Acetylcholinesterase sensor based on PANi/rGO film electrochemically grown on screen-printed electrodes

Cite this paper: Vietnam J. Chem., 2021, 59(2), 253-262 Article DOI: 10.1002/vjch.202000158 253 Wiley Online Library © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH Acetylcholinesterase sensor based on PANi/rGO film electrochemically grown on screen-printed electrodes Ly Cong Thanh 1 , Dau Thi Ngoc Nga 2 , Nguyen Viet Bao Lam 3 , Pham Do Chung 3 , Le Thi Thanh Nhi 4 , Le Hoang Sinh 4 , Vu Thi Thu 2* , Tran Dai Lam 5* 1 Hanoi University of Pharmacy (HUP), 15-17 Le Thanh Tong, Hoan Kiem, Hanoi 10000, Viet Nam 2 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 3 Hanoi National University of Education (HNUE), 134-136 Xuan Thuy, Cau Giay, Hanoi 10000, Viet Nam 4 Duy Tan University (DTU), 03 Quang Trung, Da Nang 50000, Viet Nam 5 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 as- grown 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. 1. INTRODUCTION Hybrid films which combined biocompatible polymers and highly conductive inorganic nanomaterials have recently gained many attentions in sensing and electronic applications. Among well- known 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 Vietnam Journal of Chemistry Vu Thi Thu et al. © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 254 utilized for developing high-performance supercapacitors in flexible energy storage devices. [9- 11] These hybrid composites also show high anti- corrosion 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 neuro- toxins. [16] PANi/rGO bilayer was utilized as one pH- sensitive 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. Acetylcholin- esterase 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 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. 2. 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 = 3 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 o C. 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 o C in an oven. More details on synthesis of graphene oxide (GO) were given in our previous report. [30] Vietnam Journal of Chemistry Acetylcholinesterase sensor based on © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 255 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. 1 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 min 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 as- prepared 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 5 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 4 0 C for 90 min. Amperometric responses of the acetylcholinesterase sensors based on PANi/rGO/SPE were recorded upon successive injection of acetylthiocholine solution (5 mM, 2 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). 3. 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 sp 3 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 1. 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, 1 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 1 to 4 mg.mL -1 , the concentration of GO precusor was chosen to be 1 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 1 st 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 1 st 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 Vietnam Journal of Chemistry Vu Thi Thu et al. © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 256 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 sp 2 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] 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 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 The CV curves recorded during polymerization of aniline on rGO/SPE electrode using cyclic voltammetric method is shown in Figure 2. 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 5 cycles at 0 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. SO4 2- ), 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 Vietnam Journal of Chemistry Acetylcholinesterase sensor based on © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 257 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 3 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. 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 4. The stretching vibrations of hybridized and oxygenated carbon atoms in rGO films were found at 1600 and 99