A New Strategy for Direct Electrochemical Sensing of a

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A New Strategy for Direct Electrochemical Sensing of a Organophosphorus Pesticide-Triazophos Using Coomassie Brilliant Blue Dye Surface-Confined Carbon Nano Black Modified Electrode K.S Shalini Devi, Natarajan Anusha, Sudhakaran Raja, and Annamalai Senthil Kumar ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00861 • Publication Date (Web): 20 Jul 2018 Downloaded from http://pubs.acs.org on July 20, 2018

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ACS Applied Nano Materials

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A New Strategy for Direct Electrochemical Sensing of a Organophosphorus Pesticide-Triazophos Using Coomassie Brilliant Blue Dye Surface-Confined Carbon Nano Black Modified Electrode K.S.Shalini Devi,1 Natarajan Anusha,4 Sudhakaran Raja4 and Annamalai Senthil Kumar1,2,3* 1

Nano and Bioelectrochemistry Research Laboratory, Department of Chemistry, School of Advanced Sciences, Vellore Institute of Technology University, Vellore-632 014, India

2

Carbon dioxide Research and Green Technology Centre, Vellore Institute of Technology University, Vellore-632 014, India

4

Institue of Biochemical and Biomedical Engineering, National Taipei University of Technology, Taipei 10608, Taiwan (R.O.C.) 4

Aquaculture Biotechnology Laboratory, Department of Integrative Biology, School of

Biosciences and Technology, Vellore Institute of Technology University, Vellore-632014, Tamil Nadu, India

Keywords: Triazophos; Organophosphorus pesticide; Coomassie Brilliant Blue Dye; Carbon nanoblack material; Chemically modified electrode; Electrocatalysis; Flow injection analysis. 1

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ABSTRACT:Triazophos, O,O-diethyl O-(1-phenyl-1H-1,2,4-triazol-3-yl) phosphorothioate (TPZ), is one of organophosphorus pesticides, has been widely used for the agricultural forms to control the pest, insects and some nematodes (roundworms). Unfortunately, it has been found that a significant trace of the pesticide residue enters in to the agricultural products and creates major health threat to human. In order to selectively detect the TZP pesticide in real samples, several indirect and time consuming analytical assays based on acetylcholinesterase enzyme, affinity-based antibody systems and molecularly imprinted polymer, apart from the separation coupled mass spectrophotometer, have been demonstrated. For the first time in the literature, we wish to report a direct electrochemical method for the selective and quick detection of TZP based on electrocatalytic oxidation by Coomassie brilliant blue (CoomBB) dye surface-confined carbon nanoblack modified glassy carbon electrode (GCE/CBnano@CoomBB) in pH 7 phosphate buffer solution. The GCE/CBnano@CoomBB showed a stable and highly redox active peak at Eo’ = 0.15 V vs Ag/AgCl suitable to mediate the TZP oxidation reaction selectively. The quinonoid form of CoomBB and its strong π-π interaction with the CBnano are found to be key parameters for the electrochemical TZP oxidation. Initially, amperometric i-t sensing at an applied potential, 0.1 V vs Ag/AgCl was demonstrated for the highly selective detection of TZP without any interferences from the common chemicals like uric acid, ascorbic acid, cysteine, nitrite, hydrazine, sulfide, citric acid and H2O2 and other pesticides like cypermythrin, propenofos, deltramethrin, parathion and monocrotophous. In further, this new electrochemical system was exploited to flow injection analysis of TZP with a working volume of 20 µL. The calibration plot is linear in a window of TZP concentration, 16632 ng/20µL with a detection limit value of 119 pg/20 µL. Application of this technique is further demonstrated by detecting the TZP content in a couple of simulated vegetable samples with appreciable recovery values. 2


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1. INTRODUCTION Triazophos (TZP), O,O-diethyl-1-H-1,2,4-triazol-3-yl phosphorothioate, a contact and stomach poison for insects and mites, is an approved organophosphorus (OP) by United States National Organic Program.1 Owing to the effectiveness, easy availability, low cost, biogradability and low persistence, TZP has been referred as a pest-of-choice amongst the OPs.2,3. The main-field of applications are fruits, vegetables (potatoes), maize, vegetables, cotton, coffee and ornamentals etc.1-4 Roughly, a TZP dosage amount of 600 g per hector area has been used in the agro forms.5 Unfortunately, exposure of them into the environment systems like soil,4 water bodies,6 vegetables,7,8 fruits,9-11and milk (via food-chain process)12 etc., is a major threat for public health in terms of health care, general safety and morbidity. For instance, TZP content of 20-190 µg kg-1 in green-tea12 and 79-249 µg kg of apple, citrus and cabbage7 have been detected. The Toxicity of TZP on human is significant. In general, toxicity mechanism of TZP is through the inhibition of blood serum-acetylcholinesterase (AChE) that plays a significant role in neuronal growth13,14. In further, TZP induces the production of reactive oxygen species (ROS), that lead to a condition of oxidative-stress in humans and animals.15,16 Meanwhile, pesticide exposure during pregnancy time has increased risk such as spontaneous abortion, premature birth, fetal growth retardation, congenital malformations, early childhood cancer and prenatal death17. Thus simple, rapid, selective and field effective detection protocol for TZP in real samples is a demanding research topic in the analytical chemistry. In order to detect the TZP and its related OPs selectively, separation coupled spectroscopy techniques like High Performance Liquid Chromatography with Diode Array Detection (HPLC-DAD) in association with partial least squares and principal component regression software17,

liquid chromatography electrospray time-of-flight mass spectrometry 3



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(LC-TOFMS)18, Gas chromatography–Mass spectrophometer without12,19 and with solid-phase micro-extraction techniques20,21 have been widely used. In general, these methods require tedious and time consuming off-line sample preparations, sophisticated instruments and skilled technician. Alternately, biosensors utilizing the selective inhibition of AChE enzyme by TZP and other OPs, has been referred23-25. In a typical procedure, AChE is first interacted with the target OPs which is converted to phosphorylated or carbamylated derivative of the enzyme. Later on, these secondary products were detected using conductometric23,24 and amperometric25 techniques and in turn indirectly correlated with the quantity of the OPs. Apart from that, competitive inhibition of AChE enzymatic reaction of TZP and acetylthiocholine (ATC) to thiocholine that has been monitored with electrochemical26-28 and spectroscopic techniques has been referred for the quantification assays.10,30-33 Indeed, in consideration with the selectivity and accuracy these AChE based biosensors are found to be less attractive for the real sample analysis 34,35. In aim to improve the analytical performance, affinity based imnuno-assays including enzyme-linked immune-sorption assays (ELISA) has been introduced36-41. In general, ~6.5 h working time is required to complete a detection procedure. Meanwhile, electrochemical detection based on molecularly imprinted polymer (MIP) technique, wherein, electrochemical polymerization of the monomer in presence of TZP molecule that is extracted after wards, thus leaving the cavity, has been used for the sensing purpose42-44. Herein, we report a new, simple, enzyme- and antibodyfree and direct electrocatalytic oxidation strategy for selective electrochemical sensing of TZP using Coomassie brilliant blue G-250 (CoomBB) dye immobilized carbon black nanoparticle (CBnano) glassy carbon electrode (GCE/CBnano@CoomBB) in pH 7 phosphate buffer solution (PBS).

4


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CoomBB is a well-known textile dye and has been used for staining proteins in analytical biochemistry (Bradford reagent).45,46 This dye interacts with amino and carboxylic functional groups of proteins and forms a stable {protein-dye} complex (λmax = 590-615 nm). In a recent study, the binding property of the CoomBB has been exploited for colorimetric detection of certain small amino acids such as arginine, histidine, lysine, phenylalanine, tyrosine, and tryptophan present in forensic samples to identify the biologicalsex.47 Meanwhile, owing to the poor electron-transfer property, very few electrochemical studies relating to the CoomBB have been reported in the literature. In 2008, Zhong et al used CoomBB modified carbon nanotube as a matrix for the immobilization of HRP and studied the H2O2 sensing characteristics.48 In 2015, Ganesh and Swamy have reported electrochemical polymerization of the CoomBB as poly(coomBB) on a carbon paste electrode (0.1 M NaOH) and used it for the simultaneous electrochemical sensing of hydroquinone and catechol49. Note that no faradaic response was observed with the CoomBB modified electrodes. For the first time in this work, we report a highly redox active CoomBB functionalized (apparent standard electrode potential, Eo’= 0.15 V vs Ag/AgCl) carbon nanoparticle (CBnano and pristine-multiwalled carbon nanotube (MWCNT)) modified glassy carbon electrode prepared by continuous potential cycling method for TZP sensing application. CBnano is an ultra-low cost commercial carbon material that has been prepared as a bulk from hydrocarbons (oil and natural gas) and used as ingredient in the tyre making industry.50 The modified electrode showed selective mediated oxidation current signal to TZP without any AChE enzyme. This concept is further exploited for selective and sensitive flow injection analysis (FIA) of TZP in a 20 µL test system with a detection limit of 119 tg (20 µL). As a proof of concept, trace analysis of TZP in simulated green-vegetable samples have been successfully demonstrated. 5



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2. EXPERIMENTAL SECTION 2.1 Chemicals and reagents. Carbon nano black (N330 grade) was obtained as a gift compound from Phillips Carbon Black Ltd. Kochi, India. Graphene oxide-ethanol dispersed stock solution (GO, 5 mg mL-1, ~80% carbon basis flake size-0.5-2.0 µm, thickness-0.6-1.2 nm, purity-99%), activated charcoal, coomassie brilliant blue G-250 (CoomBB, AR grade 99%), pristine-multiwalled carbon nanotubes (MWCNT, ~95% purity), carbon nanofiber (0.1×2-200 µm; ~99.9 % purity) and carboxylic functionalized MWCNT (average diameter: 9.5 nm; 8 wt.% carboxyl group) was obtained from SRL, India. All other chemicals used were all of ACScertified reagent grade reagents. Aqueous solutions were prepared using deionized and alkaline permanganate distilled water. Combination of disodium hydrogen phosphate and sodium hydrogen phosphate buffer (pH 7 PBS of ionic strength, I = 0.1 M) without any deaeration was used as a supporting electrolyte throughout this work. Experiment with aeration of the pH 7 PBS didn’t show any alteration in the CoomBB redox feature. Caution! Triazophos is toxic and causes inhalation troubles, proper care must be taken during handling. 2.2.

Apparatus.

The

electrochemical

measurements

including

hydrodynamic

voltammetric studies were carried out using a CHI 440B instrument (USA). The three-electrode system consists of Au (0.0201 cm2) and GCE (0.0707 cm2) or its chemically modified electrodes as working electrode, Ag/AgCl as a reference electrode and a platinum wire as an auxiliary electrode. The FIA system consisted of a Hitachi L-2130 solvent delivery pump (Japan), a Rehodyne model 7125 sample injection valve (20 µL loop) and conventional electrochemical cell (BAS, USA).51 Raman spectroscopic analysis was carried out using AZILTRON, PRO 532 (USA) instrument with a λ=532 nm laser excitation source. For FT-IR analysis, a Perkin 6


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Elmer(Spectrum 100, Perkin. Elmer, USA) instrument using KBR pellets was used. Transmission electron-microscope (TEM) analysis was carried out using a FEI-TECNAI, G2 20 Twin instrument (Czech republic). TEM-Energy disperse X-Ray analysis (EDX) of CBnano showed signals corresponding to the metal impurities of Ni (0.36 wt%) and Fe (2.6%) and pristine MWCNT showed impurities of Fe (2.1 wt%), Ni (0.0014 wt%) and Co (1.6×10-4 wt.%) (data not shown). 2.3 Preparation of modified electrode. In first CBnano-ethanol stock suspension was prepared by dispersing 3 mg of CBnano in 500 µL of absolute ethanol followed by 15 min sonication. Then, 5 µL of the CBnano-ethanol suspension was drop-casted onto a cleaned GCE surface and the resulting GCE/CBnano was kept for drying in room temperature for 5±1 min. In next, CoomBB modified electrode, GCE/CBnano@CoomBB was prepared by twenty continuous potential cycling of GCE/CBnano with 1 mM of CoomBB solid powder mixed 10 mL PBS in a window, −0.5 and +0.5 V at scan rate (v) = 50 mV s-1 (Scheme 1). In a similar way other carbons such as GNP, AC, CNF, pristine-MWCNT and f-MWCNT modified CoomBB were prepared (GCE/Carbon@CoomBB). Purified-MWCNT sample was prepared as per our previously reported procedure,51 wherein, known quantity of pristine-MWCNT was refluxed with 6 M dilute HNO3 in a silicone oil bath at T=140oC for 12 h, thoroughly washed with double distilled water and dried. Phyllanthus niruri-a tropical vegetable plant is chosen for real sample study. TZP simulated real vegetable-leaf samples were prepared by spraying 250 µg TZP dissolved 20 mL water solution to ~50 g of test leaf, dried in room temperature for couple of days, washed with water, crushed and grinded in a mortar pestle with known volume of pH 7 PBS, filtered using filter paper and syringe filter followed by centrifugation for 10 min. A homogenous sample solution collected was diluted with a blank pH 7 PBS and was subjected to flow injection 7



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analysis by standard addition method.

3. RESULTS AND DISCUSSION 3.1 CV of GCE/CBnano@CoomB. In first, unmodified GCE is subjected to cyclic voltammetric studies with a dilute solution of CoomBB (1 mM) in 10 mL of pH 7 PBS (Figure 1A, curve a). A poor redox peak current signal with a wider peak-to-peak potential value (∆Ep=Epc-Epa, cathodic and anodic peak potentials, Epc and Epa), 200 mV and a unstable voltammetric response in a blank pH 7 PBS was noticed. Interestingly, when the same CV experiment was performed with GCE/CBnano modified electrode with CoomBB, as in Figure 1A, curve b, a well-defined redox peak at Eo’ = 0.15±0.01 V vs Ag/AgCl (A1/C1) with growing like peak current response was noticed. Upon 20th cycle, saturation in the redox peak current response was noticed. After the experiment, the working electrode was gently washed with distilled water, medium-transferred to a black pH 7 PBS and performed the ten continuous CV again. As can be seen in Figure 1B, a sharp CV response with ∆Ep = 20±1 mV was obtained. The value of ∆Ep near to “0’ indicates fast electron-transfer feature of the modified electrode. Calculated relative standard deviation for the redox peak between the peak current values of 1st and 10th cycles is 0.9% indicating high stability of the modified electrode. The amount of electro-active CoomBB immobilized on the electrode surface (ΓCoomBB) was calculated as 4.3 nmol cm-2 using the equation, ΓCoomBB = Qpa/nFA, where n = number of electrons involved (n = 2; Scheme 1) and Qpa = charge of the anodic peak. Effect of scan rate on the CV of the GCE/CBnano@CoomBB showed a systematic increase redox peak current response (Figure 1C). A plot of anodic and cathodic peak current, ipa and ipc vs scan rate yielded a straight line starting from the origin depicting the surface-confined electron-transfer behaviour of the redox system (inset Figure 1C). A plot of 8


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anodic and cathodic peak current, ipa and ipc vs scan rate yielded a straight line starting from the origin depicting the adsorption-controlled electron-transfer behaviour of the redox system (inset Figure 1C). The mechanism of charge transfer rate constant ks was obtained by plotting the graph logarithm of scan rate against the both anodic and cathodic potentials (Epa and Epc; data not enclosed) and the transfer coefficient, α of a surface confined redox couple can be evaluated from CV experiments. According to the Laviron equation, Epa and Epc values are proportional to the logarithm of scan rate values at v>0.2 V s-1.52 The slope of the linear segments (Sa and Sc) are associated with the equation, Sa(1-α) =Scα and the calculated value for α is 0.62. To calculate the electron transfer rate constant, the following equation can be used at condition, Epa − Epc= n∆Ep>200/mV (∆Ep ~ 200 mV at v= 500 mV s-1):52,53 logks= α log(1-α)+(1-α)logα – log (RT/nFv)– α(1-α)nF∆Ep/2.3RT

--(1)

The calculated ks values for GCE/CBnano@CoomBB is found to be 1.832 s-1. The redox peak response is found to be proton-coupled electron-transfer in characteristic and follows a Epa vs pH relationship value of -40±3 mV pH-1, which is corresponding to nonNenstian electrochemical behaviour with involvement of non-stoichiometric value of H+/e(Figure 1D & E). Note that relative lesser peak current signals at acid and alkaline conditions than that of the neutral pH solution was noticed. It is expected that under acidic and alkaline conditions, the sulphonic acid of the CoomBB involves in the proton attraction and repulsions respectively rather than the proton-coupled electron-transfer reaction with the CoomBB’s quinonid site. This observation led to variation in the peak current signals against the solution pH. In further, a small peak at -0.4 V vs Ag/AgCl was noticed irrespective of solution pH on the CV responses, which may be due to irreversible oxygen reduction reaction. Regarding to the mechanism for the redox peak is unclear, it is speculated that the quinonoid structure in the 9



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CoomBB involves in the proton-coupled electron-transfer reaction as displayed in the Scheme 1 for the electron-transfer activity. Providing strong π-π interaction between the π e-s of the benzene rings of CoomBB and sp2 carbon of the CBnano is the key factor for the stability and surface-confined redox behaviour of the CoomBB modified electrode (Scheme 1). In aim to understand the carbon functional group effect on the CoomBB modification, different form of carbon materials like graphene oxide (graphitic structure with rich oxygen functional group), carbon nanofiber (nil metal impurity with graphitic structure), activated charcoal (non-graphitic material), carboxylic acid functionalized-MWCNT (f-MWCNT), purified MWCNT (p-MWCNT) and pristine MWCNT (with trace metal impurity) were subjected to CoomBB electrochemical modification in pH 7 PBS similar to the case of GCE/CBnano-@CoomBB (Figure 1A, curve b). Typical CV results of the various carbon based CoomBB modified systems were displayed in Figure 2. For comparison, Raman spectroscopic responses (D-disordered graphitic and G-Graphitic bands) of the underlying carbon systems were displayed as inset the respective plots. With respect to the anodic peak current value, the order of carbon material for CoomBB immobilization is arranged as pristine-MWCNT>CBnano>pMWCNT>functionalized-MWCNT>GO=CNF=AC. Note that purified MWCNT and oxygen functionalized showed poor CoomBB immobilization. Following are the conclusions derived from the observations: (i) graphitic structure (G-band in Raman spectroscopy) along with trace metal impurities (Ni, Fe and Co) is necessary for the effective immobilization of CoomBB. It is difficult to conclude the interaction parameters with Raman spectroscopic results alone. It is suspected that the trace metal impurity in CBnano and MWCNT forms a week coordination complex with the nitrogen terminal of the CoomBB and hence assisted for the enhanced stability and electron-transfer function. (ii) There is a strong repulsion electrostatic repulsion between the 10


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sulphonic acid of CoomBB and negative charge of the oxygen sites of GO and f-MWCNT. Although the pristine-MWCNT showed the highest peak current value, in consideration with easy availability and low cost, CBnano material is chosen as an optimal for the CoomBB modification and electroanalytical studies. 3.1 Physicochemical characterization of the CBnano@CoomB. Comparative TEM images of CBnano and CBnano@CoomBB were displayed in Figure 3A and B. A spherical particle of average size 50±10 nm was noticed with the unmodified CBnano. After the CoomBB modification, agglomerated particles like structure of CBnano was noticed, which may be due to strong interaction between the CBnano and CoomBB. This speculation was confirmed further studies. Raman spectroscopic response of the CBnano and CBnano@CoomBB showed qualitatively similar patterns with specific signals at D (disordered graphitic, sp3 carbon; 1350 cm-1) and G (ordered graphitic structure, sp2 carbon; 1575 cm-1) bands.51 Calculated intensity ratio, ID/IG are 0.67 and 0.41 respectively for the CBnano and CBnano@CoomBB samples (Figure 3C). A significant decrement in the ratio of CBnano after CoomBB immobilization was noticed. This observation indicates enhancement in the graphitic structure of the CBnano after the CoomBB modification. Strong π-π interaction between the graphitic structure of CBnano (sp2 carbon) and πe-s of CoomBB is a likely reason for the agglomeration of the particles as displayed in the TEM. Figure 3D is a comparative FTIR/KBr response of CBnano, CoomBB and CBnano@CoomBB systems. Specific IR signals at 1580, 1507, 1344 and 1173 cm-1 corresponding to the –NH and 1035 cm-1 due to the –SO3- (sulphonic acid) groups were noticed with the CoomBB. After modification with CBnano, ie., CBnano@CoomBB, these peaks were retained but with slightly shifted in the IR values (1577, 1510, 1340, 1170 and 1031 cm-1). This observation indicated that native form of the CoomBB is retained with strong interaction after its modification on CBnano. 11



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3.3. Electrocatalytic oxidation of TZP by GCE/CBnano@CoomBB: Figure 4A is a CV response of GCE/CBnano@CoomBB without (curve a) and with 500 µM of TZP (curve b) in pH 7 PBS at v=10 mV s-1. A well-defined irreversible oxidation peak current signal at an anodic peak potential, Epa = 0.2±0.005 V vs Ag/AgCl, wherein, the A1/C1 redox peak of the CoomBB exist, was noticed. Five continuous CV cycling of the TPZ showed about 15% decrement in the 2nd cycle after that an unaltered in the anodic peak current signal. Control experiment with a CoomBB unmodified electrode, i.e., GCE/CBnano with the TZP has failed to show any such irreversible oxidation peak current signal in the tested potential window indicating inability of the control electrode for the TZP oxidation (Figure 4A, curve c). The observation of marked increment in the oxidation peak at lower potential indicates the electrocatalytic oxidation function of the GCE/CBnano@CoomBB to TZP.

It is noteworthy that this is the first

electrocatalytic observation reported in the literature for the oxidation of TZP. Effect of scan rate on the electro-catalytic oxidation of TZP showed a systematic increase in the peak current (ipa) and a plot of ipa vs square root of v (v1/2) is found to be linear starting from origin suggesting the oxidation of TZP follows diffusion controlled electron-transfer reaction mechanism. In further, CV response of the modified electrode with increasing concentration of TZP was examined (Figure 4C). As can be seen, a regular increase in the ipa values were noticed. A plot of ipa vs [TZP] is linear with current sensitivity and regression coefficient values of 55.34 nA µM−1and 0.9915 respectively. Diffusion coefffient (DTPZ) of the TPZ was calculated based on following Randles-Sevick equation :53 ipa = 268600 n3/2ADTPZ1/2v1/2CTPZ

--(2)

Wherein n= total number electrons involved in the reaction, A= geometric surface area (0.0707 cm2), v = scan rate, DTPZ = diffusion coefficient and CTPZ= concentration of the TPZ 12


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sample. Calculated DTPZ value is 1.09×10-5 cm2 s-1. Regarding to the mechanism, selective binding of TZP on the GCE/CBnano@CoomBB and its mediated oxidation is proposed a possible pathway of the reaction (Scheme 1). These preliminary studies support suitability of the GCE/CBnano@CoomBB for electroanalytical assays of TZP. 3.4. Amperometric i-t analysis. The GCE/CBnano@CoomBB electrode was subjected to amperometric i-t analysis by spiking of 50 µM of TZP continuously in 10 mL pH 7 PBS at a fixed applied potential 0.1 V vs Ag/AgCl (optimized) (Figure 4D, curve a). A regular increase in current signals against TZP addition was noticed. In continuation, unmodified GCE/CBnano (control) was also tested with the TZP spikes, but, failed to show any current signal (Figure 4D, curve b). Constructed calibration plot was linear in a concentration range 50-500 µM of TZP with regression coefficient, detection limit (signal-to-noise ratio=3) and sensitivity values 0.9988, 2 µM and 314 nA µM-1 cm2 respectively. Common interferencing chemicals such as cysteine (CySH), nitrite (NO2-), uric acid (UA), ascorbic acid (AA), hydrazine (hyd), sulfide (S2) , citric acid (CA) and H2O2 and other pesticides like cypermythrin (Cyp), propenofos (Ps) (CV response; Supporting Info Figure S1), deltramethrin, parathion and monocrotophous (CV response; Supporting Info Figure S2) were also tested as in Figure 4E. Interestingly, none of the above chemicals interfered in the TPZ detection response highlighting the selective electroanalytical response of GCE/CBnano@CoomBB. Note that the monocrotophous-pesticide showed a redox peak at Eo’ = ~300 mV, which is about 150 mV positive than that of the GCE/CBnano@CoomBB redox peak system. The selectivity noticed with the Triazophos is due to followings two important reasons: (i) matching of potentials of the GCE/CB@CoomBB and Triazophos oxidation reaction and (ii) specific adsorption/orientation of Triazophos on GCE/CB@CoomBB. In further, the modified electrode stability was tested by performing 100 13



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continuous CV responses in pH 7 PBS yielded a RSD value 4.1% indicating the appreciate stability of the working electrode. Similarly, reproducibility of the modified electrode was examined by preparing four GCE/CBnano@CoomBB at different timings showed a RSD value 4.9% highlight the good reproducibility of this new sensor system (data not enclosed). 3.5 Flow injection analysis. In order to improve the analytical performance, FIA technique utilizing FIA-GCE/CBnano@CoomBB as an electrochemical detection (ECD) with sample injection volume of 20µL was adopted. ECD-GCE/CBnano@CoomBB modified was prepared similar to the preparation of GCE/CBnano@CoomBB (Figure 1A, curve b). To protect the film from hydrodynamic deformation, a dilute solution of Nafion (0.01%) was casted on the modified electrode. The Nafion film is not alternating the qualitative electrochemical property of the ECD.

In first, in reference to the peak intensity and stability, interrelated hydrodynamic

parameters such as applied potential (Eapp) and flow rate (Hf) and were optimized as Hf= 600 µL min-1 and Eapp = 0.1 V vs Ag/AgCl (data not shown). Figure 5A is a typical FIA calibration response of TZP under the optimal condition. Current-concentration linearity in a window, 16632 ng (in 20 µL) of TZP with a current sensitivity and regression coefficient values 21.02 nA/ng (20 µL) and 0.9987 respectively were noticed. Ten successive injections of 16 ng (20µL) of TZP yielded a RSD value 2.3% (inset Figure 5A). Calculated detection limit (signal-to-noise ratio = 3) is 119 pg (20 µL). Obtained detection limit value is comparable and/or lower than that of the some of the recently reported analytical procedure based on AChE biosensor combined GC-MS analysis (~5µg/L),54 luminol based electrochemiluminescent detection utilizing the poly(aminthiophenol)-MIP/gold nanoparticle/CNT GCE (3.1 ng L-1),55 poly(p-aminothiophenol) membranes-Au-nano based MIP (8.012 mg L-1),42 electrochemical displacement immunoassay coupled with oligonucleotide sensing (0.18 ng L−1),10 phage immuno-loop-mediated isothermal 14


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amplification assays detection (128 ng mL-1),37 and colorimetric sensor array sensor (10−8g L-1).30 The main advantage of this work is rapid analysis of the TZP at about 60s per injection unlike to the 6.5 h working protocol with the conventional detection procedure.10 Note that several nitrogroup containing pesticides like parathion and fenitrothion have been previously detected by electrochemical method utilizing the electro-activity of the nitro-group present in the compound.56-59 In this case, it is merely due to new electrocatalytic strategy of oxidation and sensing of TZP. Real Sample Analysis. Figure 5B and C are typical FIA of simulated vegetable leaf samples (250µg of TZP/50g vegetable), after water washing, using FIA-ECD by standard addition method. A TZP detectable FIA signal upon injecting the real samples was noticed. Addition of TZP standards mixed real samples yielded regular increase in the peak current signal confirming the applicability of the present assay for the practical analysis. Calculated detected values of TZP in the samples are 8.38 and 10.2 ng (20 µL). After the dilution factor and sample correction these values becomes 2.08 µg/50g and 2.55 µg/50g. The difference in the value between sprayed (250 µg/50g) and detected one is due to removal by water washing of the sample. Calculated recovery values are ~100±3%.

4. CONCLUSION A highly redox active and stable Coomassie brilliant blue dye surface-confined carbon nano black powder modified glassy carbon electrode was prepared by continuous potential cycling of GCE/CBnano in CoomBB containing pH 7 PBS. A well-defined redox peak corresponding to the proton-coupled electron-transfer reaction of the quinonoid form of CoomBB at Eo’= 0.15 V vs Ag/AgCl was noticed. The redox peak is adsorption-controlled 15



Page 16 of 33

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electron-transfer in feature. The modified electrode upon exposure of TZP in pH 7 PBS showed a marked oxidation signal at Epa = 2 V vs Ag/AgCl, wherein, the A1/C1 redox peak appeared. Control experiment with GCE/CBnano for the TZP oxidation failed to show any faradaic response. Thus, it is concluded that the CoomBB modified electrode mediates the TZP oxidation without any enzyme and antibody. The oxidation reaction was found to be diffusion –controlled electron-transfer in nature. In first, amperometic i-t technique was used for the quantification of TZP.

Selectivity of the working electrode towards TZP was tested by spiking common

biochemicals like cysteine, nitrite, ascorbic acid, hydrazine, uric acid, sulfide, citric acid and H2O2 and other pesticides like cypermythrin, propenofos,

deltramethrin, parathion and

monocrotophous in pH 7 PBS under an optimal experimental condition. The result showed nil interference effect to the above mentioned chemicals. Flow injection analysis coupled electrochemical detection of TZP in 20 µL working volume was further demonstrated. A linear calibration in a window 15.65632 ng (20 µL) of TZP with a detection limit 119.3 picogram (20 µL) was obtained. Applicability of the technique was further demonstrated by analyzing the TZP content in couple of simulated vegetable leaf samples. The prim advantages of the present sensor over the existing sensor systems are simplicity, rapid detection and direct detection protocol. Since, the new strategy works without any enzyme, antibody and pre-concentration procedure, it can be extendable to various practical systems.

ASSOCIATED CONTENT Supporting Information

16


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Control CV responses of GCE/CBnano@CoomBB without and with 1 mM of other pesticides, cypermythrin and propenofos in pH 7 PBS showing nil electrocatalytic responses (Figure S1A and B). The Supporting Information is available free of charge on the ACS Publications website. AUTHOR INFORMATION Corresponding Author Annamalai Senthil Kumar*, Emails; [email protected]; [email protected]; Phone: +91-416-2202754 ORCID Annamalai Senthil Kumar: 0000-0001-8800-4038 Notes The authors declare no competing financial interest.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This work was funded by Department of Science and Technology, Science Engineering Research Board, India. ACKNOWLEDGMENT The authors acknowledge the Department of Science and Technology – Science and Engineering Research Board (DST-SERB- EMR/2016/002818) Scheme. ASK acknowledges National Taipei University of Technology for the support of visiting professor program. 17



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Scheme 1. Illustration for the preparation of GCE/CBnano@CoomBB chemically modified electrode and its proton-coupled electron-transfer and electrocatalytic triazophos oxidation reactions. Inset is the illustration for the connection between the green-vegetables and insect.

26


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B.

C. b.GCE/CBnano@CoomBB

10

th

1-10 cycle

b.GCE/CBnano@CoomBB

30

10

A1

ip /µA

A.

5

I/µA

20

ipa

0 ipc

-20 0

a.GCE/CoomBB

5

15

0

0

-5

-15

-10

-30

40 80 -1 v/mV s 10-100 mV s

-1

0 -5 C1

-10 -15

v= 50 mV s

-0.4

0.0

-1

-0.4

0.4

E (vs Ag/AgCl)/V

0.0

0.4

E (vs Ag/AgCl)/V

D.

-0.4

0.0

0.4

E (vs Ag/AgCl)/V

E. pH 3-11

15

A1 Peak:

0.4 Epa/V vs Ag/AgCl

10 5

I/µA

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 -5

0.2

-10 Slope = -40±3 mV pH

-15

-1

0.0 -0.4

0.0

E (vs Ag/AgCl)/V

0.4

0

4

8

12

pH

Figure 1. (A) Twenty continuous responses of GCE (a) and GCE/CBnano with 1 mM of coomassie brillant blue dye mixed pH 7 PBS and (B) ten continuous CV response of the freshly prepared GCE/CBnano@CoomBB in a blank pH 7 PBS. Effect of CV scan rate (C) and pH (D) of GCE/CBnano@CoomBB. (E) Plot of Epa vs pH. Inset Figure 1(D) is a plot of ipa and ipc vs v1/2.

27



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Intensity/AU

20

1200

10 I/µA

D

1400

5

1600

Raman Shift/cm

C. 30 20

Intensity/AU

B.

G band

Intensity/AU

A.

1200 1400 1600

1200 1400 1600

-1

Raman Shift/cm

10

-1

0

Raman Shift/cm

-1

0 0

-10

-10 -20

-20 Graphene oxide@CoomBB

D.

200

0.0

Carbon Nanofiber@CoomBB

0.4

-0.4

E.

Intensity/AU

-0.4

-5

30 20

1400 1600 Raman Shift/cm

10

0

0

-100

-10

-200

HOOC-MWCNT@CoomBB

-20

0.4

0.0 E (vs Ag/AgCl)/V

0.4

Activated Charcoal@CoomBB -0.4

0.0

0.4

40 1200 1400 1600

20

Raman shift/cm-1

0

-20 Purified MWCNT@CoomBB

-30 -0.4

-30

F.

-1

100

0.0

Intensity/AU

-30

I/µA

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

-0.4

0.0

0.4

E (vs Ag/AgCl)/V

Pristine-CB@ CoomBB

-40 -0.4

0.0

0.4

E (vs Ag/AgCl)/V

Figure 2. CV responses of various GCE/Carbon@CoomBB modified electrodes with carbon viz, graphene oxide (A), carbon nanofiber (B), activated charcoal (C), carboxylic acid functionalized MWCNT (D), purified MWCNT (E) and Carbon nano black (F) materials, in pH 7 PBS at v=50 mV s-1. Insets are structural illustrations of the respective carbons. Inset Figures are Raman Spectroscopic responses of the respective carbon materials.

28


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A

B

C.

D. G

a. CBnano

a. CoomBB

D

1035 1507 v(-S=O) 1344 v(-NH Bending) 1580 1173

ID/IG = 0.67

T(%)

Intensity/AU

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


c. CBnano@CoomBB

1510

1031 1577 1340 1170 (-C-O stretch)

b. CBnano@CoomBB ID/IG = 0.41 b. CBnano

1730(-C=0 stretch)

1200

1400

1600 -1

Wavenumber/cm

3000

2000

1000 -1

Wavenumber/cm

Figure 3. (A and B) are TEM images, (C) Raman Spectroscopy responses and (D) FTIR responses of CBnano and CBnano@CoomBB. Control FTIR response of CoomBB is given in Figure 2D, curve a.

29



-30-

10 b.c+500 µM TZP

30

40

15

30

0

40 0

20

5 10 1/2 -1 1/2 v /(mV s )

30 500µM - 5mM 15 0

30

2 4 [TZP]/µM

0

10 -5

I/µA

I/µA

5

C. ipa/µA

B. ipa/µA

A.

10

-10

-10 c. CBnano--TZP

-15

v=10 mV s-1

0

-20

-10

-30 -0.4

0.0

-0.4

0.4

0.0

i/µA

0.0

0.4

E (vs Ag/AgCl)/ V

E.

10 a.

5

20

-0.4

0.4

E (vs Ag/AgCl)/ V

E (vs Ag/AgCl)/V

D.

20

0

a. CBnano@CoomBB

a.

#. Eapp= 0.1 V

b.

#. Each spike 100µM

15

0

TPZ TPZ -

5

#.Eapp= 0.1 V #.50 µM Each

TPZ

NO2

a. GCE/CBnano@CoomBB

Hyd 2S

10 Ps

10

CA UA AA H2O2 CySH TPZ

200 400 600 [TPZ]/µM

Cyp

0

I/µA

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

b. GCE/CB

0

0 0

300

600

t/s

900

0

300

600

900

t/s

Figure 4. (A) CV response of GCE/CBnano@CoomBB without (a) and with (b) 500 µM Triazophos dissolved pH 7 PBS at v=10 mV s-1. Curve c is a control CV of GCE/CBnano with 500 µM Triazophos. (B) Effect of scan rate of GCE/CBnano@CoomBB with 500 µM triazophos in pH 7 PBS. (C) Effect of concentration of triazophos in pH 7 PBS; inset is a plot of ipa vs [TZP], at v= 10 mV s-1. (D) Amperometric i-t response of GCE/CBnano@CoomBB (a) and GCE/CBnano (b) with 50 µM continuous spikes of TZP in pH 7 PBS (stirred condition) at Eapp = 0.1 V vs Ag/AgCl. (E) Response of GCE/CBnano@CoomBB with 100 µM of TZP, propenofos (Ps), cypermythrin (Cyp), hydrazine (hyd), sulfide (S2-), nitrite (NO2-), citric acid (CA), uric acid (UA), ascorbic acid (AA), hydrogen peroxide and cysteine (CySH).

30


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A.

B.

C.

623 ng (20µL) Eapp= 0.1 V vs Ag/AgCl 532 16 ng (20µL); RSD = 2.3%

R+47 ng (20µL)

500 nA

R+47 ng (20µL)

470 R+31

407 R+32

313 219

R+16

R+16 R

157 5 µA

R

94 31 200 nA

200 nA

200 s

400 s

400 s

Figure 5. (A) FIA response of GCE/CBnano@CoomBB with increasing concentration of TZP (20 µL injections) in pH 7 PBS. Inset is a ten continuous injections of 16 ng (20 µL) TZP. (B&C) FIA of simulated vegetable-leafs by standard addition approach. Eapp = 0.1 V vs Ag/AgCl; Hf= 600 µL min-1.

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Original Scheme 1


A New Strategy for Direct Electrochemical Sensing of a

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