Reduced Graphene Oxide Based âTurn-Onâ Fluorescence Sensor for

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Reduced Graphene Oxide based ‘Turn-On’ Fluorescence Sensor for Highly Reproducible and Sensitive Detection of Small Organic Pollutants Reetam Mitra, and Arindam Saha ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b01971 • Publication Date (Web): 10 Nov 2016 Downloaded from http://pubs.acs.org on November 13, 2016

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ACS Sustainable Chemistry & Engineering

Reduced Graphene Oxide based ‘Turn-On’ Fluorescence Sensor for Highly Reproducible and Sensitive Detection of Small Organic Pollutants

Reetam Mitra† and Arindam Saha* ‡ † Department of Chemical Engineering, Jadavpur University, 188, Raja S. C. Mallick Road, West Bengal, Kolkata-700032, India ‡ Sensor and Actuator Division, CSIR-Central Glass and Ceramic Research Institute, 196, Raja S. C. Mallick Road, West Bengal,Kolkata-700032, India

*Address correspondence to [email protected]

Abstract: Although ‘turn-on’ fluorescence based detection approach has been widely used in diverse fields, its implementation in graphene based organic pollutant detection is challenging. Water samples having low quantity of organic pollutants posing appreciable challenges in its sensitive and reproducible detection. We have developed a dextran-fluorescein functionalized graphene composite which can reproducibly detect organic pollutants like bisphenol A, 1-napthol, phenol and picric acid in nanomolar to picomolar concentration via ‘turn-on’ fluorescence approach. In this approach, dextran-fluorescein has been used as fluorescent probe whose fluorescence remains quenched on graphene surface. In presence of organic pollutants, the fluorescent probe detaches itself from graphene surface due to competitive interaction with the organic molecules for graphene surface. Recovered fluorescence is measured as the positive signal of organic pollutants. This detection approach is reproducible, sensitive and requires small amount of sample volume. We have extended this approach for on-field detection of organic pollutants. For this purpose, the composite material is coated on a silica based plate (thin layer chromatography plate) and exposed to organic pollutants. Under handheld UV lamp (365 nm excitation), recovered fluorescence can be seen in naked eye as signal of the presence of organic pollutants in the sample of interest. This approach is simple, reproducible, sensitive and reusable.

Keywords: Graphene, dextran-fluorescein, detection, sensitive, reproducible, device.

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Introduction: Scientists have been struggling since decades for the quantitative and reproducible ‘on-field’ detection of small organic pollutants. The major challenges imposed in detecting these pollutants are- (i) ultralow concentration, (ii) exact quantification, (iii) separation from aqueous phase and (iv) reproducibility of detection.1-4 Although the organic pollutants are present in small amounts in surface water, waste water, drinking water, canned food materials and baby bottles, these can generate severe health problems for living beings by creating acute diseases and destroying the ecosystem. Moreover, with the technological advancement these pollutants are amplifying day by day without being noticed. Thus the exact determination of organic pollutants has turn out to be a global concern.5-9 Among many organic pollutants, here we have focussed on four major organic pollutants Bisphenol A (BPA), 1-naphthol (NP), phenol (PH) and picric acid (PA) since these are most commonly encountered. Among these pollutants BPA is a well known endocrine disruptor compound (EDC) which can disturb the activities of hormones via interaction with hormonal receptors and interfering with the natural metabolism of the hormones in human body. These results in many diseases including cancer, neurobehavioral disorders, adverse pregnancy outcomes, diabetes etc.10-16 1-naphthol belongs to the carbaryl class of pesticides and can cause detrimental effect on human health.17-18 Phenolic compounds are another major class of organic pollutants since these are the major industrial by-product. It can easily transfer to the human body through drinking water and can cause severe liver damage.19-20 Picric acid (PA) on the other hand is a well known explosive since World War I. Other than being explosive, it has been found in industrial wastes especially in blasting, leather and pharmaceutical industries and has chronic effects on respiratory system and liver.21-22 PA detection is not only important for environmental protection but also a major concern for homeland securities and forensic science.23 Many research groups are currently involved in finding suitable methods for ultrasensitive ‘on-field’ detection of these organic pollutants. Presently many methods are available for BPA and PA detection (SI, Table S1 and S2), but on the other hand detection of NP and PH has not been attained much consideration (SI, Table S3).17, 2425

Most of the currently employed methods for BPA detection use electrochemical,26-

29

fluorescence,30-31 or chromatography-mass spectrometric methods.32-33 For PH

detection, colorimetric,34-35 electrochemical36 and chromatographic approaches37 has


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been widely used. On the other hand fluorometric and colorimetric approaches are most common for PA detection.38-39 Fluorescent conjugated polymers,40-44 metalorganic frameworks,45-46 chitosan,47 polysiloles,48 small fluorophore,49-51 graphene52-53 and fluorescent nanoparticles54-55 have been widely used for PA detection. However, these approaches are often associated to (i) Long pre-processing time with complexity (HPLC, GC, LC etc.),30,

32-33, 37

(ii) Difficult fabrication process and derivatization

processes (electrode modification, enzymatic reaction),26-29,

34-35

(iii) Complex and

large set-up (chromatographic units attached with detection unit),30, 32-33, 37 (iv) costly equipments (chromatographic set-up, enzymatic reactions etc.),30, sensitivity,

24-27, 34-37, 40-49, 51-53

27, 45-51, 54-55

(vi) Poor reproducibility

32-35, 37

(v) Low

and (vii) Lacking

‘on-field’ applications. Graphene based optical or electrochemical detection has gained enormous attention in last decade due to large surface area and unique properties of graphene.5662

Graphene and graphene based composites have been widely used to detect various

small biomolecules,61,

63-66

DNA,67-68 protein,69 enzymes70 and many other small

organic molecules including multiple pollutants.29,

52-53, 71-73

Due to its high surface

area and cyclic organic network, it can strongly interact with the small organic molecules via π-π interaction, hydrogen bonding or other hydrophobic interactions. As a result, it has been used extensively to concentrate and separate different organic pollutants in waste water treatment.74-77 Although electrochemical detection has been commonly employed in graphene based detection approaches, it requires complex electrode fabrication and often long pre processing time. On the other hand, optical detection is simple and straight-forward with high sensitivity and reliability. Graphene based optical sensor has been reported for picric acid detection.52-53 However, optical detections are mostly based on quenching of the fluorophore (turn-off) which has limited sensitivity and reproducibility. Here, we report a graphene based composite platform for optical ‘turnon’ detection of organic pollutants (BPA, NP, PH, PA). Graphene oxide (GO) is first synthesized and subsequently converted to reduced graphene oxide (RGO) via hydrazine reduction. It is then followed by polystyrene sulfonate (PSS) attachment to attain a soluble RGO-PSS composite system. Dextran-fluorescein (Dex-fl) is finally loaded on this material as the fluorophore probe under optimized condition. These composite (RGPD-fl) results in completely quenched fluorescence. This is because of the close proximity between the surface of graphene and fluorophore molecules,


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facilitating energy transfer from fluorophore to graphene. After adding organic pollutants in varying concentration (milimolar to picomolar), it strongly interacts with the graphene surface and liberate dextran-fl from graphene surface, resulting in enhanced fluorescence in solution. We have calculated the binding constants for these interactions to understand the mechanism. This approach of ‘turn-on’ detection shows high sensitivity with good reproducibility. For on-field detection of organic pollutants, this approach is further extended to fabricate silica based sensor. For this purpose, silica gel based thin layer chromatography (TLC) plates are cut into small pieces and loaded with composite solution via dip-coating technique. After air drying, drop of organic pollutant is added and enhanced fluorescence is observed in naked eye under UV-light (365 nm excitation) exposure. This approach has further extended to detect organic pollutants in various water samples. We have tested surface water, drinking water and industrial waste water for quantitative detection of organic pollutants using our approach following a standard addition method. We have detected considerable amount of BPA in waste water and surface water. Whereas, BPA content in purified drinking water is below the detection limit of this approach. We have also tested for PA and phenol in these water samples.

Experimental section: Reagents: graphite powder, dextran-fluorescein, polystyrene sulfonate, bisphenol A was purchased from Sigma-Aldrich and used as received. Sodium nitrate, concentrated sulphuric acid, potassium permanganate, phenol, 1-napthol, picric acid, TLC plates (average pore diameter ~9.5-11.5 µm, layer thickness ~175-225 µm) was purchased from Merck. Distilled water was collected from local market, surface water collected from lake and industrial waste water was collected from the industrial zone of Kolkata. Preparation of GO: Graphene oxide is prepared via modified Hummer’s method.63 In brief, 200 mg graphite powder, 100 mg sodium nitrate and 5 mL concentrated H2SO4 were mixed and cooled to 0O C under vigorous stirring condition. 600 mg KMnO4 was added to this solution slowly. The temperature of the solution was then raised to 35O C and kept there for 30 minutes. A brownish grey paste was produced which was mixed with 10 mL water. The temperature of the solution was increased to 98O C and was maintained for 15 min. The whole solution was then mixed with ~30 mL of lukewarm water followed by addition of 500 µL 3% H2O2 solutions to reduce


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the residual permanganate. The light yellow particles were washed carefully with warm water 7–8 times. The solid was air dried and dissolved in 20 mL distilled water by 15 min sonication followed by centrifugation at 3000 rpm for 30 min. The supernatant was collected and used for composite preparation. Preparation of RGO-PSS composite: 1 ml of as prepared GO (~15 mg/ml) is first diluted to 5 ml by double distilled water. Next 100 µl hydrazine hydrate was added to it followed by heating at 800C for 1 hour under stirring condition.63 Reduced graphene oxide (RGO) was prepared as indicated by complete precipitation of black solid in the reaction flask. Next 25 mg poly styrene sulfonate (PSS) dissolved in 1 ml water was added to this RGO followed by sonnication for 30 minutes. The as prepared RGOPSS composite was centrifuged at 12000 rpm for 5 minutes and the precipitate (if any) was discarded. Free PSS was separated by a precipitation-redispersion method. In brief, 1 ml of the RGO-PSS composite was mixed with 100 µl 0.1N NaCl solution followed by addition of a drop of chloroform and 4/5 drops of acetone. The resultant mixture was shaken vigorously until visible precipitate was observed. Next, the solution was centrifuged at 12000 rpm for 5 minutes and the supernatant containing unreacted PSS was discarded. The precipitate was dispersed in 1ml fresh water. This process was repeated three times. Dextran-fluorescein attachment to RGO-PSS: 6 ml RGO-PSS solution was mixed with 400 µl Dextran-fl (3mg/ml) and vigorously stirred for 12 hours. The initial fluorescence of the dextran-fl was quenched over this time period. To remove the excess dextran-fl from the solution precipitation-redispersion method was adopted as described earlier. The final RGO-PSS-dex-fl composite (RGPD-fl) was stored in 6 ml double distilled water for further use. Organic pollutant detection: 400 µl RGPD-fl composite was diluted with 500 µl water. To this solution 100 µl of organic pollutants (BPA, NP, PH, PA) of desired concentration was added separately followed by sonnication for 15 minutes and kept stand-still for half an hour. Next 200 µl of the above solution was taken in a 96 well microplate for fluorescence measurement. Different concentration of these molecules was tested from 10-4 M to 10-13 M. For control experiment, in one set 100 µl water was added in place of organic pollutants. Binding

constant calculation: For binding

concentrations (10

-11

M to 10

-5

constant calculation different

M) of each pollutants was added to the RGPD-fl

solution separately keeping the final volume fixed (1 ml). Recovered fluorescence


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was measured in each case. Control experiment was performed where no pollutant was added. Each experiment was repeated for five times and the relative standard deviation for each data point has been found below 1%. Reproducibility check for detection: For reproducibility checking, every measurement was repeated for 5 times in 5 different batches keeping other experimental conditions same. Different batch of RGPD-fl was also tested. Fluorescence measurement of each sample was carried out in a microplate reader under similar conditions. Film based approach for detection of pollutants: We have chosen silica based TLC plate for film preparation. TLC plate was first cut into 2cm X 2cm pieces and then dipped into RGPD-fl solution for overnight. RGPD-fl coated films were then rinsed with double distilled water twice and air-dried. In this film 10 µl of test sample (10-8 M) was added and checked with a hand held UV-lamp (365 nm excitation). Control experiments: To test the validity of our approach we have taken other molecules as control. The molecules tested are octadecene, cyclohexane (hydrophobic), tryptophan, tyrosine, phenyl alanine and histidine (aromatic ring containing amino acids). All the samples tested in a concentration of 10-8 M with RGPD-fl under identical conditions. We have also tested 4-nitrophenol, 1, 2, 4trichlorobenzene and 1, 2, 4, 5-tetrachlorobenzene as control molecules since they normally present in waste waters and surface waters. We have further studied the interference from common ions generally present in water samples. Practical application: For practical application of this detection approach we have collected surface water, industrial waste water and purified drinking water to test the presence of organic pollutants. All the water samples were first concentrated 10 times by evaporating the water samples followed by filtration with a Whatman 40 filter paper. Then a drop of these concentrated samples were put on the plate based device and observed under hand held UV-lamp. To determine the concentration of the organic pollutants (BPA), we have adopted a standard addition method. In this approach known amount of BPA ranging from 1 X 10-10 M to 1 X 10-7 M was added to same volume of test samples and then mixed with RGPD-fl probe followed by fluorometric detection via a microplate reader. All samples have been tested thrice and average result is plotted. From the intercept on y axis BPA, phenol and PA concentration in test samples has been calculated.


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Instrumentation: All UV-Visible spectra were measured using Shimadzu UV-2550 spectrophotometer with a quartz cell of 1 cm path length. Fluorescence measurements were performed using SynergyTM MX multi mode microplate reader. AFM was measured using VEECO DICP II autoprobe (model AP 0100). Zeta potential measurements were performed with a Zetasizer nanosystem (Malvern Instruments Ltd.). Fourier transform infrared spectra (FTIR) were measured with Nicolet 6700 (Thermo scientific) instrument using KBr pallet.

Results: (i) Synthesis of RGPD-fl: Synthesis of RGPD-fl composite is shown in scheme 1. GO has been synthesized via modified Hummer’s method. As prepared GO is hydrophilic in nature owing to its various surface functional groups (Acid: -COOH, alcohol: -OH, epoxy: -O- etc.).78-79 GO has limited electrical conductivity and fluorescence quenching efficiency due to its perturbed surface resulting from these surface functional groups.78-79 Thus it has been reduced via conventional method using hydrazine as reducing agent. This results in complete precipitation of reduced graphene oxide (RGO) owing to its loss of surface functional groups. To make it hydrophilic, polystyrene sulfonate is attached with RGO under vigorous sonnication. This attachment is due to strong π-π interaction between polystyrene backbone and graphene surface.80 Anionic sulfonate groups provide hydrophilicity to this composite. Zeta potential measurement (-30 mV) confirm its anionic nature. For dextran-fluorescein loading on this composite, optimum concentration of dextranfluorescein solution is added and stirred for overnight. Excess dextran-fl was separated from the composite by precipitating it using NaCl and few drops of chloroform/acetone mixture. (1:4 by volume) Loading amount was calculated from UV-Vis spectroscopy (SI, Figure S1) by subtracting the unloaded dye concentration from the actual amount of added dye concentration. Different samples of varying loading amount were prepared to optimize the detection sensitivity. (ii) Characterization of the composite: Optical properties of the composite have been studied by photoluminescence and UV-Visible spectroscopic measurement (Figure 1). As prepared GO show absorbance at ~230 nm due to π-π* transition of C=C bonds in sp2 bonded region and at ~300 nm due to n-π* transition of C=O bonds at sp3 bonded regions80 whereas RGO-PSS show absorbance at ~260 nm. This hypsochromic shift is due to the restoration of the electronic conjugation on graphene


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sheet.81 Moreover, RGO-PSS show higher scattering compared to as prepared GO. Although the purified composite (RGPD-fl) do not show any emission characteristics, it shows typical absorbance of fluorescein dye at ~490 nm along with the RGO absorbance at ~260 nm. Atomic force microscopic (AFM) measurement has been done to confirm the dextran-fl loading on RGO-PSS composite (Figure 2). AFM line profile of as prepared GO show average height of ~ 0.7 nm confirming the presence of single layer graphene.63 On the other hand, RGPD-fl shows average height of ~18 nm in AFM line scan. This increased height is due to the formation of the composite and layer stacking. FTIR spectroscopy has been conducted to further confirm the composite formation. (Figure 3) Strong and broad –OH stretching frequency has been observed at ~3420 cm-1 in GO which has substantially reduced in RGO-PSS and RGPD-fl composite. –C=O stretching frequency and C-O (from -COOH) stretching frequency is observed for GO at ~1742 cm-1 and ~1089 cm-1 respectively which is absent in the composites. These confirms the reduction of GO to RGO. Stretching frequency for – SO2 has been observed at ~1194 cm-1 and ~1134 cm-1 for both the composites which is missing in GO, confirms the attachment of RGO with PSS. Dextran-fl attachment has been confirmed from C-N and C=S stretching frequency at ~1044 cm-1 and ~1007 cm-1 respectively which is completely absent in GO and strong in RGPD-fl composite. C=C stretching frequency and epoxide C-H deformation has been observed at ~1630 cm-1 and ~1405 cm-1 respectively in all the three spectra. Other than that, C-H asymmetric and symmetric stretching frequency has been found at ~2925 cm-1 and ~2846 cm-1 for both the composites and is weak and suppressed (due to broad and strong –OH stretching frequency) for GO. 82 (iii) Detection of organic pollutants: Four different organic pollutants including bisphenol-A (BPA), 1-Naphthol (NP), phenol (PH) and picric acid (PA) has been detected using ‘turn-on’ fluorescence approach depicted in scheme 2. For this detection purpose RGPD-fl composite is mixed with different concentrations of these organic pollutants separately and sonnicated for 15 minutes for complete mixing and interaction between the composite and the pollutants. After half an hour, fluorescence measurement has shown increased fluorescence intensity from this solution compared to the control sample (Figure 4). This 'turn-on' fluorescence response has also been studied by lifetime measurement. With addition of organic pollutants lifetime of the fluorophore increases. (SI, Fig S2) Although all the four pollutants response in this


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approach, their extent of response is different. As for example, PA and PH offer strong responses whereas NP offers weakest response to this approach. This is evident from the fluorescence spectra of all the four pollutants with RGPD-fl (Figure 4). To better understand this trend we have calculated the binding constant of these pollutants with the composite. (SI, Fig S3) This study supports the trend we have obtained. Micromolar to picomolar concentration of all the pollutants have been tested. Fluorescence response is found to be linear with concentration in the range of 10-11 M to 10-6 M for PA and PH with limit of detection (LOD) 5 pM for PA and 7.2 pM for PH (Figure 5). In case of BPA and NP fluorescence response is found to be linear with concentration in the range of 10-10 M to 10-6 M with LOD 8 pM for BPA and 10 pM for NP (Figure 5). Linearity of detection has been confirmed by measuring other high and low concentrations of pollutants. (SI, Fig S4) Reproducibility of this sensing has been tested for all the pollutants studied. For this purpose five different sets of all the pollutants have been measured separately (10-9 M concentration) under similar experimental conditions and found to be highly reproducible. The relative standard deviation for all the measurements has been found within 1% suggesting onfield accurate detection capability of this approach (Figure 6). (iv) Application techniques: The ‘turn-on’ fluorescent detection approach has been used to detect organic pollutants in drinking water, surface water and industrial waste water. For the ease of on-field detection, we have made a small silica plate based system which is cheap, easy to carry, reusable and requires very low sample volume (1-5 µl). For this purpose, silica based TLC plate has been cut into pieces (2 cm X 2 cm) and immersed in RGPD-fl overnight. RGPD-fl coated plates are then rinsed with double-distilled water to remove unbound composites and finally air dried (Figure 7). The plates are then treated with 5-10 µl of each of the pollutants and enhanced fluorescence is observed under a hand held UV lamp (365 nm excitation). Next different test samples were detected on this plate using UV-lamp (Figure 7). Reusability of the plate has been tested with repeated detection and washing steps. It has been found that even after 5 steps detection reproducibility is restored (Figure 7). Washing has been done by ethanol since the organic pollutants are highly soluble in ethanol. Storage and stability of this plate based sensor system has been tested. For this purpose, RGPD-fl coated plate was kept for six months in dark. After six months it has been tested by putting 10 µl of BPA on it. Even after six months of storage


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recovered fluorescence is detected which supports it's excellent stability. (Figure 7) For accurate quantitative measurements we have adopted standard addition method. Here, BPA in all the three test samples have been measured. In industrial waste water ~1.075 X 10-8 M BPA has been recorded whereas surface water show ~2.3 X 10-11 M BPA. No detectable BPA has been found in purified drinking water (SI, Figure S5). Other pollutants has also been measured in the tested water samples (Table 1). According to international standard the upper limit of phenol and phenolic compounds are 5mg/l in industrial effluents, 0.001 mg/l for surface water and 0.1 µg/l for drinking water. Whereas upper limit for BPA 1.75 µg/l in industrial water and it should not be present in drinking water. Pesticides and picric acid should be below 1 mg/l in industrial water and should not be present in drinking waters according to international standard. Although, these upper limits vary depending on type of industry as well as depending on country, our detection method is suitable to detect the organic pollutants above the threshold values in different water samples.

Discussion: Our target is to develop an ultrasensitive detection approach for reproducible and ‘on-field’ detection of organic pollutants with minimum effort. Graphene has been found most suitable for this detection purpose due to its conditional fluorescence quenching efficiency and high surface area to interact with the organic molecules. Although graphene based optical detection is not new for organic pollutants, most of them are ‘turn-off’ detection approach with poor sensitivity and reproducibility.31, 52-53 (SI, Table S1-S3) Moreover ‘on-field’ detection which is very useful for monitoring health and environmental issues, is lacking. Here, we have developed a ‘turn-on’ detection approach which has been found highly sensitive and reproducible for different organic pollutants. It is superior to ‘turn-off’ approach since it strongly depends on the analyte concentration and can minimize false positive responses.83 (i) Mechanism of 'turn-on' detection approach: Our system is based on reduced graphene oxide-polystyrene sulphonate composite which is loaded with dextranfluorescein (dex-fl). Polystyrene sulfonate helps in solubilizing hydrophobic RGO whereas dex-fl serves as the fluorescent probe for ‘turn-on’ sensing. Due to efficient energy transfer (ET), fluorescence of dex-fl gets quenched as it comes in close proximity with the RGO.84 Here, dex-fl attached with the graphene surface via weak non-covalent interactions.85 On addition of organic pollutants, strong π-π interactions


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overcome the weak non-covalent interactions. This results in detachment of dex-fl from graphene surface. This minimizes the ET efficiency and the fluorescence from dex-fl reverts back depending on analyte concentrations. We have measured fluorescence lifetime of the composite before and after addition of the analyte, which in turn directly supports our claim. We have found in the absence of analyte (BPA) average fluorescence lifetime is ~1.1 ns, which is increased to ~3.5 ns in presence of analyte. This is due to enhanced separation between the fluorophore and quencher in presence of analyte, results in reduced non-radiative energy transfer and enhanced fluorescence lifetime. (SI, Figure S2) Recovered fluorescence is different for various analytes tested, since the interactions between the analytes and graphene surface are different. It has been observed that PA responses most strongly in this approach suggesting most strong interaction between graphene and PA while 1-napthal show weakest response. The mechanism of this detection can be explained by donor-acceptor interaction model. Being electron-rich, RGO is considered as electron donor and the organic molecules act as electron acceptor here. Depending on the functional groups present on organic molecules, they can be either electron-rich or electron-deficient. Electron deficient molecules can act as good acceptor and interact strongly with the RGO composite.74 In our study we have found the interaction with picric acid is strong. This is due to the presence of three electron withdrawing nitro groups which turn this molecule to behave as electron deficient. In our study we found 1-napthol responses weakly. This is due to the presence of electron donating groups which make this molecule electronrich, and thus exhibits least interaction with the modified RGO surface. This reflects in the recovered fluorescence and limit of detections (LODs) of the organic pollutants. To better understand the mechanism we have calculated the binding constants of these interactions from the recovered fluorescence using a one site binding model (SI, Figure S3).86 We have found picric acid has the highest binding constant, 9.1 X 103 M-1 whereas 1-napthal has the lowest binding constant 3.1 X 102 M-1 (Table 2). This data directly confirms our proposed mechanism of interaction based on donoracceptor model. The high reproducibility of this approach is due to modular interaction between donor-acceptor. Here the weakly bound fluorophores are easily replaced by the organic pollutants due to the strong donor-acceptor π-π interaction. (ii) Optimization of the detection approach: Detection sensitivity in ‘turn-on’ approach depends on the extent of loading of the fluorescent probe. In our optimized


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condition loading has been calculated and found around ~4 µM / mg composite. To optimize the detection, varying loading amount has been tested. Higher loading (~12 µM / mg composite) of the dye results in background fluorescence which diminishes its sensitivity. On the other hand lower loading (~1 µM / mg composite) also affects the sensitivity of detection (SI, Figure S6). Moreover, lower loading also impede the reproducibility of detection. In our optimized condition, upper limit of detection is ~2 µM. As a control, we have tested GO-dex-fl (CMP1) and RGO-PSS-fl (CMP2) for PH and BPA detection (SI, Figure S7). We have found very low sensitivity and poor reproducibility in both the cases. In CMP1 poor sensitivity and reproducibility results from covalent attachment between hydroxyl (-OH) groups of dex-fl and epoxide groups (-O-) of GO. This causes incompetent release of dex-fl from GO surface upon addition of organic pollutants. Moreover, inefficient fluorescence quenching results in high background signal, and thus hindering the detection sensitivity. It shows LOD of ~5 µM. On the other hand in CMP2, fluorescein molecules strongly absorb on RGO surface via π-π interaction which is inadequately replaced by analyte molecules, results in poor LOD of ~0.7 µM. We have also checked the response of dex-fl in the absence of GO, keeping all other conditions same. We did not notice any appreciable change in the fluorescence intensity of dex-fl in presence of organic pollutants. The reason is that GO provides the platform of π-network for interaction with organic pollutants and thus allows the pollutants and dex-fl to approach in close proximity. This data also confirms that PSS do not have any interference in the fluorescence of the fluorophore.(SI, Fig S8) We have tested different other molecules as control to study the interference in detection. We have chosen molecules as hydrophobic but devoid of π ring (cyclohexane and octadecene) and polar π ring containing molecules (tryptophan, tyrosine, phenyl alanine and histidine). We have tested 10-8 M concentration of all the control molecules with no appreciable fluorescence enhancement (SI, Figure S9). We have tested 1, 2, 4-trichlorobenzene, 1, 2, 4, 5-tetrachlorobenzene (polychlorinated benzene or PCB) and 4-nitrophenol as competing organic water pollutants. Surprisingly we have found very low response of these molecules in our approach. This is because PCBs contain multiple electron donating chlorine groups, which make these molecules electron rich, thus less interaction with modified RGO as expected (SI, Figure S9). Moreover, its strong hydrophobic nature also hinders its interaction


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with

hydrophilic

RGPD-fl composite.

In

4-nitrophenol,

although

electron

withdrawing nitro group is present, electron donating hydroxyl group in para position nullifies its effects and thus it did not show any appreciable interaction with RGPD-fl. We have also tested common inorganic ions (Na+1, K+1, Ca+2, Mg+2, Cl-1, SO4-2, NO3-1 and PO4-3) normally present is water systems. We have found no interference in detection from these ions. (SI, Figure S10) (iii) Practical applications: For rapid and easy on-field detection of organic pollutants we have developed a simple silica plate based device with RGPD-fl. For this purpose, we have chosen silica gel based TLC plate with alumina support. This is cheap, small, easy to carry, fabricate and requires low sample volume. TLC plate is first cut into pieces and then immersed in the solution of RGPD-fl overnight followed by washing with distilled water and air drying. RGPD-fl deposited on the surface of TLC plate is then employed for qualitative detection of all the organic pollutants by observing the recovered fluorescence using a hand-held UV lamp (365 nm excitation). The repeatability of the device can be tested by washing the device with ethanol after each detection step. Since the solubility of the analytes is very high in ethanol, it can be easily removed from the graphene surface by washing with ethanol. We have tested the reusability of the device with BPA and found that a single device can be reused for ~5-6 times, although their sensitivity is somewhat compromised. This is because with increasing the number of cycle removal of BPA becomes more difficult from graphene surface due to their increasing attachment tendency with graphene. Since the method is not selective for a particular pollutant, quantitative assessment can be done by standard addition method. For quantitative measurement of BPA, five different known concentrations of BPA were added to each of the test sample separately (purified drinking water, industrial waste water and surface water). Recovered fluorescence was then recorded and plotted against added BPA concentration. From the interception on y axis we can determine the concentration of BPA in test samples (SI, Figure S3). In all the cases recovery has been found between 97%-105% which shows our method can be applied for on field accurate detection and quantification of organic pollutants. We have also detected the presence of other pollutants in different water samples following similar method. (Table 1) The results obtained are comparable to the conventional techniques available. (SI, Table S4) All the conventional techniques use chromatographic procedure. Although these methods have low detection limits, they possess cumbersome technique, long time and costly


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equipments. On the other hand, our method is simple, easy, cost effective and require very small sample volume. Another advantage of our method is the long range of linearity, which is useful to detect organic pollutants in different water samples. Normally range of these pollutants in practical water samples are in the order of micromolar to nanomolar, depending on source of water. Thus for the real sample detection, the detection limit and range of linearity of our sample meet the exact requirements.

Conclusion: In conclusion we report a simple fluorescent ‘turn-on’ approach for the detection of organic pollutants in waste water. For this purpose we have developed a graphene based composite with dextran-fluorescein as fluorescent probe. Our approach for detection of bisphenol-A, 1-napthol, phenol and picric acid in solution is highly sensitive (nanomolar to picomolar concentration) and reproducible. Moreover we have developed a simple TLC plate based device which can be used for on-field detection of these organic pollutants. We believe that this approach is reliable, sensitive, reproducible and has the potential to solve the problem of detection of these minute amounts of organic pollutants in various test samples.

Acknowledgement: Authors would like to thank CSIR, government of India for financial assistance. AS acknowledges CSIR, India for providing Nehru Science PostDoctoral Fellowship. AS and RM would like to thank Dr. N. R. Jana, Indian Association for the Cultivation of Science, for his kind assistance in experimental measurements. AS would like to thank Dr. P Sujatha Devi, Central Glass and Ceramic Research Institute, for her kind assistance in scientific discussions. RM would like to thank Dr. Rina Ghosh, Jadavpur University for providing scientific assistance.

Supporting Information Available: Table summarizing the organic pollutants detection approaches, loading calculation, control experiments and description of standard addition method are available in supporting information. This material is available free of charge via Internet at http://pubs.acs.org


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Scheme 1: Preparation of RGPD-fl composite probe.


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Scheme 2: Organic pollutants detection approach (a). Digital images of the RGPD-fl composite under normal light and 365 nm UV light is also shown. Digital images of recovered fluorescence under 365 nm UV light by treating the composite with picric acid (PA), phenol (PH), bisphenol A (BPA) and 1-napthol (NP) is also shown here (b).

(a)

(b)

RGPD-fl

Under normal light

PA

PH

BPA

NP

Under 365 nm UV light


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Table 1: Quantitative estimation of phenol, bisphenol-A, 1-napthal and picric acid in industrial waste water, surface water and bottled drinking water using our method following a standard addition method. Bottled drinking water do not contain any of the pollutants tested. This means, if any of the pollutants present in the drinking water at all, that is beyond our limit of detection. We have not obtained any appreciable amount of the pollutants in it in the standard addition method.

Pollutant

Phenol (pM)

BPA (pM)

1-napthal (pM)

Picric acid (pM)

Industrial waste water

22000

10750

100

120

Surface water

95

23

16

NA

Bottled drinking water

NA

NA

NA

NA

Sample

Table 2: Binding constant values of the organic pollutants under study with RGPD-fl composite

Organic Molecules Binding Constant Values (M-1) Picric acid 9.1 X 103 Phenol 4.6 X 103 BPA 7.1 X 102 1-Napthal 3.1 X 102


25


5,000

(a)

GO RGO-PSS RGPD-fl purified

2.5 2.0 1.5 1.0 0.5

PL intensity (a.u.)

3.0

Absorbance (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

0.0 300

500

700

Wavelength (nm)

900

(b)

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GO RGO-PSS RGPD-fl RGPD-fl purified

3,000

1,000

450

550

650

750

Wavelength (nm)

Figure 1: UV-Visible absorbance spectra of graphene oxide (GO), RGO-PSS composite and purified RGPD-fl probe is shown (a). GO shows typical absorbance at ~230 nm and ~300 nm, whereas RGO-PSS composite shows absorbance at ~260 nm. In RGPD-fl probe absorbance at ~490 nm arises due to dextran-fluorescein attachment. Fluorescence spectra (b) show no emission from GO, RGO-PSS composite and purified RGPD-fl probe, although as synthesized RGPD-fl shows strong fluorescence at ~520 nm due to unbound dextran-fluorescein molecules.


26

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(a)

Line 1

(b) Line 1 Avg. Height ~0.7 nm

Line 2

(c)

(d)

(e)

Line 2 Avg. Height ~0.8 nm

Line 1 Avg. Height ~18 nm

Line 1

(f)

Line 2 Avg. Height ~20 nm

Line 2

Figure 2: Atomic force microscopy (AFM) images and line profiles of as synthesized graphene oxide (GO) (a, b, c) and RGPD-fl probe (d, e, f) show considerable differences in average height profile. Average height of GO (a) has been found ~0.70.8 nm (b, c) whereas the RGPD-fl composite probe (d) show average height of ~1820 nm (e, f) due to dextran-fluorescence attachment and layer stacking.


27


RGPD-fl

1044 cm-1 1134 cm-1

Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 33

1742 cm-1

RGO-PSS

GO

1089 cm-1

500

1500

2500

3500 -1

Wavenumber (cm ) Figure 3: FTIR spectra of graphene oxide (GO), RGO-PSS composite and RGPD-fl composite probe has shown here. –C=O and C-O stretching frequency (from –COOH of graphene oxide) at ~1742 cm-1 and 1089 cm-1 has been found prominent in GO but absent in the composites. SO2 (from poly styrene sulfonate) stretching frequency has been observed at ~1134 cm-1 in both the composites but missing in GO. C-N stretching frequency from dextran-fluorescein has been found at ~ 1044 cm-1 in RGPD-fl probe confirms dextran-fluorescein attachment.


28

35,000 28,000 21,000 14,000

Picric acid

7,000 0 475 15000

575 Wavelength (nm)

10000

6000 3000

Bisphenol A

9000

575 Wavelength (nm)

5000 Phenol 0 475

575 Wavelength (nm)

8000

-6

12000

0 475

15000

675 10 M -7 10 M -8 10 M -9 10 M -10 10 M -11 10 M Control

(c)

-6

10 M -7 10 M -8 10 M -9 10 M -10 10 M -11 10 M Control

(b) PL intensity (a.u.)

PL intensity (a.u.)

20000

-6

10 M -7 10 M -8 10 M -9 10 M -10 10 M -11 10 M Control

(a)

PL intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


675

675 -6

10 M -7 10 M -8 10 M -9 10 M -10 10 M -11 10 M Control

(d) PL intensity (a.u.)

Page 29 of 33

6000 4000 2000

1-naphthol 0 475

575 Wavelength (nm)

675

Figure 4: ‘Turn-On’ fluorescence response of four different organic pollutants, picric acid (a), phenol (b), bisphenol A (c) and 1-napthol (d) towards RGPD-fl composite probe. With increasing concentration of organic pollutants, recovered fluorescence intensity increases as evidenced from the figure. Fluorescence response differs for pollutants studied. Picric acid (a) show strongest response while 1-napthol (d) show weakest response.


29


20000

35000

(b) PL intensity (a.u.)

PL intensity (a.u.)

(a) 25000

15000 Picric acid 2 R = 0.97

5000

15000 10000 5000 Phenol 2 R = 0.99

0 -11

10

-10

10

-9

10

-8

10

10

-7

-6

-11

10

10

Log concentration (molar)

(c)

10000

6000

2000

Bisphenol A 2 R = 0.97 -11

10

-10

10

-9

10

-8

10

10

-7


-10

10

-9

10

-8

10

-7

10

-6

10


7000

PL intensity (a.u.)

14000

PL intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 33

(d)

6000

5000 1-Naphthol 2 R = 0.99

4000 -6

10

-11

10

-10

10

-9

10

-8

10

10

-7

-6

10


Figure 5: Detection sensitivity of picric acid (a), phenol (b), bisphenol A (c) and 1napthol (d) has been recorded. Recovered fluorescence intensity has been plotted with logarithm of molar concentration of organic pollutants. Good linearity has been observed in the above cases as evidenced from the R2 values within the range of detection (picomolar to micromolar). Error bars are indicated in each data point.


30

Page 31 of 33

11000

22000 PL intensity (a.u.)

19000 16000 13000 10000

(c) 4500

9000 8000 7000

3500 3000 1 2 3 4 5 Number of batches

1 2 3 4 5 Number of batches

6000

Bisphenol A RSD = ~0.2 %

4000

2500

10000

6000


5000

Phenol RSD = ~0.3 %

(b)

Picric acid RSD= ~0.4 %

1-naphthol RSD = ~0.8 %

(d) PL intensity (a.u.)

PL intensity (a.u.)

(a)

PL intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


5000 4000 3000 2000


Figure 6: Reproducibility check has been performed for all the four organic pollutants. Five different batches of organic pollutants (10-9 M) have been checked under identical conditions. Relative standard deviation has been calculated for the samples and have found within 1% for all cases. This result shows high reproducibility of the detection approach.


31


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(a)

(b)

(c)

Page 32 of 33

(d)

Detection approach (e)

(f)

(g)

Reusability

(h)

Stability

Figure 7: Digital images of TLC (thin layer chromatography) plate based detection approach. (a) Digital image of RGPD-fl composite loaded TLC plate. (b) Digital image of the composite loaded plate under 365 nm UV light without any organic pollutant. Digital image of the composite loaded plate under 365 nm UV light with bisphenol A (c) and with industrial waste water (d). Recovered fluorescence has been observed under UV light confirms the presence of organic pollutants. Reusability of this RGPD-fl loaded plate has been shown (e, f, g). It has been observed that it can be reusable upto 5 times (f), although, recovered fluorescence intensity diminished with the number of cycle compared to the initial (e). After that the recovered fluorescence is not detectable (g). Stability of the RGPD-fl loaded plate has been shown to detect BPA after six months. (h)


32

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For Table of Contents Use Only

Reduced Graphene Oxide based ‘Turn-On’ Fluorescence Sensor for Highly Reproducible and Sensitive Detection of Small Organic Pollutants

Reetam Mitra† and Arindam Saha* ‡

Synopsis: RGO based 'Turn-On' flouorescent sensor for ultrasensitive detection of small organic pollutants in solution and on paper based device platform.

Solution

Solution

Organic pollutants

Plate

Fluorescence off

Plate

Fluorescence on


33

Reduced Graphene Oxide Based âTurn-Onâ Fluorescence Sensor for

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