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Vietnmi tajraal ofChemistiy, Intenianonal Edition, 54(4): 49M95,2016 IXJI. 10.15625/0866-7144.2016-00353^'^''*?^r*''-^*u-.°'^ P°'y(1,5-diammonaphthalene) - polypyrrole nanowire bilayer for trichlorfon insecticide biosensing Ngnyen Vtu. Anb'", Ngnyen U H ^ ' . Do Ph.c Q..n=, Vnong Thi Bid. Hien"
Nguyen Tuan Dimg», Tmn Dai Lam*
^ '^'^'-^'ofOientical Engineering, Hanoi University OfScience atui Technology ''""^^f'-^-onmentalTechnologyandSustainableDevelopmenKCETASDJ-VNUHanoi
Institutefor Tropical Technology, Vietnam Academy of Science and Technology Oraduate University of Science and Technology, Vietnam Academy of Science tmd Technolog
Received 17 June 2016; Acceptiid for publication 12 August 2016 Abstract
poljm^ta nanowire imierUyer was clectrodeposit5^„„ftesSe^<tP?^^ Kieen-pnnted electrodes (SPEs). A A poMLS-diaralnonaphdialenc) outer layer ^ ^e™for S S l i l t I'llT""''''"'™'''"'''»!«'«''«"»s.
agent On the basis of die inhibition of orgai^pho^ta „St^^r?h"'''>'''^''°'"'==^^^ • ^ " e " il'««»ldehyde enzyme, die >eetyleholmesterase-immobilLd b S e r o f r I J ^ , ? "°T"" "'"'" "f ^'Wcholinesteie .«ticchemicaldetemi.ationofnichl„rfonhisec.Srone1ftp^prSSnrpli^^^^^^^
Keyword^Acetylcholmesterase, biosensors, poly(l,5^iarainonapbd«lene),polypynolenanowii.,triclil„rfon.
shown satisfactory results for pesticides analysis where die enzyme activity was employed as an"
indicator of a quantitetive measurement for such insecticides. When AChE is immobilized on die workmg electtode surface, its interaction widl die substtate of acetylfliiocholine produces die electi^oactive preduct of diiocholine. TTie inhibition on the enzyme system can be monittired by measunng tile oxidation cunent of tiiiocholine [3] - I. INTRODUCTION
Trichlorfon is a moderately toxic organophosphate pesticide. It is used to conttol insects on numerous crops. However, die contiunmation of ttichlorfon insecticide present in ae eco-systems creates a lot of pollution problems Conwit analytical metiiods such as gas or liquid ehroinatogtaphy and mass specttoscopy have been widely used for die detennination of organophosphate pesticides in common 111 Nowadays, tiie food safely conttol probleras and environmentel pollution require Uie development of such techniques diat are simpler, faster and more convenient in analysis.
Meanwhile, electtochemical biosensors in general and enzyme sensors in particular, have
"Keived considerable attention regarding Uie detection of pesticide residues owing to die
• d v a n l ^ of low cost, simplicity L d hish Knsitivity dius making diera excellent candidates lor portable detection devices. In living beings oi^ophosphate pesticides bind ineversibly to the
«ctive she of die acetylcholinesterase (AChE) enzyme [2]. Thus acetylcholinesterase (AChEl biosensois based on tiie inhibition on AChE have
Acetyldiiocholine+HjO -
2-rhiocholine—5S*^S2S52 >Thiocholine+Accticacid
^dithio-bischoline + 2H*-12e' The immobilization of enzyme to solid electtode surface Still remains a great challenge for tiie fabncation of biosensors. The use of conductine polymepolypynole (PPy) raattix for enttapmenl enzyme has received great attention in tile past few years. PPy exhibits a high conductivity, easy preparation, and excellem enviromnentel stebility However, it cannot attach enzyme directly due ti> die absence of fimctional groups such as carboxyl or ammo-groups. To resolve tills problem, a polyfl 5- diammonaphtiialene) [P(1,5DAN)] outer layer widi fimctiona amino groups was electtosyntiiesized onto polypyrrole nanowire (PPy NWs) which was
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previously template-free electrodeposited. The proposed bilayer configuration (labeled as P(l,5DAN)/PPy NWs) perfonned for covalent immobilization AChE in the design of electrochemieal biosensors in rapid determination of trichlorfon.
2. EXPERIMENTAL 2.1. Reagents and Apparatus
Pyrrole raonomer (Fluka) was purified by distillation under nitrogen atmosphere before use.
Acetyldiiocholine chloride (ATCI), AChE (VI-S, 1000 lU/rag from electtic eel), 1,5 diaminonaphthalene monomer, Trichlorfon ([2,2,2- trichloro-l-hydroxyethyl]-phosphonic acid dimethyl ester), glutaraldehyde (25%), Bovine Serum Albumin (BSA), HCIO< and LiClO, were purchased from Sigma-Aldrich.
The 0.1 M PBS solution (pH 7.4) was prepared by mbting die solutions of 0.1 M NajHPO*, 0.1 M pi2P0,, 0.137 M NaCI and 2.7 mM KCI. The de- ionized water was used tiiroughout tile experimenis.
The electtochemical experiments were canied out widl an Autolab PGSTAT302N potentiostet interfaced to a OPES 4.9 software (EcoChemie, The Nedierlands) using die screen-printed electixides (SPEs) at room temperatiire. SPEs were fabricated by tiiick-film technology with DEK Albany 247 printing machine (Weymouth, UK). SPEs widi a standard tiiree-elecfrode cell were printed onto polyediylene terephUialate film. The working and counter carbon-based screen-printed electtodes were prepared from tite carbon ink Electtodag PF-407A.
The diaraeter of die working carbon electtode was 3 mm. The reference electtode was printed from die silver ink Electtodag 418 S with E„, = -1^0.3 V vs.
standard hydrogen electtode (SHE) [4J. Finally, a last layer of a diick film curable dielecttic was covered widi PVC insulator, leaving a defined rectimgular shaped (circular well of 5 mm diameter) working area (Fig. 1).
The surface morphology was analysed by Field Emission Scanning Electton Microscope (FE-SEM) witii Hitechi S-4800.
2.2. Electotipolymerization of P(l,5DAN)/PPy NWs bilayer
Firstly, die electtopolymerization of PPy NWs on SPEs was canied out by using 0.15 M pynole monomer in 0.2 M NaiHPO, in tiie presence of vanous concenttation of LiCIO, (from 1 mM to 15 mM) [5J under potentiostetic conditions at E =• -fO 75 V m 500 s. Then, P(1,5DAN) layer was covered
Nguyen Van Anh, etal onto) PPy NWs surface in tiie mbrture 0.01 M HCIO, and 0.1 M LiClO. solution containing 5.10 ' M of 1,5 diaminonaphthalene monomer using cyclic voltammetry (CV). The parameters for CV: scan rate 50 mV/s; the potential range for electiopolymerizatton is between -0.02 and -H).75 V. Afterwards, the pnxluced electiodes (P(l,5DAN)/PPy NWs/SPEs) were washed widl deionized water and incubated in PBS solution.
Fig 1: The picttire of die integrated screen-printed electrode
2.3. Fabrication of AChE sensors
A volume of five microliters of 2.5 % glutiu-aldehyde was coated on P(l,5DAN)/PPy NWs/SPEs to form the covalent bond between amino groups on P(1,5DAN) surface widl aldehyde groups of glutaraldehyde agent. The modified electrode obtained was tteated with 10 nL AChE solution (300 mU, conttiining 5 g/L BSA to maintain die stability of AChE) and kept at room temperattne (25°C) for 2 h for covalent linkage of die AChE to die electiode surface [6-8]. Then, die obtained sensors (labeled as AChE-P(l,5DAN)/PPy NWs/SPEs) were washed witii PBS (pH 7.4) to remove the excess of AChE. The sensor was stored in the refrigerator at 4°C for further use.
2.4. Electrochemical measurement
As can be seen in Fig. 1, 50 (tL droplet deposited over the electtochemical microcell delimited by die circular dielectric layer. The AChE-P(l,5DAN)/PPy NWs/SPEs were tested by cyclic voltammetiy (CV) in PBS solution, and the anodic cunent was recorded as III. Then, 1.0 mM ATCI solution (optimized value was detennined from [3]) was injected into die microcell, the anodic cunent was recorded as I^Mrd, For tiie measurements of ttichlorfon insecticide, the PBS solution conteining die different standard concentrations of trichlorfon was injected into the microcell. The anodic cunent of ATCI on AChE-
VJC, 54(4)2016
P(l,5DAN)/PPy NWs/SPEs widi ttichlorfon mhlbition IS /„ The inhibition of ttichloribn (F/„) was calculated as [Ij;
-W = [(/»-M- mi^n,!- Ill)] X 100%
v*OT I, IS flie anodic cunont in blank solution (PBS); i m „ IS die anodic cunent in PBS solution contammg 1.0 mM ATCI substtate specificity and / IS die anodic cunent after die injection of i concentiation of tiichlorfon msecHcide into PBS conteining 1.0 mM ATCI. The anodic cunents were lecorded at -fO.65 V
3. RESULTS AND DISCUSSION
?,:' S f S f f S ^ " ' * ^ ' ' " " "°'' '^'•'•"•rt'rization of the P(14DAN)a>Py NWs bilayer
J J e P(l,5DAN)/PPy NWs bilayer-raodified electtodes were obtinned as described in Section 2 2 First, the syndiesis voltemmograms and surf-ace morphology of PPy NWs were given in Fig 2
E'earosynlhesisofapofy(l.S-diaminonaphlhalene)...
In observations on flie voltemmograms, die continuous cnnem (curve b) is slowly decreased or saturated, but die continuous cunom (curve e) is higher and it slightly increases wifli time, fhe morphology ra Fig. 2b and Fig. 2c shows fliat PPy NWs generated in 10 mM LiClO, is flie best M t ' r i o * ! ' ^ * ^- '""^ " '^O'^sP^nding to 15
? f I o ° f , * ' '•'^ ^a-Mower-like was oblrvcd Thus, 10 mM LiClO. concentiation was selected for [Wlymenzation of PPy NWs. This PPy NWs
cTndiH , 7 ' * ? ' ™ ' * " ' ' ^ ' = '" " '' ' citable candAte for flie purpose of electiode surf-ace on PPV NWs m order to improve die ability to miraobilize enqone AChE onto flie electtode smface via covalcm binding. Fig. 3 shows die cyclic voltemmograms (CVs) teken during die course of etectt^o^ymerization of P(1,5DAN) on PPy iNWs/bPEs. The anodic cunent increases at die fiist cycle due to Uie oxidation of 1.5DAN monomer S appearance of two typical redox systems (ftom die second cycle, at 0.23 V/0.07 V and 0.45 V/0 40 V) and die current continuously increased during scans reflating tiie ^owth of P1,5DAN film on L PPy tiie diameter of die nanowires ranges from 80 to 100
"'oi^^'^fr^^ versus time of polarization at :?y "10-IS M pynole and 0.2 M NajHPO, 15 mM LiClO,; and SEM images of PPy deposits
obtained, respectively
The rate of polymerization increases on fiirther U S ^ . , " ' *H- '-"''°' """"•""•iO"- At 1 S l^ro,, flie anodic cunem decreases versus tirae to a vay low value (curve a). The PPy fiin, „„ Z etewode IS so tiiin and incomplete (Fig. 2a). When - UCIO, concenttation (5 mM and 10 mM) increases m monomer solution, first die anodic cumint iraponse increases highly flien it decreases rapidly
Fig J.-Cyclic voltemmograms (CVs) registered
„ „ , „ ' ' ^ " 8 electtopolymerization of P(1,5DAN) on PPy NWs and SEM image (inset) Fig- 4 showed CVs behavior of PPv/SPEs PPv r p B r ; H ' " 7 4 ' ' > ' ' ' ? ^ * ' ^ ^ * ^ ^ ^ ^ i ^ 0^
M PBS (pH 7.4) solution. As can be seen die electiocheraical property of cauliflower-like PPv is lowerflianflie PPy NWs. It indicated PPy NWshave hi^cr electtochemical activity and/or die specific surface area. Two obseived redox couples a^e conesponding to tiie ttansfonnation of oxSati"^
VJC, 54(4) 2016
state of P(1,5DAN) [10] clearly seen when compared with CV of pure PPy NWs under the same conditions. The bilayer was formed by an inner layer of PPy NWs (high specific surfece and good conductivity) and an outer layer of P(1,5DAN) (where immobilization AChE via amino group), thereby promising enhancement sensitivity for
Nguyen Van Anh, et al 0.2284xC(ng/L) H 13.6 widi tiie coefficient of determination equals 0.9715 (Fig. 5, mset).
Fig. 4: CVs responses of cauliflower-like PPy, PPy NWs and P(l,5DAN)/PPy NWs film-coated SPEs in
0.1 M PBS (pH 7.4), scan rate: 50 raV/s 3.2. Trichlorfon biosensing based on AChE- P(l,5DAN)/PPy NWs
AChE-immobilized electtodes were studied by CV technique between -0.1 V and 0.75 V, scan rate 50 mV/s (Fig. 5). The cyclic voltammograms of die AChE-P(l,5DAN)/PPy NWs/SPEs in PBS solution (curve I) and in PBS conteining 1.0 mM ATCI (curve 2). The AChE-P(l,5DAN)/PPy NWs/SPEs exhibited significant clectixicatelysis to die oxidation of ATCI from ca -1-0.2 V to -1-0.75 V. When AChE- P(l,5DAN)/PPy NWs/SPEs were injected witii standard solutions of Trichlorf-on at given concentiations, the cunents decreased drastically (curve 3-7) compared widi die case of no inhibitor (curve 2). The cunent values at +0.65 V were decreased to 24.12% when die concenttation was 50 ngrt. (curve 3). It is due to Trichlorfon acting as one of tile ttichlorfon involved in die ineversible inhibition action on AChE, Uius reduced die enzymatic activity to its substtate. At exposure to highei Trichlorfon concenttation ftom 100 ng/L (curve 4) to 300 ng/L (curve 7), die anodic cunent decreased to 32,79% and 78.78%, lespectively. The AChE activity inhibiUon of tiichloribn (I %) and tiichlorfon concentration (ngO.) have a certain linear relationship. The regression equation was I(%) =
E(V)
Fig 5: CVs of AChE-Pl ,5DAN/PPy NWs/SPEs in PBS (1) conteining 1.0 mM ATCI (die contiol experiment) (2) with Trichlorfon concentiations of
50 ng/L (3), IOO ng/L (4), 150 ng/L (5), 200 n g i (6), and 300 ng/L (7). Inset: The calibration curve
shows Uie relation between inhibitions and concenttations of trichlorfon insecticide 4. CONCLUSION
This work successfiiUy deraonsttated two-step electtodeposition of P(l,5DAN)/PPy NWs bilayer filra on the integrated screen-printed electtode. The positive corabination of Uie most outstanding aspects of P(1,5DAN) and PPy NWs exhibits the film widi high specific surface area, relatively high conductivity, easy preparation and ability of covalent immobilization of biomolecules via amino groups. The disposable AChE biosensor base P(l,5DAN)/PPy NWs bilayer has been developed for the determination of Trichlorfon. These featiires provide scope for utilizing the mediodology proposed in the present study to immobilize odier biomolecules in the process of fabricating biosensors.
Acknowledgement. This work was financed by Ministry of Education and Training (Vietnam), code B2014-01-6S.
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Talanta, 115, 713-717 (2013). *' Corresponding aulhor. Nguyen Van Anh
School OfChemical Engineering Hanoi University ofScience and Technology 1 Da, Co VictRoad, Hai Ba Tmng, Hanoi, Vietiiam t-mail. [email protected] vn
Tel.: 04 38680109/0947314210.
