Organic Nanoparticles for Visual Detection of ... - ACS Publications

Research Article pubs.acs.org/journal/ascecg

Organic Nanoparticles for Visual Detection of Spermidine and Spermine in Vapors and Aqueous Phase Shweta Chopra,† Amanpreet Singh,‡ P. Venugopalan,§ Narinder Singh,‡ and Navneet Kaur*,†,§ †

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Centre for Nanoscience and Nanotechnology (U.I.E.A.S.T), South Campus, Block-II, Sector-25, Panjab University, Chandigarh 160014, India ‡ Department of Chemistry, Indian Institute of Technology Ropar (IIT Ropar), Rupnagar, Punjab 140001, India § Department of Chemistry, Panjab University, Chandigarh 160014, India S Supporting Information *

ABSTRACT: The rapidly emerging interest in the evaluation of biogenic amines especially spermidine and spermine has brought up new demands for the efficacious technical methods. These volatile amines are abundantly present in protein containing food items and indicate the quality of food products. An optical method relying on the efficient application of Cu2+ complex of organic nanoparticles (ONPs) for the simultaneous quantification of spermidine and spermine in vapors and aqueous phase has been detailed. The method allows visual naked-eye determination as the color of the aqueous solution of ONPs−metal complex changes, respectively, upon the additions of spermidine and spermine individually. The metal complex was further quantitatively titrated for examining a linear relation of absorbance with respect to increasing concentrations of spermidine and spermine, where corresponding detection limits for both analytes were found to be 35 and 36.2 nM, respectively. Eventually, the real sample application of proposed sensor was explored using food articles (mushroom and meat), and we observed the modulation in chromogenic signals; i.e., there was a change in color of the sensor with vapors of biogenic amines emitted from food articles on storage. KEYWORDS: Organic nanoparticles, Colorimetric detection, Biogenic amine, Vapor detection, Real sample examination

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INTRODUCTION The biologically active spermidine and spermine are considered to be the most abundant polyamines originated in living cells.1−8 At low concentrations, biogenic amines (BAs) are known to play vital role in cell growth, protein biosynthesis, maturation, and they can act as antioxidants and metabolic regulators.9,10 Moreover, these BAs also perform important physiological functions including wound healing and regeneration of intestinal mucosa.11 Normally, the level of spermidine and spermine in human blood is approximately around 6−17 nmol/mL.12 BAs are an interesting paradox to life as prescribed amounts of BAs are required for normal physiological function; however, unregulated amounts of these BAs are vasoactive, and are detrimental to health, leading to several health problems such as cardiac palpitation, intracerebral hemorrhage, and tumor growth.13−16 Another concern for BAs lies with the fact that these amines may originate during microbial decarboxylation of amino acids of fermented foods/beverages.17 In some cases, spermidine and spermine along with other biogenic amines (such as cadaverine and putrescine) may react with nitrite available in certain meat products and consequently produce carcinogenic nitrosamine compounds.18 Thus, the © 2016 American Chemical Society

consumption of food stuff with elevated levels of BAs may adversely affect the human body. Moreover, patients with different types of cancers like lung cancer, prostatic cancer, and hepatic tumors are related to higher concentrations of spermidine and spermine in their body fluids.19 Therefore, the detection of such biogenic amines can be related to hygienic food conditions and public health,20,21 and their determination becomes a matter of great concern. Traditional quantification methods, like HPLC, thin layer chromatography, GC, LC−MS, capillary electrophoresis, electroanalytical method, etc., for biogenic amine determination pose problems like expensive instrumentation, laborious sample preparation, expertise in handling, time consumption, etc.22−25 Along with these, the reported methods also offer complex sample matrix and interference from other environmental factors.26 In contrast, any system or technique capable of selectively differentiating the biogenic amines, independent of costly instrumentation and applicable in routine practice, will sound more beneficial.27−29 Received: June 10, 2016 Revised: November 26, 2016 Published: December 20, 2016 1287

DOI: 10.1021/acssuschemeng.6b01295 ACS Sustainable Chem. Eng. 2017, 5, 1287−1296

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ACS Sustainable Chemistry & Engineering

analytes.28,29 Further, the recognition of biogenic amine will depend upon the complementarity of binding sites that prevailed in biogenic amine and steric requirements of the cation coordination sphere which thus will govern the selectivity for biogenic amine.

The probe, under current study, is the metal ion complex of an organic receptor (Figure 1A), and the receptor is designed in

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RESULTS AND DISCUSSION Synthesis and Photophysical Evaluations of Receptor 1·H4. The targeted receptor 1·H4 was synthesized through twostep reactions involving amide formation between dipicolinic acid and hydrazine; the resultant amide product was purified using recrystallization.30 The product bearing amide linkages and free amine groups was subjected to condensation reaction with salicylaldehyde in ethanol to achieve the synthesis of desired dipodal receptor 1·H4 in good yield. The spectroscopic analysis with NMR, FT-IR, and mass spectrometry supports the formation of 1·H4 (Figures S1−S4). The receptor 1·H4 is fabricated of amide and imine linkages, with sp2 nitrogen binding sites from pyridine and imine linkages; oxygen donor sites form OH and carbonyl groups. This unique combination of linkages and binding sites may offer a coordination sphere for the binding of multiple cations, and the binding sites are judiciously placed in such a way that the coordination sphere of each metal ion should not be satisfied with the exclusive binding sites of the receptor; rather, the cation must exploit a monodentate anion or solvent to complete the coordination sphere. The development of the bioanalytical technique is

Figure 1. (A) Chemical structure of organic receptor 1·H4. (B) Schematic representation of proposed coordination sphere offered through the array of binding sites of receptor 1·H4.

such a way that it must offer binding of multiple cations and the coordination sphere of each metal ion should not be saturated with the binding sites of the receptor; rather, the coordination sphere must utilize monodentate anion or solvent as projected in Figure 1B. Recently, organic−inorganic hybrid polymers have been considered as promising candidates for selective absorption of analytes. Due to high thermal and chemical stability, these are used in the fields of catalysis, gas absorption, and sensing of analytes. These polymeric structures have wellshaped pores which provide selectivity toward particular

Figure 2. (A) Comparison of UV−vis absorption spectra recorded with 10 μM concentration of receptor 1·H4 in pure DMF and in the DMF/H2O (1:99, v/v) solvent system. (B) Fluorescence signatures of receptor 1·H4 (10 μM), showing enhancement in fluorescence intensity upon changing the solvent from pure DMF to the DMF/H2O (1:99, v/v) solvent system. (C) DLS histogram showing particle size analysis of organic nanoparticles of receptor 1·H4, measured using DLS. (D) TEM image showing the formation of organic nanoparticles (ONPs) of receptor 1·H4 in the DMF/H2O (1:99, v/v) solvent system. 1288


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Figure 3. (A) Fluorescence signatures of receptor 1·H4 (10 μM), showing quenching in fluorescence intensity upon successive addition of Cu2+ (0 to 22 μM) in the DMF/H2O (1:99, v/v) solvent system. (B) Modulation of UV−vis absorption bands of receptor 1·H4 (10 μM) upon successive addition of Cu2+ (0−240 μM) in the DMF/H2O (1:99, v/v) solvent system. (C) ORTEP diagram of Cu2+ complex of 1·H4 with 40% thermal ellipsoid probability and atom-numbering scheme. (D) Packing diagram of Cu2+ complex of 1·H4.

this receptor are not prone to hydrolysis even after the receptor was suspended for 5 days in a semiaqueous medium. The pH has a negligible effect on the photophysical characteristics of 1· H4 in the 6−11 pH range. Eventually, it was concluded that the emission profile of 1·H4 is highly sensitive to solvent composition; fluorescence is showing OFF−ON−OFF switching upon changing the solvent from DMF to DMF/H2O (1:99, v/v) and then again to pure DMF. It seems that the enhancement in fluorescence emission in the DMF/H2O (1:99, v/v) solvent system is due to the AIEE (aggregation induced enhanced emission) phenomenon. 32,33 It was previously studied by Tang et al. that the water enriched solvent system triggers the formation of organic nanoparticles, and due to molecular rigidity, the fluorescence emission gets enhanced through the restriction on nonradiative decay, which otherwise prevailed in pure DMF. The development of organic nanoparticles (ONPs) involves the dispersion of organic molecules dissolved in rich solvent (organic solvent) into poor solvent (water).34−36 Miscibility of both solvents generates rapid crystallization significantly due to the insolubility of organic molecules in water.37 Here, we slowly injected the organic receptor 1·H4 dissolved in DMF (1 mM) into 100 mL of doubly distilled water, and the mixture was kept for sonication, which ensures the homogeneity of the nanoparticle solution. Different ratios of solvent and water were used during the formation of ONPs; however, the DMF/ H2O (1:99, v/v) solvent system has provided the best size range. The hydrodynamic size distribution of ONPs was analyzed by DLS (dynamic light scattering), and it was in the 30−40 nm range, which is mostly higher than the size expressed from the TEM image (about 15 nm) (Figure 2C,D).

fascinating, if it operates in aqueous medium; however, the receptor 1·H4 has poor solubility in an aqueous medium. Thus, it was decided to compare the photophysical properties of receptor 1·H4 in an organic solvent such as DMF and then to compare the functioning of the receptor in a semiaqueous medium with a varied fraction of water even up to DMF/H2O (1:99, v/v). The receptor 1·H4 has shown a broad absorption band at 330 nm (ε = 1.84 × 104 M−1 cm−1) in its UV−vis absorption spectrum recorded with 10 μM concentration of receptor in DMF. The absorption spectrum recorded with 10 μM concentration of receptor in the DMF/H2O (1:99, v/v) solvent system additionally revealed the absorbance at 353 nm, a bathochromic shift in comparison to the absorption of a dilute solution of the receptor in DMF (Figure 2A). The 10 μM concentration of receptor 1·H4 in pure DMF, upon excitation at 330 nm, exhibited weak emission at 515 nm (ϕ = 0.058).31 However, the emission spectrum recorded in the DMF/H2O (1:99, v/v) solvent system using the same excitation wavelength, and same concentration (as that was used for recording the emission in pure DMF), has noticeably improved the quantum yield from ϕ = 0.058 to 0.803 (Figure 2B). In order to understand the enhanced emission in the DMF/H2O (1:99, v/ v) solvent system, we have evaluated the fate of the fluorescence signature of receptor 1·H4 under varied pH conditions and different salt strengths, and there are even timedependent variations. The salt strength assorted with 0−1000 μM concentrations of tetrabutylammonium perchlorate has no significant influence over the fluorescence profile of 1·H4. Similarly, the time-dependent emission profile of 1·H4 was recorded which remained unaltered under the tested time period up to 5 days, which signifies that the imine linkages of 1289


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ACS Sustainable Chemistry & Engineering Metal Binding Affinity of Receptor 1 and Crystal Structure. The developed fluorescent ONPs were studied for their cation binding affinity toward the library of metal ions including Li+, Na+, K+, Mg2+, Ca2+, Sr2+, Ba2+, Cr3+, Mn2+, Fe3+, Co2+, Cu2+, Zn2+, Ag+, Cd2+, Pb2+, Al3+, Hg2+, and Cs+. It was observed that, upon addition of 20 μM of Cu2+ to the 10 μM solution of ONPs in DMF/water (1:99, v/v), the fluorescence intensity of ONPs was quenched, whereas other tested metal ions could not distress the emission of ONPs so significantly (Figure S5). The complexation was quantitatively understood by carrying out titrations with successively increasing amounts of cupric ion solution (0−22 μM). The course of titration revealed a uniform pattern of quenching in the intensity owing to the paramagnetic nature of the cupric ion, thus supporting the binding event operating through the PET mechanism.38,39 Moreover, the fluorescence quenching was observed under a UV lamp, and the emission intensity was inversely proportional to the concentration of Cu2+ (inset to Figure 3A). The changes in the absorption profile of ONPs with different concentrations of Cu2+ are shown in Figure 3B. To understand the binding mode and to establish the structure of the metal complex, the solution of receptor 1·H4 mixed with Cu2+ was kept undisturbed for 20 days. The slow solvent evaporation yielded the single crystal suitable for X-ray structural determination. The complex of receptor 1·H4 with copper chloride salt crystallized in the triclinic crystal system with space group P1̅ (Table S1). It consists of a polymeric Cu2+ complex that extends through a bridged chloride ligand. Each chloride ion makes a bridge between two different Cu2+ ions. Here, each monomeric unit contains three copper metal ions having the same (+2) oxidation state, and the ORTEP diagram is shown with the atomic number scheme in Figure 3C. The complex is a 1-D coordination polymer extending parallel to the c-axis. The central copper ion Cu(1) has a distorted trigonal bipyramidal structure, having two chloride and three nitrogen atom as donor groups, and it formed two five membered chelate rings with Cu(1). On the other hand, Cu(2) and Cu(3) have acquired a distorted square pyramidal geometry. The five coordination sites of Cu(2) ion are filled by three oxygens, one nitrogens, and one chloride ion. The coordinated oxygen donors are from DMSO, phenoxide ion, and carbonyl. Here, three oxygen atoms and a nitrogen atom are coordinated in a square planar pattern, and chloride ion occupied the axial position of square pyramidal geometry. Cu(3) ion has coordination pattern similar to that of Cu(2); however, DMSO acquired the axial position instead of chloride ion in Cu(2). It is interesting that N2 and N4 got deprotonated and negative charge is delocalized on nearby carbonyl ligand. Bond lengths and bond angles are accessible in Tables S2 and S3, respectively. As shown in the packing of the complex in Figure 3D, each monomeric unit is layered over each other and extended through the bridging chloride ion. In one monomeric unit, two DMSO molecules are coordinated with Cu(2) and Cu(3), respectively. The uniformity in the quenching pattern of emission due to copper ions is represented through the regression curve (Figure 4), in which linearity is achieved with 98.9% regression. Chemosensor Activities of 1·Cu2+ Ensemble. The present paper is targeted to utilize the 1·Cu2+ ensemble for the detection of biogenic amines in the vapor phase and aqueous medium. Earlier reports revealed the determination of biogenic amines (BAs), which involves tedious sample preparation and conversion of BAs into some other chemical

Figure 4. Linear regression graph between concentration of Cu2+ ions (0−22 μM) and fluorescence intensity of receptor 1·H4 (10 μM) in the DMF/H2O (1:99, v/v) solvent system.

species.9,40 Consequently, the straightforward determination of BAs in a complex matrix is still an important lacuna for chemosensor development. In the present work, we initiated the determination of biogenic amines through the 1·Cu2+ ensemble using fixed concentrations (2 μM) of biogenic amines (BAs), and the list of amines under investigation is as follows: spermidine, spermine, tyramine, 2-phenylethylamine, histamine, 1,2-diaminopropane, and 1,4-diaminobutane. Upon addition of BAs to the 1·Cu2+ ensemble, visible color change appeared in the cases of spermidine and spermine (Figure 5B), which facilitated their qualitative and quantitative evaluation using a UV−vis absorption technique. Absorbance of the host solution was found to reduce at 400 nm, and at the same time, generation of new distinctive peaks at 602 and 555 nm were observed for spermidine and spermine, respectively (Figure 5A). In accordance to observed changes in UV−vis spectra, titration studies were performed individually with both targeted amines under same experimental conditions for confirming their recognition. The increment in the new peak at the respective wavelengths was also achieved during step by step addition of biogenic amines (Figure 5C,D). The selective behavior of the probe under study can be attributed to complementarity of binding sites that prevailed in the biogenic amine and steric requirement of the cation coordination sphere. The titration data also revealed good linearity (Figure 6) with detection limits of 35 and 36.2 nM for spermidine and spermine, respectively.41 In order to further validate the effectiveness of the probe, the competitive analysis was pursued. In addition, experiments on response time behavior of sensor for spermidine and spermine were performed with variable concentrations of selected amines. Variable concentrations of spermidine (0.4, 1, 1.4, and 2 mM) were added to the solution of the 1·Cu2+ ensemble, and a response time graph was plotted. The relation between absorbance and time was also studied for spermine with different concentrations (0.2, 0.8, 1.2, and 1.8 mM), and the interactions occurred within first 40 s in both cases (Figure S8). The authentication for the applicability of the 1·Cu2+ ensemble as a sensor in targeted environmental and biological surroundings was also checked, and this effect of pH as well as ionic strength was evaluated (Figures S9 and S10). Moderate acidic and basic solutions were used, and the aqueous solution of the 1·Cu2+ ensemble performed well under the applied conditions and remained stable in the 6−10 pH range. Similarly, a minimal effect from the addition of the tetrabutylammonium salt of perchlorate (0−100 equiv) on 1290


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Figure 5. (A) Changes in the UV−vis absorption profile of the 1·Cu2+ (10 μM) ensemble upon addition of different biogenic amines (2 μM) in the DMF/H2O (1:99, v/v) solvent system. (B) Visual changes in color of the 1·Cu2+ ensemble upon addition of 2 μM spermidine and spermine, respectively. Modulation of UV−vis absorption profile of 1·Cu2+ ensemble upon successive addition of (C) spermidine (0 to 2.2 μM) and (D) spermine (0−2.6 μM) in DMF/H2O (1:99, v/v) solvent system.

Figure 6. (A) Linear regression plot between concentration of spermidine (0−2.2 μM) and absorbance of 1·Cu2+ ensemble. (B) Linear regression plot between concentration of spermine (0−2.6 μM) and absorbance of 1·Cu2+ ensemble.

the spectral behavior of the 1·Cu2+ ensemble was examined, and results showed that, even with addition of higher concentrations of perchlorate salt, the spectral profile of the ensemble was not perturbed much, thus suggesting its practical application under real biological conditions. As described above, the trinuclear copper complex of 1·H4 has attained a polymeric structure, and each metal center has a pentavalent coordination sphere. Hence, there is a possibility of interaction with some weakly coordinating ligands through the sixth coordination site. The change in absorbance spectra with spermidine and spermine is attributed to the interaction of these amines with the organic−inorganic hybrid polymer. These amines weakly interact with the polymeric complex, resulting in a change in the photophysical properties of the receptor. The porous nature of the polymeric complex provides

selectivity for the absorption of a particular analyte that is greatly influenced by the shape and size of pores.28 Due to the aliphatic structure of spermidine, it can easily penetrate inside the pores of the organic−inorganic hybrid polymer and weakly coordinate with three metal centers (Figure S17). Other biomolecules are not as linear in shape as the biogenic amines; the large size and unsuitable shape of these biomolecules did not allow them to penetrate inside the pores and interact with the complex. The geometry and polymeric nature of the metal complex made it selective to interact with spermidine and spermine. A binding test of complex 1·Cu2+ with other biomolecules was also performed. Among these, none of the biomolecules has shown any change in the photophysical profile (Figure S18). The binding mode has been studied with an FT-IR spectroscopic technique in the 400−4000 cm−1 1291


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Figure 7. Image of culture plates containing (A) mushroom samples and (B) meat samples in the wells of the first vertical row (A1−C1) while the rest of the wells are filled with 4 mL of complex solution.

Figure 8. Image showing effect of distance on the appearance of color change of complex solution due to (A) spermidine vapors evolved from mushroom samples and (B) spermine vapors evolved from meat samples. (C) Table showing absorbance for the complex solutions kept at the mentioned distance values from the sample.

of consumers causing serious diseases like respiratory disorders, renal disorder, and even cancer.39,47,48 The food samples (mushroom/meat) were placed in the first 3 wells (A1−C1) in separate culture plates, and the rest of the wells were filled with 1·Cu2+ ensemble solution (Figure 7). Mainly, three parameters, i.e., vapor detection from distance, temperature, and response time, were studied for a period of 3 days on behalf of the colorimetric properties of our probe, which visibly predict the presence of spermidine and spermine by undergoing change in color on coming in contact with the respective vapors. We used 18 culture plates each having 12 wells to have 3 sets corresponding to each experiment in order to accomplish intraday and interday precisions. The first vertical row of wells (A1−C1) of each plate was filled with an equal amount (1 g/ well) of food sample. There were 9 plates containing a mushroom sample in the first vertical row, and there were 9 other plates containing a meat sample in the first vertical row, respectively. The wells marked as A2−A4, B2−B4, and C2−C4 of each plate were filled with 4 mL of the 1·Cu2+ ensemble solution as shown in Figure 7. Further, for the avoidance of any outside interference, the plates were covered and sealed with Teflon tape. For evaluation of the application of the 1·Cu2+ ensemble for the detection of vapors of BAs evolving from a distant sample, the studies were initially started with the observation of a distance effect on the color change of the solutions with the vapors of amines released at the maintained experimental conditions. The distance between wells of the horizontal rows (A1−A4, B1−B4, and C1−C4) was measured, and it was found to be equal to 2.5 cm between adjacent wells. As the solutions were collected after a regular time interval, and after completion of 72 h, it was noticed that solutions in wells of all the rows (A2−C2, A3−C3, and A4−C4) underwent a color change; however, a peculiar trend was noticed in the color change (Figure 8A,B). However, a gradual decrease in color change was observed in the wells with increasing distance from the first row

spectral region (Figures S14−S16, Supporting Information). The IR spectra of the copper complex with receptor (Figure S14) were first differentiated from those of the free receptor (Figure S3). The relevant changes in the IR region after complexation have been observed; for example, IR frequencies of carbonyl (CO) and imine (CN) groups of receptor 1· H4 at 1697 and 1682 cm−1 are shifted to the lower frequency of 1603 cm−1 in the infrared region of the copper complex (1· Cu2+) describing the coordination of carbonyl and imine groups with copper chloride. Additionally, new IR peaks corresponding to MN vibrations have also appeared near the 454 cm−1 position in the spectra of the copper complex. The intensified infrared band characteristic of methylene CH vibrations of polyamines was observed in IR spectra of copper complex 1·Cu2+ with polyamine [spermidine (Figure S15) and spermine (Figure S16)]. Due to coordination of the polyamine with copper ions, the bands of carbonyl and imine are further altered. The CuN vibrations are also observed near described positions in the relative spectra of complexes (1·Cu2+ + spermidine/spermine). The results suggest the coordination of polyamine through copper metal ions of the 1·Cu2+ complex. The binding and positioning of infrared frequencies has been supported with literature references.42−44 Use of 1·Cu2+ Ensemble for Real Sample Examination. During the course of study, the sensor (1·Cu2+ ensemble) was subjected to real sample determination, and two types of food items (mushroom and meat) having proteinaceous content were chosen. One of them (mushroom) is rich in spermidine with a lesser amount of spermine, and the other (meat) is rich in spermine with a smaller content of spermidine.45,46 It is a well-known fact that quality of food greatly depends on the storage condition, which further decides the amount of biogenic amine released due to microbial action on protein component of various food products. Thus, spoilage of food is immediately marked by the release of amine vapors; higher amounts of such biogenic amines deleteriously affect the health 1292


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ACS Sustainable Chemistry & Engineering (A1−C1); therefore, it can be concluded that change in the color of the complex due to vapors of biogenic amines is inversely proportional to distance of the complex from the food samples. The value of absorbance recorded for the solutions during the experiment also revealed the supportive results where the highest absorbance value was obtained corresponding to intense color change of solution in well A2 (at 2.5 cm). The order for absorbance values of solutions was A2 > A3 > A4 and is also summarized in Figure 8C. The predominant factor in storing food samples is governed by the temperature as a rise in temperature leads to faster deterioration of samples due to microbial growth accompanied by the increased amount of biogenic amines released to the atmosphere; therefore, we studied the effect of temperature conditions on our samples by keeping the samples in 3 different conditions, i.e., at 5 °C (in fridge), at ambient temperature (25−30 °C), and at summer season temperature (43−45 °C). Three sets of culture plates corresponding to each sample were kept at the three mentioned conditions to observe the rate of deterioration of samples with respect to release of biogenic amine from the samples which causes change in color of our proposed sensor (Figure 9). This experiment was also continued for 3 days. As expected, the rate of deterioration and release of amine vapors was the least for

the samples stored in the fridge and was highest for the samples exposed to higher temperatures. It was noticed that samples stored in the open atmosphere showed fast saturation with regard to color change of solution; i.e., in less than 24 h, the change was almost 3 times faster than what we observed at ambient temperature, where saturation is reached in nearly 72 h. However, the samples placed in the fridge showed negligible changes in the first two days and showed a slight change at the end of third day. It is quite evident from the above experiment that food articles undergo deterioration when stored, producing hazardous biogenic amines. Even storing food articles in the fridge for longer duration causes degradation, and eventually they release harmful biogenic amines. The comparison of behavior at different temperatures was also evaluated quantitatively by recording UV−vis absorbance spectra of complex solutions collected at various time intervals. The spectra of absorbance were recorded with respect to time under the effect of temperature. A very slow and negligible change in the spectra was perceived with solutions stored in the fridge whereas the plot obtained with solutions kept at 43−45 °C showed a fast and abrupt increase in the initial hours reaching saturation in less than 24 h. From the spectra obtained with solutions stored at room temperature, it was observed that a gradual change has taken place with exceeding intervals of time achieving its plateau after completion of 2 days. The results obtained from spermine vapors are shown in Figure 9G, and those obtained with spermidine are mentioned in Figure S11. After temperature studies were performed, it was clear that the samples stored at room temperature have shown appropriate results where a gradual change in the color of solutions contained in adjacent vertical rows was noticed. Hence, we continued the time studies with the solutions of the culture plate kept at room temperature (25−30 °C), and solutions were collected for both types of samples at the duration of 8 h for 3 days. The observable color change started appearing at completion of 16 h, and progressively, the color change started getting intense with passing time, due to rise in the amount of spermidine/spermine vapors produced during the deterioration of respective food samples (Figure 10). After 56 h, the change in color of the solution has approached the

Figure 9. Image showing change in color of the complex due to (A− C) spermidine and (D−F) spermine at different temperatures (5, 25− 30, and 43−45 °C), respectively. (G) Variation in absorbance of complex solution (1·Cu2+ensemble) in the presence of spermine at various temperatures.

Figure 10. Change in color of the complex solution observed with effect of (A) spermidine and (B) spermine at various time intervals for the samples stored at room temperature. (C) Variation in absorbance of complex solution due to spermine and spermidine individually with respect to time at room temperature. 1293


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performed to explore the effect of pH on the recognition behavior by varying the acidity and basicity of the solution. X-ray Crystallography. The details of instrumentation are provided in Supporting Information. The supplementary data for the crystallographic structure related to the present paper is contained in CCDC 1451098. The data is available for free at The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk. Real Sample Determination. The fresh samples of mushroom and meat were brought from the market and washed with distilled water to remove foreign contaminants like dust particles, etc. There were 18 culture plates (12 wells) used for carrying the experiment in three sets where samples (1 g/well) were kept in the first vertical row (A1−C1), and the remaining wells were filled with 4 mL of complex solution (1·Cu2+ ensemble). These plates were sealed with Teflon tape, and a microsyringe was used for collection of solution (1 mL). The aperture caused by the puncture was carefully sealed using Teflon tape after collection of samples to avoid any effect of outer environment on samples or solution of complex. Collected samples were stored in the fridge using glass vials. The vials were brought to room temperature before the spectra were collected on a UV−vis absorbance instrument. The effects of three parameters, i.e., distance, time, and temperature, were studied in a three day experiment on daily use food articles. The solutions for studying the distance effect were collected after 72 h. The distance between adjacent wells in horizontal rows was 2.5 cm. For the temperature effect, culture plates of samples were stored at temperature conditions of 5, 25−30, and 43−45 °C. The solutions were collected at random time intervals over a period of 72 h. However, duration of 8 h was ensured while solutions were collected for studying the time effect.

saturation level which indicates the maximum deterioration of the samples. This experiment also suggests that safety of protein rich food articles became questionable even after 2 days when kept at room temperature with the effect of biogenic amines. The spectra of solutions were recorded on a UV−vis spectrophotometer, and the obtained results are presented in the form of a graph plotted between absorbance and time (Figure 10C). A sigmoidal pattern was observed where, in the starting hours (0−16 h), only a small and gradual increase was observed, and then it crosses through a steep rise occurring between the time intervals of 24−56 h. Finally, it reached the saturation position acquired near the 64th hour until completion at 72 h.

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EXPERIMENTAL SECTION

General Information. All chemicals of analytical grade were used in the experiments and were procured from Sigma-Aldrich Co and from local vendors. A JNM-ECS400 (JEOL) instrument was used for recording 1H and 13C NMR (operating frequency of 400 MHz for 1H and 100 MHz for 13C NMR), where chemical shifts were expressed in ppm. IR spectra were obtained from a Bruker Tensor 27 spectrometer in the solid state as KBr discs or as neat samples. The UV−vis absorption spectra were taken on Spectroscan 30. Dynamic light scattering (DLS) with external probe feature of Metrohm Microtrac Ultra Nanotrac particle size analyzer was utilized for getting nanoparticle size. TEM (transmission electron microscopy) analysis was carried out on a Hitachi (H-7500) instrument to obtain images for nanoparticles. Mass spectra were taken on Waters Micromass Q-Tof model mass spectrometer, and fluorescence measurements were performed on a RF-5301 PC spectrofluorometer. Receptor Synthesis. To the solution of dipicolinic acid hydrazide (0.195 g, 1 mmol) in 50 mL of ethanol was added salicylaldehyde (0.244 g, 2.0 mmol), and the reaction was refluxed for 6 h which yielded yellow precipitate. The precipitate was filtered off and washed with ethanol. Yield: 72%. 1H NMR (400 MHz, CDCl3 + DMSO-d6, ppm) δ: 6.94−6.98 (4H, m, Ar), 7.34 (2H, t, Ar), 7.70 (2H, d, Ar), 8.29−8.33 (1H, m, Ar), 8.37−8.39 (2H, d, Ar), 8.94 (2H, s, NCH), 11.07 (2H, s, OH), 12.43 (2H, s, NH). 13C NMR (100 MHz, CDCl3 + DMSO-d6, ppm) δ: 116.5, 118.9, 119.6, 125.7, 129.0, 131.8, 140.0, 148.0, 149.2, 157.5, 159.4. IR (KBr, cm−1): 1682 (imine), 1697 (amide), 2924 (CH, str). ESI-MS [M − H]+: 402.1. Anal. Calcd for C21H17N5O4: C, 62.53; H, 4.25; N, 17.36. Found: C, 62.48; H, 4.31; N, 17.41. Synthesis of ONPs. After the reprecipitation method, organic nanoparticles were prepared by choosing the desired concentration of organic receptor among different concentrations undertaken for trial. Working solution (1 mL) of receptor (1 mM) was slowly injected into 100 mL of water and sonicated continuously for 10 min maintaining temperature conditions of 25 ± 1 °C. The procedure generated uniformly dispersed nanoparticles. Recognition Studies. UV−vis absorption and fluorimetric spectral profiles were recorded at 25 ± 1 °C. The solutions were shaken sufficiently and sonicated before recording the spectrum. The binding behavior of the organic nanoparticles formed from receptor 1 was first studied for various metal ions, and then the complex of organic nanoparticles with Cu2+ ions was used as a sensor by addition of 2 mM various biogenic amines to 5 mL of solution of receptor 1 nanoparticles taken in volumetric flasks. Before the spectra were recorded, the volumetric flasks were allowed to stand for half an hour. Titrations of the 1·Cu2+ ensemble were performed by adding solution of spermine to the volumetric flasks containing a nanoaggregate solution of the 1·Cu2+ ensemble in aqueous medium. For evaluation of possible interference due to different biogenic amines for spermine estimation, solutions of the 1·Cu2+ ensemble were prepared both with and without other interfering amines. For assessment of the effect of ionic strength, the spectrum was recorded at different concentration of TBA salt of perchlorate (0−100 equiv). The pH titrations were

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CONCLUSION The present paper describes the real sample assay of biogenic amines in aqueous as well as vapor phase to meet the terms of food and health safety. The proposed AIEE based 1·Cu2+ ensemble developed from the interactions of Cu2+ ions and imine receptor 1·H4 allowed the simultaneous chromogenic detection of spermidine and spermine. The spermidine/ spermine displaced the solvent molecules from the coordination sphere of the 1·Cu 2+ ensemble because of the complementarily between the binding sites of ensemble and biogenic amines. It also offered their naked-eye recognition vis à vis a different color change in the solution. The detection limit of 35 and 36.2 nM for spermidine and spermine, respectively, has been calculated. An experiment for examining the quality freshness of proteinaceous food items consumed frequently was performed taking mushroom and meat samples as a major source of spermidine and spermine, respectively. The naked-eye sensing helped in observing the color changes in the complex solution, produced from the effect of volatile spermidine and spermine vapors released during microbial degradation of the food samples.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.6b01295. Characterization data and other graphs related to recognition studies (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +91 9815245098. ORCID

Narinder Singh: 0000-0002-8794-8157 1294


Research Article


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Navneet Kaur: 0000-0002-0012-6151 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported with a research grant provided to N.K. from CSIR (Project 02(0216)/14/EMR-II); S.C. is thankful to UGC for a research fellowship. The authors are also thankful to SAIF, Panjab University Chandigarh, for characterizations.

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