Cite this paper: Vietnam J Chem., 2021, 59(2), 253-262 Article DOI: 10.1002/vjch.202000158 Acetylcholinesterase sensor based on PANi/rGO film electrochemically grown on screen-printed electrodes Ly Cong Thanh1, Dau Thi Ngoc Nga2, Nguyen Viet Bao Lam3, Pham Do Chung3, Le Thi Thanh Nhi4, Le Hoang Sinh4, Vu Thi Thu2*, Tran Dai Lam5* Hanoi University of Pharmacy (HUP), 15-17 Le Thanh Tong, Hoan Kiem, Hanoi 10000, Viet Nam University of Science and Technology of Hanoi (USTH), Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Hanoi 10000, Viet Nam Hanoi National University of Education (HNUE), 134-136 Xuan Thuy, Cau Giay, Hanoi 10000, Viet Nam Duy Tan University (DTU), 03 Quang Trung, Da Nang 50000, Viet Nam Institute of Tropical Technology (ITT), VAST, 18 Hoang Quoc Viet, Cau Giay, Hanoi 10000, Viet Nam Submitted September 11, 2020; Accepted February 24, 2021 Abstract In this work, the polyaniline/reduced graphene oxide (PANi/rGO) bilayer was directly electrodeposited on carbon screen-printed electrodes (SPE) Some details in growth of PANi/rGO bilayer were revealed from cyclic voltammograms and X-ray photoelectron spectra The growth of stacked rGO film at high compactness on the electrode surface is mainly accompanied with reduction of epoxy functional groups at basal planes of graphitic flakes The asgrown rGO layer with abundent hydroxyl functional groups at basal planes is preferable to attract intrinsic fibrillar-like PANi polymer chains in protonated aqueous media The as-prepared PANi/rGO hybrid bilayer has shown good conductivity, high porosity, good adhesion to biomolecules, and fast electron transfer rate (increased by 3.8 times) Herein, PANi/rGO film has been further utilized to develop disposable acetylcholinesterase sensors able to detect acetylthiocholine (ATCh) with apparent Michaelis - Menten constant of 0.728 mM These sensors provide a very promising technical solution for in-situ monitoring acetylthiocholine level in patients with neuro-diseases and determination of neuro-toxins such as sarin and pesticides Keywords Reduced graphene oxide (rGO), polyaniline (PANi), acetylcholinesterase (AChE), screen-printed electrodes (SPE), neuro-diseases, electrodeposition INTRODUCTION Hybrid films which combined biocompatible polymers and highly conductive inorganic nanomaterials have recently gained many attentions in sensing and electronic applications Among wellknown conducting nanomaterials, graphene and its derivatives with extraordinary conductivity, mechanical stability and flexibility are the best candidates that meet many critical requirements of electrochemical sensing systems.[1] Especially, reduced graphene oxide (rGO) is the most frequently used since it provides many behaviors similar with graphene and can be easily produced at large scale[2,3] through solution-based approaches and combined with other materials in composites.[4,5] Meanwhile, polyaniline (PANi) with good conductivity, high porosity, and good adhesion to biomolecules (i.e enzymes) is often utilized in electrochemical biosensors Interestingly, PANi has three different chemical states that can be tuned electrochemically[6,7] and sensitive to protonation/deprotonation process.[8] Also, the presence of amino groups in polymer chains of PANi make it becomes one favorable transducing platform to immobilize enzymes Probably, the hybrid structures based on PANi and carbonaceous materials should have inherited the mentioned benefits of these two materials Several research groups have demonstrated potential applications of hybrid films based on carbonaceous nanomaterials with PANi Depending on the purpose of the application, these hybrid films were grown either in composite structure or bilayer architecture In the beginning, composite films based on graphene derivatives and PANi were mainly 253 Wiley Online Library © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH Vietnam Journal of Chemistry utilized for developing high-performance supercapacitors in flexible energy storage devices.[911] These hybrid composites also show high anticorrosion behavior.[12] Recently, the layer-by-layer structure of hybrid films made of conducting polymers and carbonaceous materials has drawn more attentions The assembly of the two distinct materials in two separated layers allows better control in their thickness and homogeneity The use of graphitic material as one supporting layer provides the solution to overcome insulating nature and structural shrinkage of PANi in dedoping states.[13,14] Moreover, the addition of soft PANi material make carbonaceous materials become less rigid and more biocompatible For instance, the PANi ad-layer electrodeposited on graphitic electrodes has been shown to improve voltammetric signals during analysis of redox probes.[15] PANi/graphene bilayer with good conductivity and fast electron transfer has been shown to be profitable in electrochemical immunosensors for tracing neurotoxins.[16] PANi/rGO bilayer was utilized as one pHsensitive membrane to sense protons released from gene amplification process.[17] Some suggestions on structure of PANi/rGO bilayer were previously provided but the details on growth mechanism of this hybrid bilayer is still unclear until now Many neurodegenerative diseases (i.e, Alzheimer’s disease and Parkinson’s disease) are associated with the degeneration of the cholinergic system that is caused by abnormal AChE activity Therefore, it is essential to develop realiable tools for monitoring the activities of AChE enzyme as well as screening their inhibitors Acetylcholinesterase sensors based on optical approaches[18-20] offer facile preparation and visual detection which are compatible for in-situ analysis But acetylcholinesterase(AChE) electrochemical sensors are still more preferable[21] due to their good sensitivity and their ability to be integrated onto electronic devices Metallic nanoparticles with good electrical conductivity and intrinsic electrocatalytic activity have been previously employed to ensure high sensitivity of enzymatic electrochemical sensors.[22,23] Recently, carbonaceous materials are gaining more attentions due to their high conductivity and good bioacompatibility.[24-27] In our research group, we have developed AChE electrochemical sensor based on graphene flakes modified with iron oxide nanoparticles.[28] In another work, AChE sensor was manufactured from carbonnanotubes modified with thiophene polymer and gold nanoparticles.[29] In both cases, the carbonaceous materials have been synthesized using chemical vapor deposition (CVD) process which Vu Thi Thu et al requires long procedure and complex instrument In this work, rGO/PANi will be prepared on low-cost screen printed electrode (SPE) using a simple electrochemical process Some details on growth mechanism of the hybrid film will be revealed The as-prepared hybrid film will be later utilized as a transducing platform to load acetylcholinesterase (AChE) and ready for monitoring acetylthiocholine (ATCh) - one important neurotransmitter involved in nervous communication MATERIALS AND METHODS 2.1 Chemicals Graphite powder, aniline (C6H5NH2), sulfuric acid (H2SO4), potassium permanganate (KMnO4) were purchased from Sigma-Aldrich, USA Acetylthiocholine (ATCh), acetylcholinesterase (AChE), phosphate buffered saline (PBS), glutaraldehyde (GA) were also from Sigma-Aldrich, USA Screen printed carbon electrodes (SPE) (Φ = mm) were from Quansense, Thailand 2.2 Apparatus Electrochemical experiments were conducted on an AUTOLAB PGSTAT302N workstation (Metrohm, the Netherlands) FE-SEM images (Field Emission Scanning Electron Microscopy) were captured on a S-4800 system (Hitachi, Japan) ATR-FTIR spectra (Attenuated total reflection Fourier Transform Infrared spectroscopy) of the films were studied on a Shimadzu spectrometer (IR-Tracer 100) The crystalline structure of powder samples was verified by Raman spectroscopy on a Horiba spectrometer using 532 nm excitation X-ray photoelectron spectroscopy (XPS) spectra were recorded on a Thermo ESCALAB spectrometer (USA) using employing a monochromic AlKα source at 1486.6 eV 2.3 Synthesis of graphene oxide The graphitic flakes (200 mg) were oxidized using strong oxidizing agents, namely, KMnO4 (1 g) and H2SO4 (30 mL) at 60 oC After 24 hours, the reaction solution was cooled down to room temperature and left for two more days The cooled solution with dark color was centrifuged at 8000 rpm Then, the solid precipitate was thoroughly rinsed until a mild pH was obtained Finally, the gained product was dried at 60 oC in an oven More details on synthesis of graphene oxide (GO) were given in our previous report.[30] © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 254 Acetylcholinesterase sensor based on… Vietnam Journal of Chemistry 2.4 Electrodeposition of PANi/rGO films Cyclic voltammetry method is an approach able to deposit thin films with controllable thickness and uniform morphology Carbon screen-printed electrodes (on plastic substrates) which are suitable for flexible and disposable biosensors are chosen in our experiments mg.mL-1 GO dispersion in PBS (pH 7.4, 0.1x) was used as deposition solution to electrodeposit rGO film In general, the negatively charged GO flakes with abundant oxygenated functional groups (OFGs) can be easily exfoliated due to electrostatic respulsion However, the use of electrolyte containing anions and cations which is mandatory for electrodeposition process might cause π-π stacking of these exfoliated flakes For this reason, the precursor solution was sonicated for at least 30 before use in order to obtain a well-dispersed suspension of GO The GO was directly reduced and deposited on bare SPE electrode by using cyclic voltammetry method at potentials ranging from -0.2 to -1.0 V with number of cycles and scan rate were set to be 10 and 50 mV.s-1, respectively PANi was electrodeposited by sweeping asprepared rGO/SPE electrode in 0.03 M aniline solution prepared in acidic 0.5 M H2SO4 at potentials from -200 mV to +900 mV The number of cycles and scan rate were set to be and 50 mV.s-1, respectively 2.5 Sensing performances of acetylcholinesterase sensor based on PANi/rGO AChE enzyme (20 IU) was immobilized onto sensing platform using glutaraldehyde vapor (GA) as cross-linking agent at 40C for 90 Amperometric responses of the acetylcholinesterase sensors based on PANi/rGO/SPE were recorded upon successive injection of acetylthiocholine solution (5 mM, µL) to a static PBS drop (50 µL) covered totally the three electrodes of SPE The applied voltage was set to be +300 mV (vs Ag/AgCl) RESULTS AND DISCUSSION 3.1 Structural behaviors of graphene oxide The crystalline structure of GO material was examined using Raman technique (figure S1) The curves displayed two prestigious peaks at 1348 and 1593 cm-1 relevant to D mode (A1g) and G mode (E2g), respectively.[31] The two peaks relevant to 2D mode (double resonance transitions) and (D+G) mode (defect) are also observed at 2691 and 2934 cm-1 Furthermore, the ratio between intensities of two main peaks ID/IG was determined to be 0.95 This is a clear evidence to demontrate high oxidation degree of graphitic material The crystalline size of ( ) graphitic flakes (evaluated from ( ⁄ ) ) was estimated to be 20.23 nm and 190.19 nm for GO (ID/IG = 0.95) and graphite powder (ID/IG = 0.1), respectively This reduction in the average size of graphitic domains is probably resulted from structural disorder of sp3 hybridized carbon atoms during harsh oxidation process in presence of strong oxidizing agents 3.2 Growth of PANi/rGO bilayer 3.2.1 Electrodeposition of rGO film onto SPE The electroreduction and direct deposition of GO onto SPE using cyclic voltammetry (CV) method in aqueous condition is shown in figure The sweeping potentials were chosen in the range from -0.2 V to -1.0 mV in order to avoid hydrogen evolution and possible reoxidation of carbonaceous materials at more positive potentials.[32] PBS buffer (0.1 X) with neutral pH and diluted ion concentrations (13.7 mM NaCl, 0.27 mM KCl, mM Na2HPO4, 0.18 mM KH2PO4) was used as electrolyte to limit the destabilisation of suspended GO flakes at too high concentrations of ions Due to the dispersability of GO in water are typically from to mg.mL-1, the concentration of GO precusor was chosen to be mg.mL-1 The formation of black rGO thin film directly deposited on the working electrode can be easily observed by naked eyes A typical CV curve for electrodeposition of rGO was obtained with one irreversible broad reduction peak at -900 mV (vs Ag/AgCl) which occurred in the 1st cycle but disappeared in next scans (figure 1) It is well-known that this peak is relevant to the reduction of the oxygenated moieties on GO flakes The crossover (around -780 mV) in the 1st cycle during the electrodeposition of GO is resulted from intrinsically poor conductivity of carbon SPE According to the widely accepted structure model proposed by Lerf-Klinowski (figure S2), major oxygenated functional groups (OFGs) in GO materials mainly include hydroxyl and epoxy groups at basal planes, carbonyl groups at flake edges that can contribute to several irreversible electrochemical processes.[33,34] It was also reported that the reduction of carbonyl groups at the graphitic edges occurs at more negative potentials (-1050 to -1220 mV) whereas that of basal epoxy moieties occurs at © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 255 Vietnam Journal of Chemistry more positive potentials (-876 to -1120 mV).[30] As seen from cyclic voltammograms (figure 1), there is only one well-defined reduction peak which is probably assigned to the reduction of epoxy groups at basal planes This reduction process (figure S3) will probably restore more sp2 hybridized carbon atoms and might also generate more hydroxyl functional groups, thus much improve electrical conductivity as well as hydrophilicity of the electrode surface at the same time.[34] Vu Thi Thu et al surface Second, the functionalization of basal planes with these negatively charged molecules will probably facilitate the intercalation of water molecules and soluble molecules into these gaps,[33] thus accelerate once more the reduction and deposition process of carbonaceous flakes On the other hand, the grown rGO film should be very compact and durable Finally, the hydrophilization of basal planes with these hydroxyl moieties will probably provide nucleation sites that can easily adsorb aniline monomer and then facilitate the growth of polymeric ad-layer on top of GO film.[12,36] 3.2.2 Electrodepostion of PANi film onto rGO/SPE Figure 1: Cyclic voltammograms recorded during electrodeposition of GO onto SPE Figure 2: Cyclic voltammograms recorded during electrodeposition of PANi onto rGO/SPE High concentration of hydroxyl groups at basal planes of graphitic flakes provides many benefits First, the reduction of epoxy molecules to hydroxyl molecules was accompanied with direct deposition of graphitic material onto the electrode surface GO flakes accumulated on the electrode surface can be reduced and spontaneously solidified, whereas GO flakes partially reduced in electrolyte keep migrating upon the driving of electric field to electrode The CV curves recorded during polymerization of aniline on rGO/SPE electrode using cyclic voltammetric method is shown in Figure Since the protonation is essential in polymerization of aniline,[36] the electrodepostion of PANi is conducted in a diluted acidic solution The process was stopped after cycles at V to ensure the high conductivity of synthesized film by achieving a moderately thin PANi layer in emeraldine form.[37] A typical CV curve for electropolymerization of PANi was obtained with two anodic waves located at +266 mV and +752 mV relevant to transition from leucomeraldine to emeraldine salt and formation of fully doped perningraniline, respectively.[6] Similar to any electrodeposition process of conducting polymers, the intensities of those two peaks increased consecutively with number of scans It is worth to notice that the inversion current (current at switched potential of +900 mV) was found to be decreased, indicating a progressive nucleation which will lead to a porous structure of polymer film.[6] It was generally accepted that the growth of electrodeposited PANi film is a nucleation process.[36] In aqueous medium containing small doping counter ions (i.e SO42-), the electrodeposition is initiated by three-dimensional progressive nucleation and followed by prolongation of one-dimensional polymer branches Herein, the polymer chains must have been nucleated progressively on rGO modified electrodes and then grown in branch-like structure The growth of PANi film onto rGO modified electrodes should be more favorable compared to bare electrodes First of all, the carbonaceous substrate provided additional surfaces for the adsorption of aniline monomers and oligomers.[13] As mentioned above (section 3.2.1), the existence of previously deposited rGO layer with high compactness might offer more nucleation sites, thus © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 256 Vietnam Journal of Chemistry increased the disposition rate of PANi.[36] Last but not least, the adhesion of PANi with amino groups onto basal planes of graphitic flakes with abundant OFGs should be strengthened by cross-linking bonds[14,17] as well as π-π stacking interaction between these two materials 3.3 Morphological and structural behaviors of PANi/rGO/SPE Figure illustrates the surface morphologies of rGO and PANi/rGO films examined by FE-SEM It is obvious that the rGO film with multi-layered structure of stacked flakes shows a smooth surface with several wrinkles Meanwhile, PANi/rGO film shows a micropourous network which is valuable for electron transport processes The polymer chains are formed in fibrillar-like structure which is intrinsic architecture of PANi film electrodeposited in aqueous conditions This result is consistent with the progressive nucleation mechanism of PANi film as mentioned in section 3.2.2 Such a highly porous 3D architecture of PANi/rGO bilayer is very promising transducing platform in enzyme based electrochemical sensors for its accelerated electron transfer rate and improved adhesion to biomolecules Acetylcholinesterase sensor based on… casted GO film was also prepared and characterized The C:O ratio (see table 1) was determined to be 1.70, 2.044, and 2.313, and 2.025 for GO, rGO, PANi and PANi/rGO films, respectively On the other hand, the oxygen content was decreased after electrochemical reduction of GO, but slightly increased in presence of PANi top layer It is obvious that the atomic percentage of oxygen atoms must be decreased after reducing OFGs at basal planes of graphitic flakes The existance of doping counter ions SO42- on polymer chains in top layer[40] and the unhealed lattice defects in underlying graphitic flakes[41] are responsible for slight increase in atomic percentage of oxygen atoms in PANi/rGO (compared to individual rGO and PANi films) Figure 4: ATR-FTIR spectra of rGO (red) and PANi/rGO (black) films Table 1: Analysis results derived from XPS spectra Figure 3: FE-SEM images of bare SPE (A), rGO/SPE (B) and PANi/rGO/SPE films (C) IR spectra of rGO and PANi/rGO films are given in figure The stretching vibrations of hybridized and oxygenated carbon atoms in rGO films were found at 1600 and 990 cm-1 Meanwhile, the characteristic vibrations of non-nitrogenated (1572, 1489, 821 cm-1) and nitrogenated (1298 and 1113 cm-1) moieties of polyaniline in emeraldine form were also clearly observed.[37-39] XPS spectra of rGO and PANi/rGO were investigated (figure S4) For comparison, drop- Sample C/O GO rGO PANi PANi/rGO 1.699 2.044 2.313 2.025 C 1s core-shell spectrum of GO drop-casted film shows strong signals ascribed to graphitic carbon atoms (284.7 eV) and oxidized carbon species (C-O 286.9 eV, C=O 288.5 eV).[14] Upon electrochemical treatment, the peak associated with graphitic carbon atoms becomes prominent while the peaks ascertained to OFGs becomes weaker (see table 1) The most significant change in XPS spectra is observed in the concentration of C-O groups (epoxy) which is in good agreement with CV records The CO/C-C ratio was determined to be 0.925 for GO film but only 0.736 for rGO film (decreased by 20 %) In the same time, O1s spectrum of rGO film reveals © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 257 Vietnam Journal of Chemistry Vu Thi Thu et al one peak located at high energy level (534.5 eV) which is in visual in O 1s spectra of other films The appearance of this peak is probably ascribed to phenolic groups at basal planes and/or intercalated water molecules.[42] These results have provided clear evidences to demonstrate that the electrochemical reduction of GO precursor has mainly happened at basal epoxy groups N 1s signals for PANi/rGO film can be deconvoluted to assign benzenoid amine –NH(399.3 eV) and cationic radical N+ (401.3 eV).[14,43,44] The first peak relevant to amine group is located at higher binding energy compared to neutral amine (at 399.5 eV),[37] indicating the occurence of partially charged nitrogen in electrodeposited polymer chains The second peak is typical for PANi at doped state.[40] These results have confirmed the presence of PANi layer on top of rGO film The N+/N ratio in PANi/rGO film was found to be 0.492 which is slightly lower than that obtained on PANi film (0.583) This indicates that the PANi chains might have been dedoped partially in presence of negatively charged moieties from rGO layer Nevertheless, the proton doping level of PANi chains in PANi/rGO bilayer is still relatively high, and thus affords a good electrical conductivity.[40] 3.4 Electrochemical behaviors of PANi/rGO/SPE The charge transfer kinetics at modified electrodes was examined using cyclic voltammetry in 1mM Fe(CN)63-/4- solution (figure 5) For bare electrode, the two redox peaks occurred with peak separation of 440 mV which is much larger than usual glassy carbon electrodes due to the poor conductivity of carbon SPE This result is in agreement with the observation of a crossover current in the first cycle recorded during electrodeposition of rGO onto SPE (section 3.2.1) Modification of SPE electrode with rGO material has not only increased the peak intensities by 1.3 times but also much shortened peak separation by 240 mV (table 2) This is obviously resulted from good conductivity[41] and fast electron transfer rate[45] at the basal planes of rGO Also, it was generally accepted that the presence of carbonaceous nanomaterials might lower energy required for electrochemical reactions occurring at electrode surface Even the rate of charge transport at rGO film is lower by several orders compared to pristine graphene material,[41] it is still a promising candidate for electronic devices Moreover, rGO film can further be used as a supporting layer to accelerate the growth of appropriate organic ad-layer When PANi is deposited on top of rGO, the peak intensities were improved (3.8 times higher than SPE, 2.9 times higher than rGO/SPE) and the peak separation was continued to be decreased to 135 mV) It was reported that the combination of highly conductive carbonaceous materials and highly porous PANi can enhance ion diffusion and charge transport which is very profitable for further applications in electrochemical sensors as well as supercapacitors.[46] 2100 Figure 5: Electrochemical behaviors of bare SPE (black) and SPE modified with rGO (red), rGO/PANi (blue) Table 2: Electrochemical behaviors of rGO and PANi/rGO films Current (nA) 1800 1500 1200 900 600 300 Sample SPE rGO/SPE PANi/rGO/SPE Peak separation (mV) 440 200 135 Intensity (µA) 10 13 38 400 600 800 1000 1200 1400 1600 1800 2000 t (s) Figure 6: Current responses recorded on as-prepared acetylcholinesterase sensor © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 258 Acetylcholinesterase sensor based on… Vietnam Journal of Chemistry 3.5 Performances of Acetylcholinesterase sensor The sensing performance of acetylcholinesterase sensor was examined using chrono amperometric (CA) method with the applied voltage of +300 mV (figure 6) Upon the injection of an aliquot of ATCh, the current response increased rapidly with response time less than 10s The response current of the sensor was increased with the increasing concentration of ATCh according to regression equations: I (nA) = 1845.3 CATCh (mM) - 289.5 (0.192 to 1.094 mM) (figure 7) polymerization of nanofibrillar-like polyaniline films on top of rGO film The acetylcholinesterase sensor based on PANi/rGO bilayer was built as a proof-of-concept to demonstrate its potential biosensing application In our future work, more details on the effects of reduction degree on concentration of OFGs (i.e hydroxyl groups) on morphology and charge transfer kinetics of hybrid films based on rGO and several conducting polymers will be studied Table 3: Comparisons between AChE electrochemical sensors 1800 Configuration 1500 I (nA) 1200 Pd@Au/AChE 900 600 300 0.2 0.4 0.6 0.8 1.0 1.2 C (mM) Figure 7: Calibration of as-prepared acetylcholinesterase sensor Kmapp The apparent Michaelis - Menten constant was 0.728 mM (from Lineweaver-Burk relation) The limit of detection (LOD) was determined from standard deviation (after three measurements) and slope of calibration curve to be 17.5 µM These obtained values are comparable to those previously reported in other works (see table 3),[47-50] showing good affinity of immobilized enzyme AChE for the targeted substrate ATCh The developed PANi/rGO bilayer is a universal electrochemical platform that can be further applied to load many other biological elements and ready to monitor different biological processes, especially the ones that are pH sensitive CONCLUSIONS PANi/rGO film with layer-by-layer structure was successfully electro-deposited onto screen-printed electrode with significantly improved electrical conductivity and electron transfer kinetics The reduction of epoxy groups at basal planes was found to be dominant during direct electrodeposition of rGO film in aqueous medium at neutral pH Consequently, these hydrophilic flakes facilitate progressive nucleation and accelerate the Linear range 4-124 µM 0.1-9.0 mM 2-272 µM GCE/rGO/CS@ TiO2-CS/AChE GCE/Pd@AuN Rs/AChECS/Nafion Graphite 0.125electrode/poly(F 2.6 BThF)/MNPs/A mM ChE GCE/PDDA/PS µMS/AChE 10 mM 12.5GCE/Gr112.5 MNPs/AChE µM 0.192SPE/rGO/PANi/ 1.094 AChE mM Dection limit (µM) Km (mM) Ref - 0.19 [20] - 3.1 [37] - 0.207 [38] 6.66 0.731 [39] - 2.16 [40] 8.35 - [41] 17.5 0.728 This work Note: CS = chitosan, NRs = nanorods; FBThF = 4,7-di(furan-2-yl)benzo[c][1,2,5]thiadiazole; MNPs = magnetic nanoparticles; PDDA = poly(diallyldimethylammonium chloride), PSS = polystyrene sulfonate Declaration of interest The authors have no financial interests to declare Acknowledgment This research is funded by Vietnam National Foundation for Science and Technology Development NAFOSTED (grant number 104.03-2018.344 and 103.02-2018.360) The authors also express great thanks to our colleagues at Hanoi National University of Education (Hanoi, Vietnam) for their supports in Raman measurements and our colleagues at University of Paris-Sarclay (Paris, France) for their supports in XPS measurements © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 259 Vietnam Journal of Chemistry Electrochem Commun., 2019, 98, 110-114 REFERENCES Y Song, Y Luo, C Zhu, H Li, D Du, Y Lin Recent advances in electrochemical biosensors based on graphene 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Lam Institute of Tropical Technology (ITT) Vietnam Academy of Science and Technology (VAST) 18 Hoang Quoc Viet, Cau Giay, Hanoi 10000, Viet Nam E-mail: trandailam@gmail.com © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 262 ... Growth of PANi/ rGO bilayer 3.2.1 Electrodeposition of rGO film onto SPE The electroreduction and direct deposition of GO onto SPE using cyclic voltammetry (CV) method in aqueous condition is shown... SPE (A), rGO/SPE (B) and PANi/ rGO/SPE films (C) IR spectra of rGO and PANi/ rGO films are given in figure The stretching vibrations of hybridized and oxygenated carbon atoms in rGO films were... Acetylcholinesterase sensor based on? ?? casted GO film was also prepared and characterized The C:O ratio (see table 1) was determined to be 1.70, 2.044, and 2.313, and 2.025 for GO, rGO, PANi and PANi/ rGO films,