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Detection of Sudan Dyes Based on Inner Filter Effect with Reusable Conjugated Polymer Fibrous Membranes Ming Wu, Lijuan Sun, Kesong Miao, Yingzhong Wu, and Li-Juan Fan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b00164 • Publication Date (Web): 13 Feb 2018 Downloaded from http://pubs.acs.org on February 14, 2018

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ACS Applied Materials & Interfaces

Detection of Sudan Dyes Based on Inner Filter Effect with Reusable Conjugated Polymer Fibrous Membranes Ming Wu, Lijuan Sun, Kesong Miao, Yingzhong Wu and Li-Juan Fan* State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Department of Polymer Science and Engineering, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, Jiangsu 215123, P. R. China KEYWORDS: Sudan Dyes, Inner Filter Effect, Fluorescence Sensing, Reusability, Conjugated Polymer

ABSTRACT: Developing effective methods for detecting illegal additives in food or seasoning is of great significance. In this study, a sensing strategy for selective detection of Sudan dyes was designed based on fluorescence inner filter effect (IFE) by using poly(phenylenevinylene) (PPV) solid materials and combing with optimized experimental protocol. Two types of fluorescent solid materials, electrospun fibrous membranes and drop-cast films, were fabricated with PPV as the fluorophore and poly(vinyl alcohol) (PVA) as the matrix. Sudan dyes greatly quenched the fluorescence of membrane and film, while other food colorings or possible food ingredients displayed much smaller or negligible quenching effect. The sensing mechanism was studied and 1 Environment ACS Paragon Plus

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the selectivity was ascribed to IFE, which requires the overlap between the absorption of analyte and absorption/emission of the sensing material. The form of materials (membrane or film), the content of PPV and crosslink or not, did not have much influence on the selectivity and sensitivity, which is consistent with the IFE mechanism and demonstrats the advantage of no requirement for strict control of preparative process. All the crosslinked materials were found to be stable against water/humidity, and displayed good reversibility in sensing and can be reused at least for 10 cycles with negligible influence on the sensing performance. Crosslinked membrane was selected for detecting Sudan dyes in chili powder since folding didn’t affect mechanical stability of the membrane. Two different protocols were used to pre-treat the chili samples, which allowed the detection of Sudan dyes in chili powder as well as discrimination of Sudan dyes from synthetic food coloring such as allura red. This study provides a facile and costeffective method for preparing reusable sensing materials for detecting some dyes in commercial foods or food seasonings.

1. INTRODUCTION Sudan dyes are one class of dyes containing phenyl azo and are widely used as coloring agent for many chemical products and daily supplies, such as hydrocarbon solvents, waxes, oils, inks, paints, plastics, cosmetics, polishes, textiles and so on. They were also employed as additives for foods/seasonings previously but now are prohibited. Sudan dyes are water-insoluble and difficult to be excreted once get into the digestive system; and they are potential human carcinogen and mutagen due to the release of aniline during decomposition.1-3 However, sometimes Sudan dyes are still used illegally to enhance the appearance of food, such as chili powder, curry and chili sauces, due to their bright red color but low cost. Therefore, it is necessary to develop a fast and sensitive method to detect Sudan dyes in foods or food seasonings.

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There have been many methods for detecting Sudan dyes in the past ten years, such as chromatography technique coupled with other techniques,4-8 enzyme linked immunosorbent assay (ELISA),9-11 electroanalytical technique12,

13

and surface-enhanced Raman scattering

technique.14, 15 Most of those methods have been demonstrated to be sensitive and quantitative but usually require complicated procedure, expensive instrument and/or time-consuming process, which limit their application for real-time and in situ detection of Sudan dyes. Fluorescencebased sensing has attracted great interest due to short response time and easy operation.16, 17 Recently, various fluorescent materials, such as micelles,18,

19

upconversion nanoparticles,20

quantum dots,21 silicon nanoparticles22 and metal nanoclusters,23 have been used in fluorescence sensing systems for detecting Sudan dyes. These solution or colloidal solution systems may offer rapid and sensitive detection, but lack reusability and are not very cost-effective. Fluorescence sensing based on solid-materials is promising in meeting the great demand for portable and reusable sensory devices,24-27 which has advantages over solution-based sensing systems in realizing real-time detection and reducing the waste of reagent. However, to our knowledge, only very few reports were about employing fluorescent solid materials for detecting Sudan dyes but the reusability was not mentioned.19, 28 Therefore, developing reusable fluorescent solid materials for rapid, convenient detection of Sudan dyes is of practical significance. However, challenges exist for sensing applications based on fluorescent solid materials. Most fluorescent solid sensing materials are prepared by introducing fluorophores onto/into solid matrixes.29-33 The sensing process is usually accomplished over three steps: recognition, transduction and signal read-out.16, 17, 34 Most transduction mechanisms employed to convert the recognition

event

into

signal

read-out,

aggregation/conformation change which

are

electron/energy/charge

transfer

or

induce intensity quenching or enhancing. In such



sensing systems, the interaction between analytes and the sensing materials is a prerequisite and the initial fluorescence intensity of the material should maintain even and stable. There are several weaknesses for fluorescence sensing system based on solid materials employing above mechanisms. First, the inherent nonuniformity of solid materials in regard to surface morphology, thickness, surface-to-volume ratio and the fluorophore distribution, may affect the reproducibility of the sensing result. Second, the distribution of the fluorophores may also change over time/temperature due to the phase separation or other reasons, which also makes the fluorescence of sensing materials not stable over a long time and thus affects the sensing performance. Therefore, the sensitivity for solid material-based systems usually have relatively greater deviation from batch to batch, or from time to time even with the same material, than that for the solution-based systems. Seeking some alternative strategy to avoid or circumvent such weaknesses will promote the practical sensing application based on the fluorescent solid materials. Here we advanced a new strategy for detecting Sudan dyes. The fluorescence inner filter effect (IFE) was employed as the sensing mechanism. IFE results from the absorption of the excitation and/or emission light of fluorophores by absorbers in the sensing systems.35 Recently, IFE has attracted increasing attention in the fluorescence-sensing field (Figure S1),36-42 although it was used to be regarded as an adverse factor that should be avoided during emission spectra measurement.35, 43-45 In the IFE-based sensing system, if the absorption band of the analyte (or compound produced in the presence of the analyte) overlaps with the absorption (or excitation) band and/or emission band of the fluorophore, the fluorescence will be quenched.46-49 There are several possible advantages for IFE-based sensing system. First, compared with other fluorescence assays, such as fluorescence resonance energy transfer (FRET) which requires the


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distance between the donor and the acceptor less than 10 nm and appropriate orientation,35 the IFE based system does not require the direct interaction and short distance between analyte and the sensing material. Thus the introduction of receptor for recognition is not necessary which reduces the synthetic challenge. Second, the form of materials and the percentage/distribution of fluorophore may not affect the sensitivity in detecting analyte, which make the preparation of the material much easier. Therefore, florescence sensing systems based on IFE mechanism may be promising in real applications, especially when reusable fluorescent solid materials are employed. Our group has been dedicated to employing conjugated polymers as sensing materials.50-55 Conjugated polymers have distinctive advantages for fluorescence sensing, such as great sensitivity and good processability.56-58 In this study, poly(phenylenevinylene) (PPV), a class of highly fluorescent and stable conjugated polymers, was selected as the fluorophore due to the large overlap between the absorption band of Sudan dyes and excitation/emission band of PPV. First, the precursor of PPV (pre-PPV) and poly(vinyl alcohol) (PVA) was co-dissolved in water, and PPV/PVA fibrous membranes and PPV/PVA films were prepared through electrospinning and drop-casting followed by thermal elimination, respectively (Figure 1). Then, the crosslinked fibrous membranes (PPV/CPVA membranes) and films (PPV/CPVA films) were obtained after post treatment. The morphology, flexibility and photophysics of the above membranes and films were characterized, and the sensing behavior toward Sudan dyes, food colorings and some possible compounds in food was tested. The preliminary mechanism study was carried out, and the influences on sensing performance from material form, PPV content and post treatment were investigated in respect of selectivity, sensitivity and reversibility. Finally, the real application of this sensing strategy in detecting Sudan dyes in different brands of chili powder was carried out.



Figure 1. Schematic diagram for preparing PPV/PVA and PPV/CPVA fibrous membranes and films, and the chemical structures of the polymers involved. The heating was carried out at 105 °C for 1 hour; the crosslinking process was accomplished by placing fibrous membranes or films in an acetone solution of glutaraldehyde and HCl for 24 hours at room temperature. 2. EXPERIMENTAL SECTION 2.1 Reagents and materials Tetrahydrothiophene and α, α’-dichloro-p-xylene were purchased from J&K Co., Ltd. pphenylenebis(tetrahydrothiophenium) dichloride was synthesized according to the literature ഥ௡ =80000, 88% hydrolyzed) was purchased from method.59 Poly(vinyl alcohol) (PVA-1788, ‫ܯ‬ Beijing Eastern Petrochemical Co., Ltd. Sudan I-IV were obtained from Adamas reagent Co., Ltd. Vitamin C, sodium benzoate, glucose, methanol and ethanol were purchased from


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Sinopharm Chemical Reagent Co., Ltd. D-fructose, L-lysine, allura red, pyrosine B, new coccine, amaranth and capsanthin (75 wt%) were purchased from Shanghai yuanye BioTechnology Co., Ltd. All reagents and materials were used as received without further purification. 2.2 Synthesis of pre-PPV The precursor of PPV (pre-PPV) was synthesized with slight modification to the literature report.59 The mass percentage of as-obtained pre-PPV in aqueous solution was calculated to be 0.6%. The synthetic and work-up procedure and calculation details are shown in Supporting Information. 2.3 Preparation of fibrous membrane and film The aqueous solution containing pre-PPV and PVA was prepared according to the following procedure: PVA was added into deionized water and the mixture was stirred at 50 °C until the PVA was completely dissolved. After the mixture was cooled down to room temperature, the asstored pre-PPV solution was added into the PVA solution. The mixture was stirred for another 1 hour to form pre-PPV/PVA mixture solution before electrospinning or drop-casting. For electrospinning, about 3 mL of the pre-PPV/PVA solution was filled into a 10 mL syringe with a stainless steel nozzle (1.2 mm internal diameter). The nozzle was connected to a stable high voltage DC power supply set to 12 kV with copper wire. A constant volume flow rate (0.3 mL/h) was maintained via an injection pump. The aluminum foil was used as the collecting screen and the distance between the nozzle tip and the collecting screen was about 10 cm. The electrospinning process lasted for 4 h under ambient temperature (about 20-25 °C) with humidity below 40% to produce white and flexible pre-PPV/PVA fibrous membranes with a round shape on the aluminum foil, and the effective area was about 7×7 cm2. Heating the membrane on the



aluminum foil at 105 °C for 1 hour to convert pre-PPV into PPV, and then the aluminum foil was removed to give self-standing PPV/PVA fibrous membrane with light green color under ambient light and emitting blue-green fluorescence under 365 nm UV lamp. For further use, the membrane was cut into serval smaller pieces (about 1.5×0.8 cm2). For drop-casting, about 1 mL of the above solution was filled into a 1 mL syringe, and 0.1 mL of pre-PPV/PVA solution was dropped onto a neat aluminum foil. Heating above pre-PPV/PVA solution on aluminum foil at 105 °C for 1 hour to convert pre-PPV into PPV, and then the aluminum foil was removed to give self-standing light green and flexible PPV/PVA film with an elliptical shape, and the effective area was about 0.7×0.7 cm2. To make the materials more stable against water, further crosslinking treatment was carried out. The above membrane/film was placed in 25 mL of glutaraldehyde (GA)/HCl acetone solution (0.01 M HCl, HCl/GA = 1:10 in molar ratio) for 24 h. The membrane and film were then rinsed with acetone and dried under ambient environment overnight to give the crosslinked PPV/CPVA fibrous membrane and film. The crosslinked PPV/CPVA membrane/film was stable against water, but the PPV/CPVA fibrous membrane was flexible while the PPV/CPVA film was brittle. For comparison, different feeding ratios were used for preparing pre-PPV/PVA mixture solution (shown in Table S1). With different initial mass percentages of pre-PPV in all polymer materials (Mpre-PPV/(Mpre-PPV+MPVA)), the resulting fibrous membranes were named as PPV/PVA1 membrane, PPV/PVA-2 membrane and PPV/PVA-3 membrane, respectively, and the films were named as PPV/PVA-1 film, PPV/PVA-2 film and PPV/PVA-3 film, respectively. Correspondingly, the crosslinked fibrous membranes were named as PPV/CPVA-1 membrane,


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PPV/CPVA-2 membrane and PPV/CPVA-2 membrane, and the crosslinked films were accordingly named as PPV/CPVA-1 film, PPV/CPVA-2 film and PPV/CPVA-3 film. 2.4 Instruments and methods The electrospinning process was carried out with a variable high voltage power supply (DWP303-1ACFO, Tianjin Dongwen High Voltage Power Supply Co., Ltd.) and an injection pump (TJ-3A/W0109-1B, Baoding Longer Precision Pump Co., Ltd.). The morphology of the fibrous membranes and films was obtained from a scanning electron microscope (SEM, S-4700, Hitachi, Japan) with an operating voltage of 15.0 kV. The UV-vis absorption spectra were recorded with a Shimadzu UV-1800 spectrophotometer. Digital photos of the fibrous membranes and films under natural light or 365 nm UV light were taken with a Nikon D5100 camera (35 mm, 1:11.8G).

The

fluorescence

spectra

were

collected

with

a

Shimadzu

RF-5301PC

spectrophotometer using excitation at 390 nm. The laser confocal microscopy images were obtained on a Leica TCS SP5 with excitation at 405 nm. The fluorescence sensing experiments were carried out using the following protocol. The pieces of fibrous membranes and films were pasted onto a quartz slide (4.5×1.2×0.1 cm) using double-sided adhesive tape on the top, and the quartz slide with the fibrous membrane/film was placed into a 1 cm quartz cuvette, at about 30° to the exciting light. Analytes in ethanol solution in various concentrations were prepared in advance. 2 mL of pure ethanol was carefully added to the quartz cuvette to immerse the entire membrane or film and the emission spectrum was recorded after 1 minute. After pipetting pure ethanol out of the cuvette, 2 mL of analyte solution was pipetted into the cuvette and then the emission spectrum was recorded after 1 minute. The fluorescence response upon addition of analyte was calculated based on I0/I, where I0 was the emission maximum intensity of membrane/film after adding 2 mL of pure ethanol to the quartz



cuvette, and I was the maximum intensity after adding 2 mL of analyte ethanol solution to the quartz cuvette after the above pure ethanol was removed. All sensing experiments were repeated at least three times, and the results were reproducible. During the practical application, the pretreatment of chili powder sample was carried out using different protocols. First, four centrifugal tube (Tube 1, Tube 2, Tube 3 and Tube 4) were placed on the desk in parallel and 0.1 g of chili powders were added into each tube. Then 1.0 mg of allura red was added into the Tube 2, 1.0 mg of Sudan I was added into the Tube 3, and 1.0 mg of allura red and 1.0 mg of Sudan I were added into the Tube 4, respectively. All the tubes were shaken by hand to mix chili powder with Sudan I or/and allura red. Then the samples in the tubes were pretreated using two different protocols as list below. Protocol 1: 5 mL of ethanol was added into each tube, and then all the tubes were oscillated for 5 min with an oscillating rate of 1500 rpm. Then the supernatant (extract in ethanol solution) in the tubes was collected after the tubes was centrifuged for 5 min at 2500 rpm. Afterwards, 40 µL of the extract solution was diluted to 8 mL with ethanol, which is ready for fluorescence measurement. Protocol 2: First, 5 mL of deionized water was added into each tube and all the tubes were oscillated for 5 min with an oscillating rate of 1500 rpm, then were centrifuged for 5 min at 2500 rpm. After removing the supernatant (aqueous phase) and keeping the solid, all the tubes were placed back to the desk, followed by treatment as same as described in protocol 1. The experiment to investigate the relationship between the quenching effect and the amount of Sudan I added in the chili power was carried out by varying the amount of Sudan I (from 1 mg to 5 mg) added into 100 mg of chili powder. 3. RESULTS AND DISCUSSION


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3.1 Preparation and Characterization The fibrous membranes and films were prepared according to the procedures as described in the experimental section. Here are several points to be noted. First, fibrous membrane and film were prepared in parallel, aiming to investigate if the material form will affect the fluorescence sensing performance. Second, a series of fibrous membranes and films were prepared with different PPV contents to see if the content of fluorophore matters greatly in the sensing behavior. Third, the crosslinking step was to make the fibrous membrane/film more stable against water or humidity, since non-crosslinked PVA is soluble in water. The general characterizations of the fibrous membranes and films are shown in Figure S2-S5 (for all materials) and Figure 2 (using PPV/CPVA-2 membrane and film for representative). All membranes displayed a clear fibrous structure with diameters of around 250 nm, while the surface of the crosslinked/non-crosslinked films was relatively plain and structureless with slight unevenness. No significant difference was observed in the microscopic morphology of the membranes and films when varying the PPV content, or before/after the crosslinking. The digital photos show that both membranes and films emit blue fluorescence under 365 nm UV light and are of green color under natural light, which are consistent with the excitation/emission spectra in general. Slight red-shift in the emission peak can be observed, with the increase of PPV content in both membrane and film, or changing the form of materials from membrane to film when the PPV content is the same (Table S2). Such red-shift should be reasonable, since more PPV in the material can enlarge the conjugated system. In addition, in the drop-cast film, PPV chains should have more time for reorganization to reach equilibrium and form larger conjugation system, compared with electrospun fibrous membrane.



The stability of the solid materials against ethanol, water and humidity was also investigated by immersing the materials pasted on a glass slide into ethanol, pure water, ethanol/water mixture for 5 minutes and then getting dried in an oven at 60 °C; or immersing the materials in ethanol for 5 minutes and then naturally dried in humid environment (humidity around 50%-60%) under room temperature. Figure S6 shows the pictures of all the materials after above treatments. In general, all the crosslinked membrane and films displayed no significant change in macroscopic morphology after all the treatment. However, for the non-crosslinked membranes and films, damage or/and contraction to various degrees were observed. The fibrous membranes displayed more obvious damage than the films. Very likely the larger surface-to-volume ratio of the membrane facilitates the fast dissolution of PVA, while the dissolution of PVA in film needs more time. The above results suggest that in practical applications, crosslinked materials are more preferred. However, the crosslinked membrane remained unchanged even after 10 folding, while the crosslinked film is very brittle and was broken after one folding (Figure 1c). The membranes has the advantage in flexibility which may be attributed to the nano-micro fibrous structure. Therefore, crosslinked fibrous membrane should be more desirable to be used as reusable sensing materials or be incorporated into sensing device even if there is no significant difference in photophysics and the sensing performance.


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Figure 2. SEM images (a), confocal fluorescence microscopy images (b), digital photos (c), and solid state fluorescence spectra of excitation (emission at 480 nm) and emission (excitation at 390 nm) spectra (d) for PPV/CPVA-2 film and PPV/CPVA-2 membrane. 3.2 Sensing application Crosslinked PPV/CPVA-2 membrane was selected as the main sensing materials for the sensing application. However, comparisons among different materials were carried out to get a comprehensive investigation of possible influence on the sensing performance from the material form, PPV content and the crosslinking process. 3.2.1 Selectivity study The fluorescence responses of PPV/CPVA-2 membrane toward Sudan dyes were investigated. For comparison, the study was also carried out with food colorings and possible compounds in food as the analytes. Capsanthin (a natural pigment of red-color in red pepers) and red water13 Environment ACS Paragon Plus


soluble synthetic food colorings including allura red, new coccine, amaranth and pyrosine B are used as the representative food colorings. Sodium benzoate (a commonly used food preservative) and D-fructose, vitamin C, L-lysine and glucose (some natural compounds existing in foods) are used as the representative compounds in food. The chemical structures of all analytes were shown in Figure S7. The emission spectra were shown in Figure S8 and Figure S9. Figure 3 showed the intensity changes at the emission maxima of PPV/CPVA-2 membrane toward these solutions. Fluorescence quenching was very obvious upon Sudan dyes, relatively smaller towards synthetic food colorings but almost negligible upon capsanthin. Interestingly, other compounds with much higher concentration (two orders of magnitude higher than Sudan dyes and food colorings) have very little influence on the emission of the membrane. Therefore, with the experimental conditions adopt here, this membrane showed selectivity toward to Sudan dyes, with very small or negligible interference from food colorings and some compounds existing in food. For comparison, exactly same experiments were carried out with PPV/CPVA-2 film. The film showed very similar results as membrane (Figure S10). Therefore, the form of solid materials, fibrous membrane or film in this study, seems to have no significant influence on the sensing performance, such as extent of fluorescence quenching and selectivity.


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Figure 3. Fluorescence response (I0/I) of PPV/CPVA-2 membrane upon immersed into various ethanol solutions of analytes. The concentration of Sudan dyes and food coloring are 5 µg/mL, and other possible compounds in food (sodium benzoate, D-fructose, vitamin C, L-lysine, glucose) concentrations were 500 µg/mL. As mentioned in the introduction, inner filter effect (IFE) was employed as the sensing transduction mechanism. To verify that IFE worked in above sensing experiments, preliminary mechanism study was carried out. All the absorption spectra were measured (Figure S11) for the analytes with the same concentrations corresponding to those in sensing experiments in Figure 3. The excitation and emission spectra of PPV/CPVA-2 membrane which fall in between 290 nm to 600 nm are also shown in Figure S11. All the other compounds in food and capsanthin absorbed little in this range. The absorption of the synthetic food coloring has some overlap with the excitation and emission range of PPV/CPVA membrane, but some of the absorption are relatively low and some of the absorption are partly outside of excitation/emission spectra of the membrane. However, absorptions of all Sudan dyes have very large overlap with the excitation and emission spectra of PPV/CPVA membrane. Such difference in the absorption of the analytes may result in varied degree of IFE and thus different extent of fluorescence quenching, since the absorption of the excitation light and/or emission light of fluorophores by different anaytes are different. In all, the observed fluorescence quenching and selectivity in Figure 2 are generally consistent with the IFE, but further quantitatively analyzing the IFE may need more photophysical investigation in detail. In addition, some other characteristics displayed by this sensing system are also consistent with IFE mechanism. First, the form of materials, such as membrane or film, did not significantly affect the extent of quenching and selectivity, suggesting the specific surface area of



the materials does not influence the sensing behavior. Although the spectral overlap is also one of the characteristics of FRET, the distance between the donor and the acceptor (less than 10 nm) with appropriate orientation were very critical for the FRET.35 In the specific surface area of sensing materials should affect the sensitivity based on FRET mechanism since it affects the local concentration and orientation of the dyes adsorbed on the materials, and thus affects the average distance between the donor and acceptor. FRET cannot be the sensing mechanism in this study. Moreover, the quenching effect will be not greatly affected even if the material has some defects and thus the preparative process becomes much easier with no need for strict control. Second, the fluorescence response was very fast for both membrane and film, which measured only one minute after adding the analytes. Such fast response is very likely because that IFE doesn’t require physically direct interaction between the sensing materials and analytes, which usually is diffusion controlled and time-consuming, especially for thin film. Different from sensing systems based on other mechanisms where the analyst must be in proximity to the fluorophores, the sensing systems based on IFE possess advantages of only requiring spectra overlap. 3.2.2 Sensitivity study To get more accurate evaluation about the sensing performance of the materials towards Sudan dyes, the fluorescence response toward various concentrations of Sudan dyes were measured, using Sudan I as the representative analyte. Figure 4a shows the spectra of PPV/CPVA-2 membrane. As shown in the inset, a good linear relationship can be found for I0/I vs concentration of Sudan I in the range of 0-20 µM (0-5 µg/mL). The similar phenomenon was also found when using other crosslinked and non-crosslinked membranes and films as sensing materials (Figure S12). At low concentrations, the emission intensity at peak decreased rapidly


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by increasing the concentration of Sudan I. About 35% of the initial peak intensity was quenched upon 20 µM (5 µg/mL) of Sudan I. Stern-Volmer equation, I0/I = Ksv[A]+1, is commonly used for identifying the fluorescence quenching mechanism, which can be dynamic collision at excited state of fluorophore, static complexation at ground state of fluorophore, or a combination of both.35, 60 In this equation, [A] is the molar concentration of the analyte and Ksv is the Stern-Volmer constant. The larger Ksv, more efficient fluorescence quenching achieved. Similar Stern-Volmer analysis was performed for evaluating the quenching efficiency in this study. As shown in Figure 4(b) and Figure S13, all the Ksv values are very similar for membrane and film, of very little variation with the increase of PPV content and with the crosslinking treatment. These results indicate that the PPV content, different matrix (PVA and CPVA) and the form of fluorescent solid-material (or the surface-to-volume ratio) have negligible effect on the sensitivity toward Sudan I. In general, IFE-based fluorescence quenching is much less perturbed from some influencing factors which used to affect the performance of the sensing systems based on dynamic or static quenching mechanism. Therefore, IFE-based sensing strategy seems to offer considerable flexibility and simplicity in the preparation of the fluorescent solid materials as well as the fluorescent measurement without requiring very strict control of the process.



Figure 4. (a) Fluorescence spectra of PPV/CPVA-2 membrane upon Sudan I in ethanol solution of various molar concentrations (from top to bottom:0, 0.04, 0.2, 0.4, 0.8, 2.0, 4.0, 8.0, 20.0, 40.0, 80.0, 161.0, 242.0 and 322.0 µM); the excitation was set at 390 nm. The inset shows the plot of I0/I vs molar concentration of Sudan I, and all values of Ksv for different materials were obtained from slope of the linear part of the plot. (b) The Ksv of crosslinked membranes and films upon Sudan I. The error bars shown are based on the calculated standard deviations from the average value of at least three measurements. The limits of detection (LODs) for Sudan dyes were calculated (calculation detail shown in Supporting Information) and are very similar for PPV/PVA-2 membrane, PPV/CPVA membrane-2, PPV/PVA-2 film and PPC/CPVA-2 film (Table S3), with the values about 64.32,


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73.39, 90.35 and 71.68 ng/mL, respectively. The difference in polymer matrix, PPV fluorophore and form of materials has no significant influence on the LOD, which is consistent with the IFEbased mechanism. As shown in Table S3, LODs in this study are slightly higher but comparable with the LODs reported by literatures for detecting Sudan dyes,20-23, 28, 61 especially very close to the LOD reported using egg-shell membrane. However, our crosslinked membranes possess the advantage over the reported sensing systems in the reusability as will be discussed below. 3.2.3 Reusability study The reusability of the sensing materials is preferred, especially when the material is incorporated into a sensor device. The photostability was important for reusing these materials. Here PPV/CPVA-2 membrane was selected for representative. The membrane was continuously exposed to the Xe lamp and the slit width of excitation was set at 3 nm and the slit width of emission was set at 1.5 nm on the same instrument we used for sensing experiment. The excitation was set at 390 nm, and the emission spectra of the membrane over time were recorded, as shown in Figure S14. With the 5 minutes’ irradiation, only 3.4% of intensity at the maximum decreased and even after 30 minutes’ irradiation, only 15.9% of intensity at the maximum decreased. Such photostability is enough for reusing this sensing material. There are several reasons that such slight decrease in the intensity after long irradiation will not affect the sensing performance. First, in the sensing experiment, the membrane will not be exposed to the Xe lamp for a long time, but only about a few seconds for excitation before recording the emission spectrum. Second, the initial fluorescence intensity of the membrane seems to have little effect on the selectivity and sensitivity based on IFE mechanism. In addition, to investigate if the sensing performance changes over time/temperature, the responses upon Sudan dyes were compared, using the same batch of PPV/CPVA-2 membrane



right after preparation (in summer) and about 6 months late (in winter). As shown in Figure S17, there was no significant change on the extent of fluorescence quenching after six months. . This result confirmed that the sensing performance of the solid materials in our study maintains similar over time/temperature (kept in a room without air conditioning for 6 months, with temperature ranging from over 35 oC to 5 oC). This result confirmed that the sensing performance of the solid materials in our study maintained similar over time/temperature. Then, the reversibility study of detecting Sudan I was carried out using the crosslinked membranes and films. After each measurement, the Sudan dyes could be washed off by ethanol easily since there are no receptors in solid materials for interacting with them. As shown in Figure 5, the emission intensity of PPV/CPVA-2 membrane recovered to the same level as the initial one which suggested that the membrane displayed very good reversibility for fluorescence quenching upon Sudan I, even after 10 cycles, and the digital photos of the material (Figure S15(a)) also shows that negligible change was observed in the macroscopic morphology. Other membranes and films displayed similar performance (see Figure S15-S16). Interestingly, all the materials were found to have similar sensing reversibility which further demonstrated the form of the materials and content of the fluorophore didn’t greatly affect the fluorescence sensing performance based on IFE. The reversibility study suggests that the crosslinked materials can be reused many times which can reduce the use and waste of solvents, compared with many reported fluorescence sensing systems based on solution or colloidal solution with IFE as the transduction mechanism. Moreover, combing the reusability of the crosslinked membrane without breaking after folding, the fluorescent PPV/CPVA membrane is very promising for practical sensing application based on IFE mechanism.


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Figure 5. Reversibility study of PPV/CPVA-2 membrane for sensing 10 µg/mL of Sudan I in ethanol solution for 10 cycles. The red circle represents the initial state of the membrane immersed in ethanol (intensity change I/I0 = 1); the triangle represents the intensity change of the membrane immersed in Sudan I after removing ethanol; the square represents the intensity change of the membrane immersed in ethanol again after removing Sudan I. The excitation was set at 390 nm. 3.2.4 Detection of Sudan dye in chili powder The ultimate goal for this study is to employ above sensing materials for real application of detecting the possible existence of Sudan dyes in commercial foods or food seasonings. Since PPV/CPVA films were found to be brittle, PPV/CPVA membrane, as the most ideal solid sensing material, was used for practical applications. PPV/CPVA-2 membrane and Sudan I were selected as the representative florescence sensing material and the dye, respectively. Four commercial brands of chili powders were used in this study (digital photos shown in Figure 6(a) and Figure S18). The results from the previous selectivity study shows that the synthetic food coloring, allura red, may have some interference to Sudan dyes, using the sensing protocol by dissolving dye,



food colorings or other compounds in ethanol. In current practical application study, small amount of Sudan I or/and allura red was mixed with chili powder to mimic the real situation. It is to be noted that Sudan dyes are soluble in ethanol but not in water, but synthetic food colorings are soluble in both ethanol and water. The water-soluble characteristic makes synthetic food colorings nontoxic within the limited dosage and allowed to be added in food since they can be excluded out of body. Thus different protocols were adopted for the pre-treatment based on consideration of the difference in solubility. The first protocol is to wash the mixture with ethanol at first, then collect the supernatant (or also called extracted solution) which is further diluted for fluorescence measurement. In the second protocol, the mixture was washed by water first and then centrifuged. The resulting solid after centrifugation was treated following exactly the same procedure as the first protocol to give another extracted solution which also was diluted. The fluorescence response of PPV/CPVA-2 membrane upon above diluted extracted solutions was obtained according to the regular steps described in the experimental section. Figure 6(b) and Figure 6(c) show the intensity changes at the emission maximum of the membrane upon different diluted extracted solutions. As shown in Figure 6(b), when all the chili powder samples were treated with protocol l, the extract of the pure chili powder samples had a very small effect on the fluorescence intensity of the membrane, the extract of the sample with allura red could slightly quench the fluorescence of the membrane because allura red is slightly soluble in ethanol. Obvious fluorescence quenching was observed in both extracts of the sample only having Sudan I, and the sample having Sudan I and allura red, with relatively greater quenching for the latter. These results suggest that no Sudan dyes were detected in the four brands of chili powder using protocol 1. However, when


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employing protocol 1, Sudan dyes added into the chili powder can be easily detected, while the presence of allura red may slightly interfere the detection of Sudan dyes. Therefore, protocol 2 was employed to eliminate the interference from allura red by pretreatment with water first, since most synthetic food colorings are water-soluble, while Sudan dyes are oil-soluble dyes and poorly soluble in water. As shown in Figure 6(c), both the extract of the pure chili powder sample and the extract of the sample with allura red had a little effect on the fluorescence intensity of the membrane, very likely most of allura red was removed by washing with water. The extract of the sample with Sudan I, and the extract of the sample with Sudan I and allura red, have a similar and obvious quenching effect on the fluorescence intensity of the membrane. These results suggest that no Sudan dyes were detected in the four brands of chili powder using protocol 2. However, when employing protocol 2, Sudan dyes added into the chili powder can be easily detected, and the interference of allura red can be reduced by water washing. Therefore, and protocol 2 we proposed is very promising for the detection of Sudan dyes in real samples without interference from food colorings.



Figure 6. Digital photos of four commercial chili powders (a). Fluorescence response of PPV/CPVA-2 membrane upon four extracts of the chili powders using protocol 1 by pre-treating


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chili powders with ethanol (b) and protocol 2 by pre-treating chili powders with water first and then with ethanol (c) (the amount of Sudan I added was 1 mg per 100 mg chili powder). The relationship between I0/I vs the amount of Sudan I (0-5 mg per 100 mg chili powder) added in chili powder 1 using protocol 1 and protocol 2 (d). To investigate if the real sample detection is only qualitative or can be quantitative, more experiments were carried out. Chili powder 1 was selected as the representative and different amount (0-5 mg per 100 mg chili powder) of Sudan I was added. In both protocols, linear relationship can be found for I0/I vs amount of Sudan I’s added. The difference in the values of I0/I between the two protocols might be due to the protocol 2 has one more step (washed by water and followed by centrifugation without drying) that may take away some amount of Sudan dyes. Although it seems that the detection can be quantitative for using one brand of chili powder in the range of amount of Sudan dye added, different brands of chili powder may have different degree of interferences. Therefore, detection of Sudan dyes in real samples can only be semiquantitative analysis up to now in our study. Further study is needed to improve the sensing strategy and protocol to provide a complete and universal quantitative analysis for Sudan dyes. 4. CONCLUSION In this work, fluorescent PPV/PVA fibrous membranes and films have been successfully prepared. The following crosslinking treatment made the resulting PPV/CPVA materials more stable against water compared with the non-crosslinked materials. The crosslinked film was found to brittle upon folding while the crosslinked fibrous membrane was very flexible and remained intact against many times of folding. Sudan dyes displayed more obvious fluorescence quenching effect on all the fibrous membranes and films than food colorings and some possible compounds in food. Photophysical study showed the absorption spectra of Sudan dyes have



much larger overlap with the excitation/emission spectra of PPV than other analytes and thus the mechanism for the selectively quenching can be attributed to the inner filter effect (IFE). Systematic study showed that the material form, PPV content and crosslinking treatment or not had little effect on the sensitivity, which are generally consistent with the characteristics of IFE. In addition, the crosslinked membrane can be reused for many times and the sensing performance can maintain similar even after long storage. Finally, combining with certain protocols for pre-treatment of the chili powder based on the different solubility of the dyes, the selective detection of Sudan dyes in chili powder was successfully realized by using the flexible crosslinked membranes. The facile preparation and good reusability of the membrane, and effectiveness in sensing, suggest the overall strategy presented in this work is cost-effective and very promising to be extended for other sensing applications. ASSOCIATED CONTENT Supporting Information Some additional information, figures and tables for experiment, calculation, characterizations and discussions. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (Fan L. J.) Notes The authors declare no competing financial interests. ACKNOWLEDEMENTS


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TOC



Figure 1. Schematic diagram for preparing PPV/PVA and PPV/CPVA fibrous membranes and films, and the chemical structures of the polymers involved. The heating was carried out at 105 °C for 1 hour; the crosslinking process was accomplished by placing fibrous membranes or films in an acetone solution of glutaraldehyde and HCl for 24 hours at room temperature. 108x140mm (300 x 300 DPI)

ACS Paragon Plus Environment

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Figure 2. SEM images (a), confocal fluorescence microscopy images (b), digital photos (c), and solid state fluorescence spectra of excitation (emission at 480 nm) and emission (excitation at 390 nm) spectra (d) for PPV/CPVA-2 film and PPV/CPVA-2 membrane. 103x128mm (300 x 300 DPI)



Figure 3. Fluorescence response (I0/I) of PPV/CPVA-2 membrane upon immersed into various ethanol solutions of analytes. The concentration of Sudan dyes and food coloring are 5 µg/mL, and other possible compounds in food (sodium benzoate, D-fructose, vitamin C, L-lysine, glucose) concentrations were 500 µg/mL. 62x45mm (300 x 300 DPI)


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Figure 4. (a) Fluorescence spectra of PPV/CPVA-2 membrane upon Sudan I in ethanol solution of various molar concentrations (from top to bottom:0, 0.04, 0.2, 0.4, 0.8, 2.0, 4.0, 8.0, 20.0, 40.0, 80.0, 161.0, 242.0 and 322.0 µM); the excitation was set at 390 nm. The inset shows the plot of I0/I vs molar concentration of Sudan I, and all values of Ksv for different materials were obtained from slope of the linear part of the plot. (b) The Ksv of crosslinked membranes and films upon Sudan I. The error bars shown are based on the calculated standard deviations from the average value of at least three measurements. 119x171mm (300 x 300 DPI)



Figure 5. Reversibility study of PPV/CPVA-2 membrane for sensing 10 µg/mL of Sudan I in ethanol solution for 10 cycles. The red circle represents the initial state of the membrane immersed in ethanol (intensity change I/I0 = 1); the triangle represents the intensity change of the membrane immersed in Sudan I after removing ethanol; the square represents the intensity change of the membrane immersed in ethanol again after removing Sudan I. The excitation was set at 390 nm. 63x48mm (300 x 300 DPI)


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Figure 6. Digital photos of four commercial chili powders (a). Fluorescence response of PPV/CPVA-2 membrane upon four extracts of the chili powders using protocol 1 by pre-treating chili powders with ethanol (b) and protocol 2 by pre-treating chili powders with water first and then with ethanol (c) (the amount of Sudan I added was 1 mg per 100 mg chili powder). The relationship between I0/I vs the amount of Sudan I (0-5 mg per 100 mg chili powder) added in chili powder 1 using protocol 1 and protocol 2 (d). 213x545mm (300 x 300 DPI)



TOC 68x55mm (300 x 300 DPI)


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Detection of Sudan Dyes Based on Inner-Filter ... - ACS Publications

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