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Acetylcholinesterase inhibition-based ultrasensitive fluorometric detection of malathion using unmodified silver nanoparticles
Colloids and Surfaces A: Physicochem. Eng. Aspects 485 (2015) 111–117
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Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Acetylcholinesterase inhibition-based ultrasensitive fluorometric detection of malathion using unmodified silver nanoparticles D. Nanda Kumar, S.A. Alex, R.S. Suresh Kumar, N. Chandrasekaran, A. Mukherjee ∗ Centre for Nanobiotechnology, VIT University, Vellore, India
h i g h l i g h t s
g r a p h i c a l
• Ultrasensitive fluorometric detection
Ultrasensitive readout probe for the detection of malathion was developed using unmodified silver nanoparticles in the presence of AChE and ATCh.
method for malathion developed. • AChE inhibition-based detection strategy using AgNPs. • No external fluorophore or additional surface functionalization of AgNPs required. • Very low LOD of 0.556 × 10−15 M compared to existing methods.
a r t i c l e
i n f o
Article history: Received 25 July 2015 Received in revised form 2 September 2015 Accepted 4 September 2015 Available online 8 September 2015 Keywords: Silver nanoparticles Acetylcholinesterase Acetylthiocholine Malathion Fluorescence spectrophotometry
a b s t r a c t
a b s t r a c t An ultrasensitive sensor was developed for the detection of an organophosphorus compound using unmodified silver nanoparticles (AgNPs). The hydrolysis of acetylthiocholine (ATCh) by acetylcholinesterase (AChE) generates a thiol-containing compound, thiocholine, which influences the AgNP aggregation. Herein, we describe an indirect enzyme-based detection of malathion with AChE and ATCh in the presence of AgNPs using the fluorometric method. The addition of malathion to the system decreased the enzyme hydrolysis and thiocholine generation, and thus was able to quench the AgNPs, with a corresponding decrease in the emission intensity at 423 nm (ex = 340 nm). The proposed system showed linearity in the range, 2–100 fM, with an excellent limit of detection of 0.556 fM by fluorescence spectroscopy. The aggregation and disaggregation of AgNPs were also supported by transmission electron microscopy and dynamic light scattering analyses. The potential application of the system was demonstrated by determination of malathion in real samples like agricultural runoff water, lake water, cabbage, and apple samples using fluorometric method, and the obtained results were further validated with HPLC. © 2015 Elsevier B.V. All rights reserved.
1. Introduction ∗ Corresponding author. Fax: +91 416 2243092. E-mail addresses: [email protected], [email protected] (A. Mukherjee). http://dx.doi.org/10.1016/j.colsurfa.2015.09.013 0927-7757/© 2015 Elsevier B.V. All rights reserved.
The use of organophosphorus compounds (OPs) in pesticides for agricultural activity is widely increasing throughout the world [1].
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Their extensive usage can pose as a potential toxicant for human beings through the ingestion of treated food sources like vegetables, fruits, and through water bodies [2]. Organophosphates (OP) are commonly found in herbicides, insecticides, and warfare agents [3]. Malathion belongs to the phosphorodithioate group and has sulfur atoms bound to the phosphorus atoms by single and double bonds [4]. In the central nervous system of the human body, acetylcholinesterase (AChE) can enzymatically hydrolyze acetylcholine (ACh) into choline (Ch) and acetic group. OP compounds are attributed to cause the irreversible inhibition of AChE and decrease the production of Ch. The cholinergic manifestations can finally cause respiratory paralysis and death [5]. For this reason, there are increasing demands to develop a rapid, highly sensitive and effective strategy for the detection of malathion in environmental samples. The conventionally used methods for OP analysis include, high-performance liquid chromatography (HPLC) [6], gas chromatography (GC), mass spectrometry (MS), cyclic voltammetry (CV), and Raman spectrometry [7–9]. Although these methods exhibit high sensitivity, they are very expensive, involve timeconsuming pretreatments, and require highly skilled personnel. On the other hand, the application of colorimetric and fluorometric principles for the sensing of OP compounds using nanoparticles (NPs) has drawn more attraction due to their optical, electrical, and sensing properties [10]. Nanomaterials, including ZrO2 NPs [11], AuNPs [12], multiwalled carbon nanotubes coated with AuNPs (MWCNTs-AuNPs) [13], and photoluminescent CdTe quantum dots (QDs) [14], have been developed with high sensitivity towards OP pesticides and nerve agents. Unlike other NPs, gold and silver NPs (AuNPs and AgNPs) have excellent electrical and optical properties [15]. Recently, Liu et al. (2012) employed Rhodamine B-capped gold NPs (RB-AuNPs) to obtain dual readouts (colorimetric and fluorometric) for detecting OPs, by preventing the thiocholine generation, which in turn unquenches the fluorescence of RB simultaneously [16]; the minimum detectable concentration of malathion for this method is 0.908 nM. The described work requires complex functionalization and time involved for functionalization and preparation of NPs (2 h). Vasimalai and John used chitosan-capped AgNPs as fluorophore probe to detect malathion in the range of 1 nM to 10 pM with an LOD of 94 fM based on aggregation of AgNPs, and they tested the assay in polluted lake water, grape, and mango samples [17]. In another approach, Guo et al. utilized a highly conjugated phenanthroline derivative, which showed high fluorescence quantum yield for developing a dual mode (fluorescent and electrochemical) sensor for OP detection with an LOD of 1 nM [18]. However, most of these methods required additional surface functionalization with NPs. The significant advantage of the current work is the ability of the system to detect malathion in the presence of AChE and acetylthiocholine (ATCh) without using any functionalization over as-prepared citrate-stabilized particles (without the aid of any external linkers), unlike the other methods requiring definite functionalization agents (Table 1 benchmarks the current study with the already existing ones). There are only a limited number of studies elucidating the detection of OP compounds utilizing unmodified AgNPs and AuNPs. Recently, Li et al. (2013) developed a system, wherein the thiocholine-induced aggregation of AgNPs in the presence of AChE was found to be reversed by the addition of dipterex in the range 0.97–145 nM (0.25–37.5 ng mL−1 ), and the assay has an LOD of 0.69 nM using UV–vis spectroscopy [19]. Similarly, Li et al. demonstrated the visual detection of methamidophos by AChE inhibition method using AuNPs as a probe with an LOD of 9.92 nM [20]. In this work, we have proposed an indirect enzyme-based biosensor using AgNPs as a probe in the presence of AChE and ATCh for fluorometric detection of malathion in aqueous samples. The
Table 1 Detection of malathion using various nanoparticle-based methods. Reagent b
MWCNTs-AuNPs Chitosan capped AgNPsd Using phenanthroline derivatives CuO NWs-SWCNTs hybrid nanocompositec Nanosturctured Ag surfaces Nanostructured Ag surface by electrochemical method Unmodified AgNPs in presence of AChE and ATChd a b c d
LOD (fM)a
Ref.
1.81 × 106 94 1 × 106 3 × 105 10 × 103 100 × 109 0.556
[13] [17] [18] [25] [30] [31] This wok
Limit of detection. Multi-walled carbon nanotubes. Copper oxide nanowire—single-walled carbon nanotubes. By fluorescence spectroscopy.
proposed indirect biosensing method was found to achieve a very low LOD of 0.556 fM with fluorescence spectroscopy, which is very low when compared with the nanoparticles based existing methods either using UV–vis or fluorescence spectroscopic techniques. The major advantages of the current methods lie in ultra-low detection limit and rapid response time. The detection of malathion in agricultural runoff water, lake water, cabbage, and apple samples was successfully performed. 2. Materials and methods 2.1. Chemicals and materials Acetylcholinesterase (AChE, from Electrophorus electricus) was obtained from Sigma–Aldrich, India. Analytical grade pesticides, malathion (98.7%), trichlorfon (96.7%), paraoxon-methyl (95.3%), temephos (95.6%), and permethrin (98.3%) were purchased from Sigma–Aldrich, India. Tris (hydroxymethyl) aminomethane (tris buffer), acetylthiocholine iodide (ATChI) and other metal salts were obtained from Himedia Laboratories Pvt. Ltd (India). Ethanol (99.9%) was purchased from SD Fine Chemicals Ltd (India). Silver nitrate (AgNO3 ), trisodium citrate dihydrate (C6 H5 Na3 O7 ·2H2 O), and sodium borohydride (NaBH4 ) were procured from SRL Pvt. Ltd. (India). AChE (400 mU mL−1 ) and ATChI (10 mM) stock solution were prepared using tris buffer solution (10 mM) and stored in the refrigerator at 4–5 ◦ C when not in use. The presence of iodide ion in ATChI stock solution was removed by using AgNO3 and further treated with NaCl to remove excess of Ag+ ions from the solution. A stock solution of malathion (10−3 M) was prepared freshly in ethanol, and further, the solution was diluted with deionized water (Milli-Q) to the appropriate dilution for further experimental use. Ultrapure deionized water (Milli-Q) obtained from Cascada Bio water (Pall Corporation, USA) was used throughout the experiments, unless stated otherwise. Aquaregia solution was used for the cleaning of all glassware apparatus, which was finally rinsed with Milli-Q water at least two times and dried in a hot-air oven. These analytical grade chemical reagents were used in all the experiments without further purification. 2.2. Preparation of silver nanoparticles (AgNPs) An aqueous solution of citrated-capped AgNPs was prepared by the reduction of AgNO3 using NaBH4 (strong reducing agent). The colloidal AgNPs were prepared according to the literature [21] with slight modifications that were intended for enhancing their sensing applications. Briefly, 1.0 mL of sodium citrate (50 mM) solution was added to a flat-bottomed flask immersed in an ice-water bath (∼5 ◦ C) that contained 39 mL of AgNO3 (0.64 mM) solution under vigorous stirring. After 20 min, 10 mL of NaBH4 (25.11 mM) was added to the above solution, and the stirring was continued for another 30 min at room temperature (25 ± 1 ◦ C). The addition of
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NaBH4 yielded a bright yellow solution, which confirmed the formation of citrate-capped AgNPs. The colloidal solution was stored in dark condition for 24 h at room temperature until further use. Then, UV–vis absorption spectrum was recorded for freshly prepared AgNPs, and it shows a strong surface plasmon resonance (SPR) at 396 nm (for details see ESI in Fig. S1). The stability of AgNPs was confirmed by observing the nanoparticles for a period of 4 months for any variations in their characteristic absorption peak. No significant variation was observed in the absorption peak at 396 nm for up to 4 months (Fig. S1). 2.3. Detection of malathion with AgNPs using AChE and ATCh Different concentrations of malathion (2–100 fM) were prepared from the stock solution, and 50 L of each concentration of malathion was added to 10 L of AChE (400 mU mL−1 ) and 20 L ATCh (80 M) and incubated at room temperature (27 ± 1 ◦ C) for 5 min. Subsequently, 1420 L of Milli-Q water was added, and finally, 500 L of the prepared AgNPs was added to the reaction mixture. Then, the fluorescence emission intensity at 423 nm was reported for each sample concentration. The linear calibration curve was plotted between the fluorescence intensity (F423 ) of AgNPs and malathion concentration. 2.4. Real sample preparation For the analysis of malathion that is spiked in environmental matrices, real samples like agricultural runoff water, lake water, vegetable, and fruit samples were tested. The lake water used was collected form Vellore Institute of Technology (VIT) University Lake (Vellore, India) during the spring season (November). The agriculture runoff water was collected during the same season (November) from the paddy field (Vellore, India). The collection was done during the vegetative growth phase of the paddy when the application of pesticides is maximum. The agricultural runoff water and lake water were filtered by simple filtration method to remove any foreign materials [17] and then spiked with different concentrations of malathion. The cabbages and apples were purchased from a local market (Vellore, India). For the preparation of fruit/vegetable samples, different concentrations of malathion was sprayed onto the apples and cabbage by using a pesticide sprayer on the same day of purchase and were allowed to incubate for 1 h at room temperature. Then, the edible parts of the samples were taken and crushed well. 20 g of both crushed apple and cabbage were taken separately and mixed vigorously with 50 mL of Milli-Q water in a beaker for 30 min. Finally, the sample mixture was filtered to remove any residues. For all samples, a control sample (without spiking malathion) was prepared by using the same procedure. All the spiked samples were analyzed by fluorescence spectroscopic method, and the obtained results were validated with HPLC method. The prepared stock solution of samples was stored in the refrigerator at 4-5 ◦ C when not in use. 2.5. Instrumentation 2.5.1. UV–visible spectrophotometry The preliminary characterization of as-synthesized AgNPs was performed using a UV–vis spectrophotometer (UV-2600, Shimadzu, Tokyo, Japan). All the measurements were made in the spectral range from 200 to 800 nm. 2.5.2. Fluorescence spectrophotometry The fluorescence spectra were obtained by employing Cary eclipse fluorescence spectrophotometer (Agilent Technologies,
Scheme 1. Ultrasensitive detection of malathion using unmodified AgNPs in the presence of AChE and ATCh.
Model-G9800A) at a slit width of 10 nm. The excitation and emission wavelengths used were 340 and 423 nm, respectively, and the spectral range being studied was from 400 to 450 nm. The additional tests were carried out to confirm that the emission peak of AgNPs at 423 nm was not due to Rayleigh scattering phenomenon by varying the excitation peaks from 300 to 380 nm and the excitation peak of AgNPs was obtained at 340 nm (for details see ESI in Fig. S2). 2.5.3. Transmission electron microscope The morphological characteristics of AgNPs and the particle size of the dispersed and aggregated AgNPs were obtained using Transmission electron microscopy (TEM, FEI Company TecnaiTM , G2 Spirit, BioTWIN) at an accelerating voltage of around 120 kV. 2.5.4. Particle size and surface charge analysis Dynamic light scattering (DLS) and zeta potential were performed for as-synthesized AgNPs, with and without addition of malathion to AChE, ATCh, and AgNPs using a particle size analyzer (90 Plus Particle Analyzer, Brookhaven instruments Corporation, USA). 2.5.5. Malathion estimation by HPLC The detection of malathion in spiked real samples like fruits and vegetables were further validated by using High-performance liquid chromatography (HPLC) (Hitachi LaChrom Elite) having a UV detector with 220 nm wavelength. The column used was Reliant C18 5-m column (250 × 4.6 mm), which was maintained at a constant column temperature of 40 ◦ C.A solution of acetonitrile and water (60:40) was used the eluent with a flow rate of 1 mL min−1 at retention time 10.36 min. 10 L of the real samples were injected in to the HPLC at a periodic interval of 15 min. 3. Results and discussion 3.1. Reaction scheme The proposed mechanism for the detection of OP assay is described in the reaction Scheme 1. In the control experiment, AChE causes the enzymatic hydrolysis of ATCh into thiocholine (TCh) and acetic acid. The produced TCh molecule is positively charged at neutral pH, which can electrostatically bind with the negatively charged citrate-capped AgNPs [19]. The excess generation of thiol group replaces the citrate layer and results in the decrease in repulsive force between the NPs, which thereby results
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Fig. 1. TEM micrographs of (A) well-dispersed AgNPs in colloidal solution, (B) Citrate-capped AgNPs after addition of AChE (400 mU mL−1 ), ATCh (80 M) and (C) AgNPs in presence of malathion (1000 fM), AChE (400 mU mL−1 ), and ATCh (80 M).
in the AgNP aggregation. OP compounds can cause the nucleophilic attack through the phosphorylation of the serine group in AChE and forms a covalent bond with its active site [22]. So, the phosphorylation of AChE causes the inhibition of the enzyme activity, which in turn results in the decreased production of TCh. Thus, the aggregation of AgNPs would be proportional to the amount of thiocholine produced. Since malathion is an OP compound, the detection of malathion using this scheme has been investigated fluorescence spectroscopy. 3.2. Preliminary characterization of AgNPs The mean hydrodynamic size distribution of AgNPs and their stability were studied by performing dynamic light scattering (DLS) and zeta potential analyses. The mean hydrodynamic particle size was 23 ± 1 nm and their zeta potential value was −38.42 mV. Further, the size and shape of as-synthesized AgNPs was confirmed by Transmission electron microscopy (TEM) (Fig. 1A), and the average particle diameter of AgNPs was found to be 22 ± 1 nm. The synthesized AgNPs were used for the detection of malathion using AChE and ATCh with the aid of fluorimetry 3.3. Development of assay for malathion detection using fluorescence spectroscopy 3.3.1. Fluorescence spectral changes in AgNPs with malathion For fluorometric method, the emission intensity at 423 nm (excitation wavelength at 340 nm) had significantly increased when AChE and ATCh (without malathion; control experiment) were added to the reaction mixture (for details see ESI in Fig. S3). There is no substantial increase in the fluorescence intensity of AgNPs when AChE, ATCh, or malathion alone were added. The same was observed when malathion was added in the presence of either AChE or ATCh alone. The increase in the emission intensity of AgNPs upon aggregation of AgNPs in the presence of AChE and ATCh is due to the energy transfer between the NPs and TCh. As the size of the NPs increases, the fluorescence intensity increases due to the enhancement in its scattering. When malathion (100 fM) was added to this, there was a decrease in the fluorescence intensity due to the reduction in the size of AgNPs (for details see ESI in Fig. S3.A). After a reaction time of 5 min, the color of the colloidal solution changed from yellow to grey (for details see ESI in Fig. S4). Further, the control experiment was performed to study if malathion alone can precipitate or adsorb onto the AgNP surfaces upon interaction for a long time. The fluorescence spectra (for details see ESI in Fig. S3.B) reveals that the direct addition of malathion alone to the synthesized AgNPs could not cause aggregation or precipitation upon interaction for a long time (after 4 h). The TEM micrograph (Fig. 1A) indicated that the NPs were well dispersed in colloidal solution,
with diameters ranging from 10 to 50 nm and average particle diameter of 22 ± 1 nm as statistically determined by using “ImageJ” software (Image Processing and Analysis in Java). The TEM image (Fig. 1B) confirmed the aggregation of AgNPs after the addition of AChE and ATCh to the system. Further, the addition of malathion (1000 fM) to AgNPs in presence of AChE and ATCh prevents the aggregation of AgNPs, and the average particle diameter was found by TEM to be 35 ± 1 nm (Fig. 1C). The aggregation and disaggregation of AgNPs were further confirmed by DLS and zeta potential measurements. The size distribution of as-synthesized AgNPs and in the absence and presence of malathion (100 and 1000 fM) with AChE and ATCh confirms the aggregation and disaggregation of AgNPs, and the mean hydrodynamic sizes were found to be 23 ± 1, 251 ± 1, 179 ± 1, and 115 ± 1 nm, respectively (for details see ESI in Fig. S5). Correspondingly, the zeta potential for the as-synthesized AgNPs was analyzed to be −38.42 mV. For the control experiment, the zeta potential had increased to 67.59 mV, which confirmed the aggregation of AgNPs after the addition of AChE and ATCh. The addition of malathion (100 and 1000 fM) again increased the stability of AgNPs, which was indicated by the decrease in zeta potential to 13.71 and 5.45 mV respectively (for details see ESI in Table S1). 3.3.2. Process optimization for fluorometric detection The effect of changing the concentration of AChE and ATCh was studied for fluorescence optimization. To determine the concentration of ATCh, AChE was kept at a fixed concentration of 400 mU mL−1 . As the concentration of ATCh was increased from 0 to 100 M, the amount of TCh produced increases; however, 80 M of ATCh was chosen as the optimum concentration as maximum emission intensity was obtained with this concentration. Further increase in ATCh concentration did not vary the response significantly (for details see ESI in Fig. S6). Similarly, with fixed ATCh (80 M), the concentration of AChE was increased from 0 to 500 mU mL−1 . A linear increase in the fluorescence intensity (for details see ESI in Fig. S7) was noted and 400 mU mL−1 of AChE was taken as the optimized concentration as maximum activity was observed for the chosen concentration. The pH of the system, 6.5 to 7.5, was maintained throughout the experiment, and no significant differences were observed before and after interaction with malathion in the presence of AChE, ATCh, and AgNPs. 3.3.3. Sensitivity of the fluorometric detection The sensitivity of the system was assessed for the quantitative detection of malathion in the range, 2–100 fM, with the optimized conditions. With increasing malathion concentration, there was a proportional increase in the hindrance of the AChE activity, thus reducing the AgNP aggregation. Since the fluorescence intensity is proportional to the AgNP size, the fluorescence
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Fig. 4. Stern–Volmer plot showing the AgNP quenching with different concentrations of malathion (2–100 fM). Fig. 2. Emission spectra of AgNPs in a system containing AChE (400 mU mL−1 ) and ATCh (80 M) with different concentrations of malathion (2–100 fM) at em = 423 nm.
quenching observed (i.e., decrease in emission intensity at 423 nm) was found to be proportional to the malathion concentration (Fig. 2). The obtained emission peak intensities for the different concentrations were utilized to plot a linear calibration curve (regression coefficient, R2 = 0.9784) with malathion concentration versus fluorescence emission intensity (F423 ) of AgNPs (Fig. 3). The limit of detection obtained was 0.556 fM, which was determined by using the formula 3/s, where is the standard deviation of ten blank responses and s is the slope of the calibration curve. The sensitivity of the developed method was found to be higher when compared with earlier detection methods, which involve AChE inhibition using a pH-sensitive fluorescence probe [2], AChEbased dipstick assay [23], emissive core-shell silica particles with tetraphenylethylene [24], and CuO nanowires—single-walled carbon nanotubes (CuO-SWCNTs) (Table 1) [25]. For statistical analysis of the obtained data, the Student’s t-test was performed, and a value 2.364 at a confidence level of 95% was calculated. This shows that there was no significant variation when the experiment was repeated. All the experiments were carried out in triplicates, and a p value < 0.0001 obtained using one-way ANOVA ensures the sensitivity of the assay.
3.3.4. Fluorescence quenching mechanism The fluorescence quenching of AgNPs can be explained based on the mechanism below. As the concentration of malathion increased in the presence of the AChE and ATCh, the AgNP aggregation decreased. In the control experiment in presence of AChE, the energy transfer (Förster resonance energy transfer) occurs between TCh and AgNPs, which causes the increase in the fluorescence intensity of AgNPs [26]. The addition of malathion to the system would bind to the AChE and decrease the energy transfer in the process. This would result in fluorescence quenching of AgNPs. Thus, the decrease in the fluorescence intensity of AgNPs would be proportional to the malathion concentration. The quenching of fluorescent intensity of AgNPs with increase in the concentration of malathion (2–100 fM) can be estimated from the Stern-Volmer relation (Eq. (1)) [27]: Fo = 1 + Ksv [Q ] F
where F0 is the fluorescence intensity of citrate-capped AgNPs with AChE and ATCh, and F is the fluorescence intensity of AgNPs in the presence of malathion with AChE and ATCh, KSV is the Stern–Volmer quenching constant, and [Q] is the concentration of malathion. KSV was calculated to be 3.22 × 1013 L moL−1 . The Stern–Volmer plot obtained was found to be linear (Fig. 4), and the positive deviation observed for the higher concentration suggests the presence of both dynamic and static quenching [28]. The obtained Stern-Volmer constant suggested the strong fluorescence quenching effect of malathion on the emission intensity of AgNPs. The number of binding sites (n) and binding constant (K) on AChE for interaction with malathion were calculated using the following Eq. (2) [27].
log Fo −
Fig. 3. Linear calibration curve plotted with the emission intensities at 423 nm against the different concentrations of malathion (2–100 fM).
(1)
F Fo
= logK + nlog [Q ]
(2)
where F0 and F are the fluorescence intensities of citrate-capped AgNPs in absence and presence of malathion with AChE and ATCh, respectively. K and n represent the binding constant and number of binding sites, and Q is the concentration of malathion. By plotting the linear calibration curve between log [F0 − F/F0 ] and log [Q], the number of binding sites (n) and binding constant (K) were found to be 0.6732 and 8.7223 from the slope and y-intercept, respectively (for details see ESI in Fig. S8). The obtained binding constant value reveals that there is a strong binding force between AChE and malathion, which thereby affects the fluorescence intensity of AgNPs.
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D.N. Kumar et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 485 (2015) 111–117 Table 2 Detection of malathion in spiked real samples by fluorescence spectroscopy. Sample
Added (fM)
Found (fM)
Recovery (%)
RSD (%)
Agricultural runoff water Lake water
5.5 7.5 11.5 5.5 7.5 11.5 5.5 7.5 11.5 5.5 7.5 11.5
5.14 7.76 11.53 5.46 7.49 11.48 5.66 7.41 11.59 5.31 7.89 11.47
107 96.65 99.74 100.73 100.13 100.17 97.17 101.21 99.22 103.58 95.06 100.26
6.54 1.95 1.97 1.48 0.84 0.3 4.77 5.41 2.26 4.23 2.88 1.55
Cabbage
Apple
Fig. 5. Comparison of fluorescence intensity ratios of AgNPs obtained with malathion (1fM) and other common interferences of pesticides (100 fM).
3.3.5. Effect of interfering substances The selectivity of the present method was further examined with other common interfering OP and pyrethroid compounds. The response of the assay to malathion at a concentration (1 fM) 100 times lesser than other common interferences such as trichlorfon, paraoxon-methyl, temephos, and permethrin (100 fM). No significant variation in the AgNP emission intensity was observed with the other interferents tested. The quenching of the fluorescence intensity at 423 nm was observed only with malathion, indicating its selective action in inhibiting AChE. Thus, the obtained results support that the assay exhibits high selectivity towards malathion as compared to other interferences (Fig. 5). The effect of commonly interfering cations and anions was also investigated as water samples normally contain many organic and inorganic substances, which can hinder the analysis of OPs. Therefore, the effect of common interfering inorganic ions like Cu2+ , Hg2+ , Fe3+ , Cd2+ , Pb2+ , Mg2+ , Na+ , K+ and Cl– that may exist together with the OPs in water samples were tested. The fluorescence spectra of AgNPs (with AChE and ATCh) were recorded in the presence of malathion alone (control) and malathion in the presence of other interfering metal ions (binary mixture). The resulting spectra (for details see ESI in Fig. S9) indicates that almost no change occurred in the presence of most of the selected inorganic ions, except Hg2+ which showed slight interference in presence of malathion, as compared with the other binary mixtures of metal ions used. This is because Hg2+ possess high affinity for -SH group compounds [29] (i.e., thiocholine), and it decreases the fluorescence signal even at a lower concentration. 3.3.6. Real sample analysis using fluorometric detection The validation of the fluorometric assay was done by studying the response of the system for detecting malathion in real samples like agricultural runoff water, lake water, apple, and cabbage samples. Higher concentrations of stock solution were prepared for each sample, and the required concentrations of solution were prepared by serial dilution with the respective real samples. Three different concentrations of malathion, 5.5, 7.5, and 11.5 fM, were spiked in the real samples. The spiked samples were analyzed with the proposed assay, and the fluorescent intensities of AgNPs
were measured at 423 nm in the presence of real samples. Table 2 indicates recovery percentages of 96.65–107%, 100.13–100.73%, 97.17–101.21%, and 95.06–103.58% for agricultural runoff water, lake water, cabbage, and apple samples, respectively. The spiked and measured malathion concentrations were noticed to be in good agreement. RSD% values were found to be in the range of 1.95–6.54%, 0.3–1.48%, 2.26–5.41%, and 1.55–4.23%, respectively, indicating the repeatability of the measurements. The validation in real samples was also performed with HPLC method. Two different samples (cabbage and lake water) were spiked with a concentration of malathion 3 M (Table 3) and the recoveries was noted to be above 90.0%.
4. Conclusion In this work, we proposed a fluorescence based biosensor for the indirect detection of malathion with AChE and ATCh using as-synthesized and unmodified AgNP-based probe. The detection limits accomplished by the proposed probe was 0.556 fM. As compared with other conventional methods, the system was found to achieve good reproducibility, high sensitivity, and selectivity against other common pesticides. The designed system was successfully demonstrated for the detection of malathion in spiked real samples with good recovery percentage.
Acknowledgement The authors would like to acknowledge the funding from the Defence Research & Development Organization (ERIP/ER/1103964/M/01/1485), Ministry of Defence, Government of India. We are also acknowledge the MCB department, IISC Bangalore for providing TEM facility.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfa.2015.09. 013.
Table 3 Validation of the present method for detection of malathion with HPLC method. Sample
Cabbage Lake water
Malathion spiked (M)
3 3
Malathion measured (M) Fluorescence method
HPLC method
Recovery (%)
RSD (%)
2.9 2.7
2.8 2.9
95 93
2.4 5.0
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Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Acetylcholinesterase inhibition-based ultrasensitive fluorometric detection of malathion using unmodified silver nanoparticles D. Nanda Kumar, S.A. Alex, R.S. Suresh Kumar, N. Chandrasekaran, A. Mukherjee ∗ Centre for Nanobiotechnology, VIT University, Vellore, India
h i g h l i g h t s
g r a p h i c a l
• Ultrasensitive fluorometric detection
Ultrasensitive readout probe for the detection of malathion was developed using unmodified silver nanoparticles in the presence of AChE and ATCh.
method for malathion developed. • AChE inhibition-based detection strategy using AgNPs. • No external fluorophore or additional surface functionalization of AgNPs required. • Very low LOD of 0.556 × 10−15 M compared to existing methods.
a r t i c l e
i n f o
Article history: Received 25 July 2015 Received in revised form 2 September 2015 Accepted 4 September 2015 Available online 8 September 2015 Keywords: Silver nanoparticles Acetylcholinesterase Acetylthiocholine Malathion Fluorescence spectrophotometry
a b s t r a c t
a b s t r a c t An ultrasensitive sensor was developed for the detection of an organophosphorus compound using unmodified silver nanoparticles (AgNPs). The hydrolysis of acetylthiocholine (ATCh) by acetylcholinesterase (AChE) generates a thiol-containing compound, thiocholine, which influences the AgNP aggregation. Herein, we describe an indirect enzyme-based detection of malathion with AChE and ATCh in the presence of AgNPs using the fluorometric method. The addition of malathion to the system decreased the enzyme hydrolysis and thiocholine generation, and thus was able to quench the AgNPs, with a corresponding decrease in the emission intensity at 423 nm (ex = 340 nm). The proposed system showed linearity in the range, 2–100 fM, with an excellent limit of detection of 0.556 fM by fluorescence spectroscopy. The aggregation and disaggregation of AgNPs were also supported by transmission electron microscopy and dynamic light scattering analyses. The potential application of the system was demonstrated by determination of malathion in real samples like agricultural runoff water, lake water, cabbage, and apple samples using fluorometric method, and the obtained results were further validated with HPLC. © 2015 Elsevier B.V. All rights reserved.
1. Introduction ∗ Corresponding author. Fax: +91 416 2243092. E-mail addresses: [email protected], [email protected] (A. Mukherjee). http://dx.doi.org/10.1016/j.colsurfa.2015.09.013 0927-7757/© 2015 Elsevier B.V. All rights reserved.
The use of organophosphorus compounds (OPs) in pesticides for agricultural activity is widely increasing throughout the world [1].
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Their extensive usage can pose as a potential toxicant for human beings through the ingestion of treated food sources like vegetables, fruits, and through water bodies [2]. Organophosphates (OP) are commonly found in herbicides, insecticides, and warfare agents [3]. Malathion belongs to the phosphorodithioate group and has sulfur atoms bound to the phosphorus atoms by single and double bonds [4]. In the central nervous system of the human body, acetylcholinesterase (AChE) can enzymatically hydrolyze acetylcholine (ACh) into choline (Ch) and acetic group. OP compounds are attributed to cause the irreversible inhibition of AChE and decrease the production of Ch. The cholinergic manifestations can finally cause respiratory paralysis and death [5]. For this reason, there are increasing demands to develop a rapid, highly sensitive and effective strategy for the detection of malathion in environmental samples. The conventionally used methods for OP analysis include, high-performance liquid chromatography (HPLC) [6], gas chromatography (GC), mass spectrometry (MS), cyclic voltammetry (CV), and Raman spectrometry [7–9]. Although these methods exhibit high sensitivity, they are very expensive, involve timeconsuming pretreatments, and require highly skilled personnel. On the other hand, the application of colorimetric and fluorometric principles for the sensing of OP compounds using nanoparticles (NPs) has drawn more attraction due to their optical, electrical, and sensing properties [10]. Nanomaterials, including ZrO2 NPs [11], AuNPs [12], multiwalled carbon nanotubes coated with AuNPs (MWCNTs-AuNPs) [13], and photoluminescent CdTe quantum dots (QDs) [14], have been developed with high sensitivity towards OP pesticides and nerve agents. Unlike other NPs, gold and silver NPs (AuNPs and AgNPs) have excellent electrical and optical properties [15]. Recently, Liu et al. (2012) employed Rhodamine B-capped gold NPs (RB-AuNPs) to obtain dual readouts (colorimetric and fluorometric) for detecting OPs, by preventing the thiocholine generation, which in turn unquenches the fluorescence of RB simultaneously [16]; the minimum detectable concentration of malathion for this method is 0.908 nM. The described work requires complex functionalization and time involved for functionalization and preparation of NPs (2 h). Vasimalai and John used chitosan-capped AgNPs as fluorophore probe to detect malathion in the range of 1 nM to 10 pM with an LOD of 94 fM based on aggregation of AgNPs, and they tested the assay in polluted lake water, grape, and mango samples [17]. In another approach, Guo et al. utilized a highly conjugated phenanthroline derivative, which showed high fluorescence quantum yield for developing a dual mode (fluorescent and electrochemical) sensor for OP detection with an LOD of 1 nM [18]. However, most of these methods required additional surface functionalization with NPs. The significant advantage of the current work is the ability of the system to detect malathion in the presence of AChE and acetylthiocholine (ATCh) without using any functionalization over as-prepared citrate-stabilized particles (without the aid of any external linkers), unlike the other methods requiring definite functionalization agents (Table 1 benchmarks the current study with the already existing ones). There are only a limited number of studies elucidating the detection of OP compounds utilizing unmodified AgNPs and AuNPs. Recently, Li et al. (2013) developed a system, wherein the thiocholine-induced aggregation of AgNPs in the presence of AChE was found to be reversed by the addition of dipterex in the range 0.97–145 nM (0.25–37.5 ng mL−1 ), and the assay has an LOD of 0.69 nM using UV–vis spectroscopy [19]. Similarly, Li et al. demonstrated the visual detection of methamidophos by AChE inhibition method using AuNPs as a probe with an LOD of 9.92 nM [20]. In this work, we have proposed an indirect enzyme-based biosensor using AgNPs as a probe in the presence of AChE and ATCh for fluorometric detection of malathion in aqueous samples. The
Table 1 Detection of malathion using various nanoparticle-based methods. Reagent b
MWCNTs-AuNPs Chitosan capped AgNPsd Using phenanthroline derivatives CuO NWs-SWCNTs hybrid nanocompositec Nanosturctured Ag surfaces Nanostructured Ag surface by electrochemical method Unmodified AgNPs in presence of AChE and ATChd a b c d
LOD (fM)a
Ref.
1.81 × 106 94 1 × 106 3 × 105 10 × 103 100 × 109 0.556
[13] [17] [18] [25] [30] [31] This wok
Limit of detection. Multi-walled carbon nanotubes. Copper oxide nanowire—single-walled carbon nanotubes. By fluorescence spectroscopy.
proposed indirect biosensing method was found to achieve a very low LOD of 0.556 fM with fluorescence spectroscopy, which is very low when compared with the nanoparticles based existing methods either using UV–vis or fluorescence spectroscopic techniques. The major advantages of the current methods lie in ultra-low detection limit and rapid response time. The detection of malathion in agricultural runoff water, lake water, cabbage, and apple samples was successfully performed. 2. Materials and methods 2.1. Chemicals and materials Acetylcholinesterase (AChE, from Electrophorus electricus) was obtained from Sigma–Aldrich, India. Analytical grade pesticides, malathion (98.7%), trichlorfon (96.7%), paraoxon-methyl (95.3%), temephos (95.6%), and permethrin (98.3%) were purchased from Sigma–Aldrich, India. Tris (hydroxymethyl) aminomethane (tris buffer), acetylthiocholine iodide (ATChI) and other metal salts were obtained from Himedia Laboratories Pvt. Ltd (India). Ethanol (99.9%) was purchased from SD Fine Chemicals Ltd (India). Silver nitrate (AgNO3 ), trisodium citrate dihydrate (C6 H5 Na3 O7 ·2H2 O), and sodium borohydride (NaBH4 ) were procured from SRL Pvt. Ltd. (India). AChE (400 mU mL−1 ) and ATChI (10 mM) stock solution were prepared using tris buffer solution (10 mM) and stored in the refrigerator at 4–5 ◦ C when not in use. The presence of iodide ion in ATChI stock solution was removed by using AgNO3 and further treated with NaCl to remove excess of Ag+ ions from the solution. A stock solution of malathion (10−3 M) was prepared freshly in ethanol, and further, the solution was diluted with deionized water (Milli-Q) to the appropriate dilution for further experimental use. Ultrapure deionized water (Milli-Q) obtained from Cascada Bio water (Pall Corporation, USA) was used throughout the experiments, unless stated otherwise. Aquaregia solution was used for the cleaning of all glassware apparatus, which was finally rinsed with Milli-Q water at least two times and dried in a hot-air oven. These analytical grade chemical reagents were used in all the experiments without further purification. 2.2. Preparation of silver nanoparticles (AgNPs) An aqueous solution of citrated-capped AgNPs was prepared by the reduction of AgNO3 using NaBH4 (strong reducing agent). The colloidal AgNPs were prepared according to the literature [21] with slight modifications that were intended for enhancing their sensing applications. Briefly, 1.0 mL of sodium citrate (50 mM) solution was added to a flat-bottomed flask immersed in an ice-water bath (∼5 ◦ C) that contained 39 mL of AgNO3 (0.64 mM) solution under vigorous stirring. After 20 min, 10 mL of NaBH4 (25.11 mM) was added to the above solution, and the stirring was continued for another 30 min at room temperature (25 ± 1 ◦ C). The addition of
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NaBH4 yielded a bright yellow solution, which confirmed the formation of citrate-capped AgNPs. The colloidal solution was stored in dark condition for 24 h at room temperature until further use. Then, UV–vis absorption spectrum was recorded for freshly prepared AgNPs, and it shows a strong surface plasmon resonance (SPR) at 396 nm (for details see ESI in Fig. S1). The stability of AgNPs was confirmed by observing the nanoparticles for a period of 4 months for any variations in their characteristic absorption peak. No significant variation was observed in the absorption peak at 396 nm for up to 4 months (Fig. S1). 2.3. Detection of malathion with AgNPs using AChE and ATCh Different concentrations of malathion (2–100 fM) were prepared from the stock solution, and 50 L of each concentration of malathion was added to 10 L of AChE (400 mU mL−1 ) and 20 L ATCh (80 M) and incubated at room temperature (27 ± 1 ◦ C) for 5 min. Subsequently, 1420 L of Milli-Q water was added, and finally, 500 L of the prepared AgNPs was added to the reaction mixture. Then, the fluorescence emission intensity at 423 nm was reported for each sample concentration. The linear calibration curve was plotted between the fluorescence intensity (F423 ) of AgNPs and malathion concentration. 2.4. Real sample preparation For the analysis of malathion that is spiked in environmental matrices, real samples like agricultural runoff water, lake water, vegetable, and fruit samples were tested. The lake water used was collected form Vellore Institute of Technology (VIT) University Lake (Vellore, India) during the spring season (November). The agriculture runoff water was collected during the same season (November) from the paddy field (Vellore, India). The collection was done during the vegetative growth phase of the paddy when the application of pesticides is maximum. The agricultural runoff water and lake water were filtered by simple filtration method to remove any foreign materials [17] and then spiked with different concentrations of malathion. The cabbages and apples were purchased from a local market (Vellore, India). For the preparation of fruit/vegetable samples, different concentrations of malathion was sprayed onto the apples and cabbage by using a pesticide sprayer on the same day of purchase and were allowed to incubate for 1 h at room temperature. Then, the edible parts of the samples were taken and crushed well. 20 g of both crushed apple and cabbage were taken separately and mixed vigorously with 50 mL of Milli-Q water in a beaker for 30 min. Finally, the sample mixture was filtered to remove any residues. For all samples, a control sample (without spiking malathion) was prepared by using the same procedure. All the spiked samples were analyzed by fluorescence spectroscopic method, and the obtained results were validated with HPLC method. The prepared stock solution of samples was stored in the refrigerator at 4-5 ◦ C when not in use. 2.5. Instrumentation 2.5.1. UV–visible spectrophotometry The preliminary characterization of as-synthesized AgNPs was performed using a UV–vis spectrophotometer (UV-2600, Shimadzu, Tokyo, Japan). All the measurements were made in the spectral range from 200 to 800 nm. 2.5.2. Fluorescence spectrophotometry The fluorescence spectra were obtained by employing Cary eclipse fluorescence spectrophotometer (Agilent Technologies,
Scheme 1. Ultrasensitive detection of malathion using unmodified AgNPs in the presence of AChE and ATCh.
Model-G9800A) at a slit width of 10 nm. The excitation and emission wavelengths used were 340 and 423 nm, respectively, and the spectral range being studied was from 400 to 450 nm. The additional tests were carried out to confirm that the emission peak of AgNPs at 423 nm was not due to Rayleigh scattering phenomenon by varying the excitation peaks from 300 to 380 nm and the excitation peak of AgNPs was obtained at 340 nm (for details see ESI in Fig. S2). 2.5.3. Transmission electron microscope The morphological characteristics of AgNPs and the particle size of the dispersed and aggregated AgNPs were obtained using Transmission electron microscopy (TEM, FEI Company TecnaiTM , G2 Spirit, BioTWIN) at an accelerating voltage of around 120 kV. 2.5.4. Particle size and surface charge analysis Dynamic light scattering (DLS) and zeta potential were performed for as-synthesized AgNPs, with and without addition of malathion to AChE, ATCh, and AgNPs using a particle size analyzer (90 Plus Particle Analyzer, Brookhaven instruments Corporation, USA). 2.5.5. Malathion estimation by HPLC The detection of malathion in spiked real samples like fruits and vegetables were further validated by using High-performance liquid chromatography (HPLC) (Hitachi LaChrom Elite) having a UV detector with 220 nm wavelength. The column used was Reliant C18 5-m column (250 × 4.6 mm), which was maintained at a constant column temperature of 40 ◦ C.A solution of acetonitrile and water (60:40) was used the eluent with a flow rate of 1 mL min−1 at retention time 10.36 min. 10 L of the real samples were injected in to the HPLC at a periodic interval of 15 min. 3. Results and discussion 3.1. Reaction scheme The proposed mechanism for the detection of OP assay is described in the reaction Scheme 1. In the control experiment, AChE causes the enzymatic hydrolysis of ATCh into thiocholine (TCh) and acetic acid. The produced TCh molecule is positively charged at neutral pH, which can electrostatically bind with the negatively charged citrate-capped AgNPs [19]. The excess generation of thiol group replaces the citrate layer and results in the decrease in repulsive force between the NPs, which thereby results
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Fig. 1. TEM micrographs of (A) well-dispersed AgNPs in colloidal solution, (B) Citrate-capped AgNPs after addition of AChE (400 mU mL−1 ), ATCh (80 M) and (C) AgNPs in presence of malathion (1000 fM), AChE (400 mU mL−1 ), and ATCh (80 M).
in the AgNP aggregation. OP compounds can cause the nucleophilic attack through the phosphorylation of the serine group in AChE and forms a covalent bond with its active site [22]. So, the phosphorylation of AChE causes the inhibition of the enzyme activity, which in turn results in the decreased production of TCh. Thus, the aggregation of AgNPs would be proportional to the amount of thiocholine produced. Since malathion is an OP compound, the detection of malathion using this scheme has been investigated fluorescence spectroscopy. 3.2. Preliminary characterization of AgNPs The mean hydrodynamic size distribution of AgNPs and their stability were studied by performing dynamic light scattering (DLS) and zeta potential analyses. The mean hydrodynamic particle size was 23 ± 1 nm and their zeta potential value was −38.42 mV. Further, the size and shape of as-synthesized AgNPs was confirmed by Transmission electron microscopy (TEM) (Fig. 1A), and the average particle diameter of AgNPs was found to be 22 ± 1 nm. The synthesized AgNPs were used for the detection of malathion using AChE and ATCh with the aid of fluorimetry 3.3. Development of assay for malathion detection using fluorescence spectroscopy 3.3.1. Fluorescence spectral changes in AgNPs with malathion For fluorometric method, the emission intensity at 423 nm (excitation wavelength at 340 nm) had significantly increased when AChE and ATCh (without malathion; control experiment) were added to the reaction mixture (for details see ESI in Fig. S3). There is no substantial increase in the fluorescence intensity of AgNPs when AChE, ATCh, or malathion alone were added. The same was observed when malathion was added in the presence of either AChE or ATCh alone. The increase in the emission intensity of AgNPs upon aggregation of AgNPs in the presence of AChE and ATCh is due to the energy transfer between the NPs and TCh. As the size of the NPs increases, the fluorescence intensity increases due to the enhancement in its scattering. When malathion (100 fM) was added to this, there was a decrease in the fluorescence intensity due to the reduction in the size of AgNPs (for details see ESI in Fig. S3.A). After a reaction time of 5 min, the color of the colloidal solution changed from yellow to grey (for details see ESI in Fig. S4). Further, the control experiment was performed to study if malathion alone can precipitate or adsorb onto the AgNP surfaces upon interaction for a long time. The fluorescence spectra (for details see ESI in Fig. S3.B) reveals that the direct addition of malathion alone to the synthesized AgNPs could not cause aggregation or precipitation upon interaction for a long time (after 4 h). The TEM micrograph (Fig. 1A) indicated that the NPs were well dispersed in colloidal solution,
with diameters ranging from 10 to 50 nm and average particle diameter of 22 ± 1 nm as statistically determined by using “ImageJ” software (Image Processing and Analysis in Java). The TEM image (Fig. 1B) confirmed the aggregation of AgNPs after the addition of AChE and ATCh to the system. Further, the addition of malathion (1000 fM) to AgNPs in presence of AChE and ATCh prevents the aggregation of AgNPs, and the average particle diameter was found by TEM to be 35 ± 1 nm (Fig. 1C). The aggregation and disaggregation of AgNPs were further confirmed by DLS and zeta potential measurements. The size distribution of as-synthesized AgNPs and in the absence and presence of malathion (100 and 1000 fM) with AChE and ATCh confirms the aggregation and disaggregation of AgNPs, and the mean hydrodynamic sizes were found to be 23 ± 1, 251 ± 1, 179 ± 1, and 115 ± 1 nm, respectively (for details see ESI in Fig. S5). Correspondingly, the zeta potential for the as-synthesized AgNPs was analyzed to be −38.42 mV. For the control experiment, the zeta potential had increased to 67.59 mV, which confirmed the aggregation of AgNPs after the addition of AChE and ATCh. The addition of malathion (100 and 1000 fM) again increased the stability of AgNPs, which was indicated by the decrease in zeta potential to 13.71 and 5.45 mV respectively (for details see ESI in Table S1). 3.3.2. Process optimization for fluorometric detection The effect of changing the concentration of AChE and ATCh was studied for fluorescence optimization. To determine the concentration of ATCh, AChE was kept at a fixed concentration of 400 mU mL−1 . As the concentration of ATCh was increased from 0 to 100 M, the amount of TCh produced increases; however, 80 M of ATCh was chosen as the optimum concentration as maximum emission intensity was obtained with this concentration. Further increase in ATCh concentration did not vary the response significantly (for details see ESI in Fig. S6). Similarly, with fixed ATCh (80 M), the concentration of AChE was increased from 0 to 500 mU mL−1 . A linear increase in the fluorescence intensity (for details see ESI in Fig. S7) was noted and 400 mU mL−1 of AChE was taken as the optimized concentration as maximum activity was observed for the chosen concentration. The pH of the system, 6.5 to 7.5, was maintained throughout the experiment, and no significant differences were observed before and after interaction with malathion in the presence of AChE, ATCh, and AgNPs. 3.3.3. Sensitivity of the fluorometric detection The sensitivity of the system was assessed for the quantitative detection of malathion in the range, 2–100 fM, with the optimized conditions. With increasing malathion concentration, there was a proportional increase in the hindrance of the AChE activity, thus reducing the AgNP aggregation. Since the fluorescence intensity is proportional to the AgNP size, the fluorescence
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Fig. 4. Stern–Volmer plot showing the AgNP quenching with different concentrations of malathion (2–100 fM). Fig. 2. Emission spectra of AgNPs in a system containing AChE (400 mU mL−1 ) and ATCh (80 M) with different concentrations of malathion (2–100 fM) at em = 423 nm.
quenching observed (i.e., decrease in emission intensity at 423 nm) was found to be proportional to the malathion concentration (Fig. 2). The obtained emission peak intensities for the different concentrations were utilized to plot a linear calibration curve (regression coefficient, R2 = 0.9784) with malathion concentration versus fluorescence emission intensity (F423 ) of AgNPs (Fig. 3). The limit of detection obtained was 0.556 fM, which was determined by using the formula 3/s, where is the standard deviation of ten blank responses and s is the slope of the calibration curve. The sensitivity of the developed method was found to be higher when compared with earlier detection methods, which involve AChE inhibition using a pH-sensitive fluorescence probe [2], AChEbased dipstick assay [23], emissive core-shell silica particles with tetraphenylethylene [24], and CuO nanowires—single-walled carbon nanotubes (CuO-SWCNTs) (Table 1) [25]. For statistical analysis of the obtained data, the Student’s t-test was performed, and a value 2.364 at a confidence level of 95% was calculated. This shows that there was no significant variation when the experiment was repeated. All the experiments were carried out in triplicates, and a p value < 0.0001 obtained using one-way ANOVA ensures the sensitivity of the assay.
3.3.4. Fluorescence quenching mechanism The fluorescence quenching of AgNPs can be explained based on the mechanism below. As the concentration of malathion increased in the presence of the AChE and ATCh, the AgNP aggregation decreased. In the control experiment in presence of AChE, the energy transfer (Förster resonance energy transfer) occurs between TCh and AgNPs, which causes the increase in the fluorescence intensity of AgNPs [26]. The addition of malathion to the system would bind to the AChE and decrease the energy transfer in the process. This would result in fluorescence quenching of AgNPs. Thus, the decrease in the fluorescence intensity of AgNPs would be proportional to the malathion concentration. The quenching of fluorescent intensity of AgNPs with increase in the concentration of malathion (2–100 fM) can be estimated from the Stern-Volmer relation (Eq. (1)) [27]: Fo = 1 + Ksv [Q ] F
where F0 is the fluorescence intensity of citrate-capped AgNPs with AChE and ATCh, and F is the fluorescence intensity of AgNPs in the presence of malathion with AChE and ATCh, KSV is the Stern–Volmer quenching constant, and [Q] is the concentration of malathion. KSV was calculated to be 3.22 × 1013 L moL−1 . The Stern–Volmer plot obtained was found to be linear (Fig. 4), and the positive deviation observed for the higher concentration suggests the presence of both dynamic and static quenching [28]. The obtained Stern-Volmer constant suggested the strong fluorescence quenching effect of malathion on the emission intensity of AgNPs. The number of binding sites (n) and binding constant (K) on AChE for interaction with malathion were calculated using the following Eq. (2) [27].
log Fo −
Fig. 3. Linear calibration curve plotted with the emission intensities at 423 nm against the different concentrations of malathion (2–100 fM).
(1)
F Fo
= logK + nlog [Q ]
(2)
where F0 and F are the fluorescence intensities of citrate-capped AgNPs in absence and presence of malathion with AChE and ATCh, respectively. K and n represent the binding constant and number of binding sites, and Q is the concentration of malathion. By plotting the linear calibration curve between log [F0 − F/F0 ] and log [Q], the number of binding sites (n) and binding constant (K) were found to be 0.6732 and 8.7223 from the slope and y-intercept, respectively (for details see ESI in Fig. S8). The obtained binding constant value reveals that there is a strong binding force between AChE and malathion, which thereby affects the fluorescence intensity of AgNPs.
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D.N. Kumar et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 485 (2015) 111–117 Table 2 Detection of malathion in spiked real samples by fluorescence spectroscopy. Sample
Added (fM)
Found (fM)
Recovery (%)
RSD (%)
Agricultural runoff water Lake water
5.5 7.5 11.5 5.5 7.5 11.5 5.5 7.5 11.5 5.5 7.5 11.5
5.14 7.76 11.53 5.46 7.49 11.48 5.66 7.41 11.59 5.31 7.89 11.47
107 96.65 99.74 100.73 100.13 100.17 97.17 101.21 99.22 103.58 95.06 100.26
6.54 1.95 1.97 1.48 0.84 0.3 4.77 5.41 2.26 4.23 2.88 1.55
Cabbage
Apple
Fig. 5. Comparison of fluorescence intensity ratios of AgNPs obtained with malathion (1fM) and other common interferences of pesticides (100 fM).
3.3.5. Effect of interfering substances The selectivity of the present method was further examined with other common interfering OP and pyrethroid compounds. The response of the assay to malathion at a concentration (1 fM) 100 times lesser than other common interferences such as trichlorfon, paraoxon-methyl, temephos, and permethrin (100 fM). No significant variation in the AgNP emission intensity was observed with the other interferents tested. The quenching of the fluorescence intensity at 423 nm was observed only with malathion, indicating its selective action in inhibiting AChE. Thus, the obtained results support that the assay exhibits high selectivity towards malathion as compared to other interferences (Fig. 5). The effect of commonly interfering cations and anions was also investigated as water samples normally contain many organic and inorganic substances, which can hinder the analysis of OPs. Therefore, the effect of common interfering inorganic ions like Cu2+ , Hg2+ , Fe3+ , Cd2+ , Pb2+ , Mg2+ , Na+ , K+ and Cl– that may exist together with the OPs in water samples were tested. The fluorescence spectra of AgNPs (with AChE and ATCh) were recorded in the presence of malathion alone (control) and malathion in the presence of other interfering metal ions (binary mixture). The resulting spectra (for details see ESI in Fig. S9) indicates that almost no change occurred in the presence of most of the selected inorganic ions, except Hg2+ which showed slight interference in presence of malathion, as compared with the other binary mixtures of metal ions used. This is because Hg2+ possess high affinity for -SH group compounds [29] (i.e., thiocholine), and it decreases the fluorescence signal even at a lower concentration. 3.3.6. Real sample analysis using fluorometric detection The validation of the fluorometric assay was done by studying the response of the system for detecting malathion in real samples like agricultural runoff water, lake water, apple, and cabbage samples. Higher concentrations of stock solution were prepared for each sample, and the required concentrations of solution were prepared by serial dilution with the respective real samples. Three different concentrations of malathion, 5.5, 7.5, and 11.5 fM, were spiked in the real samples. The spiked samples were analyzed with the proposed assay, and the fluorescent intensities of AgNPs
were measured at 423 nm in the presence of real samples. Table 2 indicates recovery percentages of 96.65–107%, 100.13–100.73%, 97.17–101.21%, and 95.06–103.58% for agricultural runoff water, lake water, cabbage, and apple samples, respectively. The spiked and measured malathion concentrations were noticed to be in good agreement. RSD% values were found to be in the range of 1.95–6.54%, 0.3–1.48%, 2.26–5.41%, and 1.55–4.23%, respectively, indicating the repeatability of the measurements. The validation in real samples was also performed with HPLC method. Two different samples (cabbage and lake water) were spiked with a concentration of malathion 3 M (Table 3) and the recoveries was noted to be above 90.0%.
4. Conclusion In this work, we proposed a fluorescence based biosensor for the indirect detection of malathion with AChE and ATCh using as-synthesized and unmodified AgNP-based probe. The detection limits accomplished by the proposed probe was 0.556 fM. As compared with other conventional methods, the system was found to achieve good reproducibility, high sensitivity, and selectivity against other common pesticides. The designed system was successfully demonstrated for the detection of malathion in spiked real samples with good recovery percentage.
Acknowledgement The authors would like to acknowledge the funding from the Defence Research & Development Organization (ERIP/ER/1103964/M/01/1485), Ministry of Defence, Government of India. We are also acknowledge the MCB department, IISC Bangalore for providing TEM facility.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfa.2015.09. 013.
Table 3 Validation of the present method for detection of malathion with HPLC method. Sample
Cabbage Lake water
Malathion spiked (M)
3 3
Malathion measured (M) Fluorescence method
HPLC method
Recovery (%)
RSD (%)
2.9 2.7
2.8 2.9
95 93
2.4 5.0
D.N. Kumar et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 485 (2015) 111–117
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