interfering analytes. The sensitivity was examined by adding 100 µM of Na 2 S to the nanorod suspension containing other interfering analytes.
For paper-based test-strip fabrication, 10 cm × 10 cm clean Whatman filter papers were immersed in 1 mM aqueous solution of APBI-K solution. The paper was dried in an oven and then immersed in 10 mM aqueous solution of Cu(NO 3 ) 2 . Then, the paper was washed with water to remove the loosely bound complex on the surface. The APBI-Cu coated paper was dried under vacuum and stored in a dark environment prior to use.

Detection of Sulfide in Chicken Sample
Fresh chicken mince (~5 g) was placed in two separate 250 mL round bottom flasks and sealed with septa. One flask was kept refrigerated at −4 • C and the other was kept at room temperature (~25 • C). Gas was collected from the headspace of the round bottom flask using a 25 mL syringe with 24 h intervals. The collected gas was then slowly bubbled in the aqueous suspension of APBI-Cu nanorods.

Design and Synthesis of APBI-Cu Nanorods
The designed fluorophore molecule, APBI-K, is highly water soluble due to its salt form (Scheme 1). Such high-water solubility is essential for the sensor to be used in a pure water system. The optical properties of free APBI-K were studied by measuring absorption and emission spectra in water at room temperature. As shown in Figure 1, the normalized spectra of APBI-K show two strong absorption bands near 532 nm, 495 nm along with a broad shoulder peak around 465 nm. This typical characteristic absorbance could be assigned to the 0-0, 0-1, and 0-2 transition energy [36][37][38]. The emission spectra of the same solution portray the similar structural features with nearly mirror images of the absorption spectra, and the emission peaks appear at 547 nm, 587 nm, and a weak shoulder peak near 638 nm. Coordination induced self-assembly was studied by using aqueous solutions of APBI-K and Cu(NO3)2. When Cu 2+ was introduced into the APBI-K solution at a 10:1 molar ratio, a rapid precipitate formation was observed, which confirms the water-soluble fluorophore molecules undergo a fast coordination complex formation with Cu 2+ ion. A clear supernatant solution was observed after standing overnight, confirming the completion of the process (Figure 2a,b). The SEM imaging confirms the nanorod morphology of the self-assembled APBI-Cu precipitates (Figure 2c,d). Another set of self-assembled precipitates was obtained from the same APBI-K solution by evaporating the solution without adding any Cu 2+ ion solution. No distinct morphology was found for only APBI-K material ( Figure S1, supporting information). This result suggests that Cu 2+ ion coordination is important to obtain monodispersed nanorods. Coordination induced self-assembly was studied by using aqueous solutions of APBI-K and Cu(NO 3 ) 2 . When Cu 2+ was introduced into the APBI-K solution at a 10:1 molar ratio, a rapid precipitate formation was observed, which confirms the water-soluble fluorophore molecules undergo a fast coordination complex formation with Cu 2+ ion. A clear supernatant solution was observed after standing overnight, confirming the completion of the process (Figure 2a,b). The SEM imaging confirms the nanorod morphology of the self-assembled APBI-Cu precipitates (Figure 2c,d). Another set of self-assembled precipitates was obtained from the same APBI-K solution by evaporating the solution without adding any Cu 2+ ion solution. No distinct morphology was found for only APBI-K material ( Figure S1, Supplementary Materials). This result suggests that Cu 2+ ion coordination is important to obtain monodispersed nanorods.
The designed fluorophore molecule, APBI-K, is highly water soluble due to its salt form (Scheme 1). Such high-water solubility is essential for the sensor to be used in a pure water system. The optical properties of free APBI-K were studied by measuring absorption and emission spectra in water at room temperature. As shown in Figure 1, the normalized spectra of APBI-K show two strong absorption bands near 532 nm, 495 nm along with a broad shoulder peak around 465 nm. This typical characteristic absorbance could be assigned to the 0-0, 0-1, and 0-2 transition energy [36][37][38]. The emission spectra of the same solution portray the similar structural features with nearly mirror images of the absorption spectra, and the emission peaks appear at 547 nm, 587 nm, and a weak shoulder peak near 638 nm.  Coordination induced self-assembly was studied by using aqueous solutions of APBI-K and Cu(NO3)2. When Cu 2+ was introduced into the APBI-K solution at a 10:1 molar ratio, a rapid precipitate formation was observed, which confirms the water-soluble fluorophore molecules undergo a fast coordination complex formation with Cu 2+ ion. A clear supernatant solution was observed after standing overnight, confirming the completion of the process (Figure 2a,b). The SEM imaging confirms the nanorod morphology of the self-assembled APBI-Cu precipitates (Figure 2c,d). Another set of self-assembled precipitates was obtained from the same APBI-K solution by evaporating the solution without adding any Cu 2+ ion solution. No distinct morphology was found for only APBI-K material ( Figure S1, supporting information). This result suggests that Cu 2+ ion coordination is important to obtain monodispersed nanorods. Copper coordinating self-assembly was further investigated spectroscopically. A 10:1 equivalent of Cu 2+ ion was incrementally added in APBI-K solution, and both absorption and emission spectral changes were monitored. The aqueous solution of APBI-K shows the absorption ratio of 0-0 and 0-1 band (A0-0/A0-1) as nearly 1.52 ( Figure 3a). It is worth mentioning that the A0-0/A0-1 ratio obtained from absorption spectra is considered a tool to monitor the aggregation behavior of perylene diimides (PDIs). Franck-Condon progression with a ratio A0-0/A0-1 of ~1.6 is considered for free monomeric PDIs [39]. Thus, APBI-K solution primarily exists in monomeric form in solution. The ratio of A0-0/A0-1 changed Copper coordinating self-assembly was further investigated spectroscopically. A 10:1 equivalent of Cu 2+ ion was incrementally added in APBI-K solution, and both absorption and emission spectral changes were monitored. The aqueous solution of APBI-K shows the absorption ratio of 0-0 and 0-1 band (A 0-0 /A 0-1 ) as nearly 1.52 (Figure 3a). It is worth mentioning that the A 0-0 /A 0-1 ratio obtained from absorption spectra is considered a tool to monitor the aggregation behavior of perylene diimides (PDIs). Franck-Condon progression with a ratio A 0-0 /A 0-1 of~1.6 is considered for free monomeric PDIs [39]. Thus, APBI-K solution primarily exists in monomeric form in solution. The ratio of A 0-0 /A 0-1 changed rapidly with the addition of Cu 2+ and shows A 0-0 /A 0-1 value of~0.94. This result indicates the formation of an aggregated state in the presence of Cu 2+ ion [40]. A new broad shoulder near 570 nm was also observed with increasing Cu 2+ , which also suggests the formation of a new species via coordinating self-assembly of APBI-K molecules [41,42]. A clear isosbestic point was observed at 550 nm, indicating quantitative conversion of APBI-K Chemosensors 2022, 10, 500 5 of 13 from free molecular state to the aggregate. The emission behavior of nanorods is also drastically different compared to the free APBI-K molecules (Figure 3b). A 98% quenching was observed upon formation of a self-assembled compound. These results suggest that the addition of Cu 2+ ions boost the π-π stacking among PDI core and facilitates the formation of coordinating self-assembled nanorods. molecular state to the aggregate. The emission behavior of nanorods is also drastically different compared to the free APBI-K molecules (Figure 3b). A 98% quenching was observed upon formation of a self-assembled compound. These results suggest that the addition of Cu 2+ ions boost the π-π stacking among PDI core and facilitates the formation of coordinating self-assembled nanorods.
To obtain further structural information of APBI-Cu nanorods, ATR-IR spectra were also recorded ( Figure S2, supporting information). Free APBI-K displays strong absorption bands in the areas of 1570 and 1343 cm −1 . These bands can be assigned to the asymmetric and symmetric CO2 stretching vibrations of the potassium coordinated APBI-K fluorophore [33,43]. The peak corresponding to CO2 stretching vibrations diminished in intensity and broadened upon nanorod formation. This result validates the coordination bond formation between Cu 2+ and APBI-K via the carboxylate functional group during self-assembly [44,45].

Detection of Sulfide in Water
It is well known that copper has stronger affinity to sulfides compared to carboxylates [46,47], consistent with the dramatically different solubility product constants of CuS (7.9 × 10 −37 ) and CuCO3 (1.4 × 10 −10 ) [34,48]. Based on this metal ion displacement approach (MDA), various colorimetric and fluorometric probes have been reported [2,30,[49][50][51][52][53][54][55]. However, the use of perylene diimide (PDI) based metal coordinated self-assembled materials as a sulfide sensor is rare. Recently, Yao et al. reported H2S sensing using a similar mechanism with a 0.41 μM detection limit [53]. However, the use of highly toxic cadmium metal could restrict the sensor application in real world applications. Thus, the selfassembled APBI-Cu nanorods could be a better option for sulfide sensing considering their real-world application potential.
To test the sulfide sensing ability of APBI-Cu nanorods, the nanorods were dispersed in water and an aqueous solution of Na2S was added incrementally. As depicted in Figure  4a, a rapid turn-on fluorescence signal was observed in the presence of sulfide in the system. The spectral pattern is the exact same as free APBI-K molecules. A time-dependent study shows that the turn-on signal gets saturated within 120 s ( Figure S3, supporting To obtain further structural information of APBI-Cu nanorods, ATR-IR spectra were also recorded ( Figure S2, Supplementary Materials). Free APBI-K displays strong absorption bands in the areas of 1570 and 1343 cm −1 . These bands can be assigned to the asymmetric and symmetric CO 2 stretching vibrations of the potassium coordinated APBI-K fluorophore [33,43]. The peak corresponding to CO 2 stretching vibrations diminished in intensity and broadened upon nanorod formation. This result validates the coordination bond formation between Cu 2+ and APBI-K via the carboxylate functional group during self-assembly [44,45].

Detection of Sulfide in Water
It is well known that copper has stronger affinity to sulfides compared to carboxylates [46,47], consistent with the dramatically different solubility product constants of CuS (7.9 × 10 −37 ) and CuCO 3 (1.4 × 10 −10 ) [34,48]. Based on this metal ion displacement approach (MDA), various colorimetric and fluorometric probes have been reported [2,30,[49][50][51][52][53][54][55]. However, the use of perylene diimide (PDI) based metal coordinated self-assembled materials as a sulfide sensor is rare. Recently, Yao et al. reported H 2 S sensing using a similar mechanism with a 0.41 µM detection limit [53]. However, the use of highly toxic cadmium metal could restrict the sensor application in real world applications. Thus, the self-assembled APBI-Cu nanorods could be a better option for sulfide sensing considering their real-world application potential.
To test the sulfide sensing ability of APBI-Cu nanorods, the nanorods were dispersed in water and an aqueous solution of Na 2 S was added incrementally. As depicted in Figure 4a, a rapid turn-on fluorescence signal was observed in the presence of sulfide in the system. The spectral pattern is the exact same as free APBI-K molecules. A time-dependent study shows that the turn-on signal gets saturated within 120 s ( Figure S3, Supplementary Materials). Such a fast response is desirable for rapid onsite sensor development. In the presence of sulfide, the color of the solution also changes to red with the reappearance of typical 0-0 and 0-1 transition peaks in UV-Vis spectra ( Figure S4, Supplementary Materials). A change of pH was also observed during the sensing experiments. The pH of the system Chemosensors 2022, 10, 500 6 of 13 changes from 5.4 to 7.8. Thus, to stabilize the pH of the system, a same experiment was carried out in 10 mM HEPES buffer at pH 7.4. As shown in Figure S5, APBI-Cu nanorods can detect the sulfide in buffer medium efficiently. Hence, the spectral study in both water and buffer medium confirm the sulfide sensing ability of APBI-Cu nanorods. amino acids and other thiol containing biomolecules could show false positive responses. To test the selectivity towards sulfide over other interfering substances, APBI-Cu nanorods were treated with different thiol-containing biomolecules (cysteine (Cys), homocysteine (Hcys), glutathione (GSH)), various anions (NaF, NaCl, NaBr, NaI, NaNO3, NaNO2, NaHSO3, NaS2O3, Na2SO4, NaHCO3 and Na3PO4) and meat spoilage-associated volatile organic compounds (ethanol, hexanol, phenol, acetic acid, butanoic acid and hexanal) [62]. Figures 4b and S6, and supporting information show that no strong response was found from other interfering substances. Concentration dependent fluorescence studies show that the APBI-Cu nanorods are capable enough to distinguish among sulfide and other interfering molecules and anions ( Figure S7, supporting information). Thus, APBI-Cu nanorods are highly selective towards sulfide over common thiol containing biomolecules and various anions.
NaF, (6) NaCl, (7) NaBr, (8) NaI, (9) NaNO2, (10) NaNO3, (11) NaHSO3, (12) Na2SO3, (13) Na2SO4, An efficient sensor must work in a complicated system where the target analyte coexists with other interfering substances. To check the sensitivity of APBI-Cu nanorods, another set of experiments was designed. Here, the interfering molecules and anions were added to an equal amount of sulfide. As shown in Figures 5 and S8-S27, supporting information, a similar turn-on signal was observed from APBI-Cu nanorods and no strong interference was noticed even with other molecules present. Based on these results, we can conclude that the self-assembled APBI-Cu nanorods are not only selective towards Different thiol-containing amino acids are one of the main sources of sulfide contamination in food and other biological samples [56][57][58]. In addition, the affinity of such thiol-containing amino acids towards copper ion is also high [59][60][61]. Thus, thiolcontaining amino acids and other thiol containing biomolecules could show false positive responses. To test the selectivity towards sulfide over other interfering substances, APBI-Cu nanorods were treated with different thiol-containing biomolecules (cysteine (Cys), homocysteine (Hcys), glutathione (GSH)), various anions (NaF, NaCl, NaBr, NaI, NaNO 3 , NaNO 2 , NaHSO 3 , NaS 2 O 3 , Na 2 SO 4 , NaHCO 3 and Na 3 PO 4 ) and meat spoilageassociated volatile organic compounds (ethanol, hexanol, phenol, acetic acid, butanoic acid and hexanal) [62]. Figure 4b and Figure S6 and Supplementary Materials show that no strong response was found from other interfering substances. Concentration dependent fluorescence studies show that the APBI-Cu nanorods are capable enough to distinguish among sulfide and other interfering molecules and anions ( Figure S7, Supplementary Materials). Thus, APBI-Cu nanorods are highly selective towards sulfide over common thiol containing biomolecules and various anions.
An efficient sensor must work in a complicated system where the target analyte coexists with other interfering substances. To check the sensitivity of APBI-Cu nanorods, another set of experiments was designed. Here, the interfering molecules and anions were added to an equal amount of sulfide. As shown in Figure 5 and Figures S8-S27, Supplementary Materials, a similar turn-on signal was observed from APBI-Cu nanorods and no strong interference was noticed even with other molecules present. Based on these results, we can conclude that the self-assembled APBI-Cu nanorods are not only selective towards sulfide, but also highly sensitive. Further, the limit of detection was calculated in the concentration range of 0-12 µM and the plot of the fluorescence intensity of APBI-Cu nanorods vs. the concentration of Na 2 S revealed a linear relationship ( Figure S28, Supplementary Materials). The LOD for the detection of sulfide was calculated at a signalto-noise (S/N) ratio of 3 and has been estimated to be 0.181 µM.
Chemosensors 2022, 10, x FOR PEER REVIEW 7 of 13 sulfide, but also highly sensitive. Further, the limit of detection was calculated in the concentration range of 0-12 μM and the plot of the fluorescence intensity of APBI-Cu nanorods vs. the concentration of Na2S revealed a linear relationship ( Figure S28, supporting information). The LOD for the detection of sulfide was calculated at a signal-to-noise (S/N) ratio of 3 and has been estimated to be 0.181 μM. Metal ion displacement approach (MDA) is a common mechanism for sulfide sensors [63][64][65] (Table S1, supporting information). In the presence of sulfide, metals convert to metal sulfides. The fluorophore then shows turn-on signals as it is released in solution. As discussed earlier, we observed similar emission and absorption signals from APBI-Cu nanorods in the presence of sulfide. In addition, 1 H-NMR of APBI-Cu nanorods were recorded before and after the treatment of Na2S in D2O ( Figures S29-S31, supporting information). No obvious peaks were found in 1 H-NMR for only APBI-Cu nanorods as they are insoluble in water. On the other hand, Na2S treated APBI-Cu nanorods show a similar 1 H-NMR signal compered to free APBI-K [33]. The peaks at 7.96 and 8.02 ppm could be assigned to aromatic protons of the PDI core of APBI molecules. The peak around 3.14 ppm shifted to 2.65 ppm, which might occur from the different metal environments around the carboxylate functionalized molecule [66,67]. The SEM images of APBI-Cu nanorods were taken after the treatment of Na2S to observe any changes in morphology. Figure S32, supporting information confirms that the nanorods lost their shape after the sensing event, which also supports the copper ion displacement mechanism. The detection mechanism is shown in Scheme 2. Metal ion displacement approach (MDA) is a common mechanism for sulfide sensors [63][64][65] (Table S1, Supplementary Materials). In the presence of sulfide, metals convert to metal sulfides. The fluorophore then shows turn-on signals as it is released in solution. As discussed earlier, we observed similar emission and absorption signals from APBI-Cu nanorods in the presence of sulfide. In addition, 1 H-NMR of APBI-Cu nanorods were recorded before and after the treatment of Na 2 S in D 2 O (Figures S29-S31, Supplementary Materials). No obvious peaks were found in 1 H-NMR for only APBI-Cu nanorods as they are insoluble in water. On the other hand, Na 2 S treated APBI-Cu nanorods show a similar 1 H-NMR signal compered to free APBI-K [33]. The peaks at 7.96 and 8.02 ppm could be assigned to aromatic protons of the PDI core of APBI molecules. The peak around 3.14 ppm shifted to 2.65 ppm, which might occur from the different metal environments around the carboxylate functionalized molecule [66,67]. The SEM images of APBI-Cu nanorods were taken after the treatment of Na 2 S to observe any changes in morphology. Figure S32, Supplementary Materials confirms that the nanorods lost their shape after the sensing event, which also supports the copper ion displacement mechanism. The detection mechanism is shown in Scheme 2.

Detection of Sulfide in Water and Meat
'Sulfur bacteria' can produce toxic sulfide in groundwater, wells, and even plumbing [68,69]. Use or consumption of sulfide contaminated water is bad for human health. Con-Scheme 2. Schematic presentation of detection of sulfide based on the APBI-Cu nanorods.

Detection of Sulfide in Water and Meat
'Sulfur bacteria' can produce toxic sulfide in groundwater, wells, and even plumbing [68,69]. Use or consumption of sulfide contaminated water is bad for human health. Considering the possible use of the APBI-Cu nanorods for on-site detection of sulfide without using any complex device, a paper test strip (~3 cm × 5 cm) was utilized. Paper strips were dipped in various concentrations of Na 2 S solution made by laboratory tap water. The APBI-Cu nanorods-coated paper test strip showed gradually enhanced emission under UV lamp with increasing concentration from 0 µM to 100 µM ( Figure 6). These observations indicate that the APBI-Cu nanorods coated paper can be used for the on-site detection of sulfide in water. Scheme 2. Schematic presentation of detection of sulfide based on the APBI-Cu nanorods.

Detection of Sulfide in Water and Meat
'Sulfur bacteria' can produce toxic sulfide in groundwater, wells, and even plumbing [68,69]. Use or consumption of sulfide contaminated water is bad for human health. Considering the possible use of the APBI-Cu nanorods for on-site detection of sulfide without using any complex device, a paper test strip (~3 cm × 5 cm) was utilized. Paper strips were dipped in various concentrations of Na2S solution made by laboratory tap water. The APBI-Cu nanorods-coated paper test strip showed gradually enhanced emission under UV lamp with increasing concentration from 0 μM to 100 μM ( Figure 6). These observations indicate that the APBI-Cu nanorods coated paper can be used for the on-site detection of sulfide in water. As it spoils, meat produces a high amount of sulfides via the degradation of proteins [70,71]. In such cases, sulfide can act as a marker of meat spoilage [72]. Chicken mince was kept at room temperature and another set of meat was stored at −4 °C. The collected gas from the headspace of the flask was slowly bubbled in the aqueous suspension of APBI-Cu nanorods. Emission spectra were then recorded. No remarkable turn-on signal was observed in the initial 2 days from both samples. A detectable turn-on signal was observed from the meat stored at room temperature after 3 days. As shown in Figures 7 and S33, supporting information, the turn on signal increased with time. On the other hand, no turn-on signal was observed for the gas collected from the headspace of the chicken-containing flask stored at −4 °C, which indicates that the meat remained fresh and was not undergoing spoilage. No turn-on signal was observed when only air was bubbled in the aqueous suspension of APBI-Cu nanorods as a control experiment. In addition, when APBI-Cu nanorods coated paper was placed on fully spoiled meat, a rapid color change was observed with an enhanced fluorescence signal under UV-lamp ( Figure S34, supporting information). These observations suggest that the APBI-Cu nanorods can be utilized as an indicator of raw meat freshness via monitoring the released sulfide from the meat sample. As it spoils, meat produces a high amount of sulfides via the degradation of proteins [70,71]. In such cases, sulfide can act as a marker of meat spoilage [72]. Chicken mince was kept at room temperature and another set of meat was stored at −4 • C. The collected gas from the headspace of the flask was slowly bubbled in the aqueous suspension of APBI-Cu nanorods. Emission spectra were then recorded. No remarkable turn-on signal was observed in the initial 2 days from both samples. A detectable turn-on signal was observed from the meat stored at room temperature after 3 days. As shown in Figure 7 and Figure S33, Supplementary Materials, the turn on signal increased with time. On the other hand, no turn-on signal was observed for the gas collected from the headspace of the chicken-containing flask stored at −4 • C, which indicates that the meat remained fresh and was not undergoing spoilage. No turn-on signal was observed when only air was bubbled in the aqueous suspension of APBI-Cu nanorods as a control experiment. In addition, when APBI-Cu nanorods coated paper was placed on fully spoiled meat, a rapid color change was observed with an enhanced fluorescence signal under UV-lamp ( Figure S34, Supplementary Materials). These observations suggest that the APBI-Cu nanorods can be utilized as an indicator of raw meat freshness via monitoring the released sulfide from the meat sample.

Conclusions
In short, an APBI-Cu nanorod based colorimetric and fluorometric probe was developed. The APBI-Cu nanorods can selectively detect the toxic sulfide without interference from common thiol-containing biomolecules like cysteine, homocysteine, and glutathione. Water soluble APBI molecules undergo controlled coordination-induced self-assembly with copper and show a fluorescent turn-off signal. In the presence of sulfide, APBI-Cu nanorods begin disassembling. This phenomenon was made evident by spectroscopic investigation, morphology evaluation, and observation of a distinct turn-on signal. The APBI-Cu nanorod probe showed sensitive and selective detection of sulfide with a low detection limit of 0.181 µM in a purely aqueous medium, which is significantly lower than those reported recently with other perylene diimide fluorescent probes. Finally, it has been successfully utilized to detect sulfide in both water and meat samples. Hence, this newly developed colorimetric and fluorometric sulfide sensor has great potential in the fields of water and meat spoilage monitoring.

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