Elsevier

Applied Surface Science

Volume 474, 30 April 2019, Pages 154-160
Applied Surface Science

Full Length Article
Bienzymatic assembly formed @ Pt nano sensing framework detecting acetylcholine in aqueous phase

https://doi.org/10.1016/j.apsusc.2018.04.056Get rights and content

Highlights

  • Platinum nanoparticles (PtNPs) encapsulated metallic organic framework (MOF) modified Au electrode was fabricated.

  • Acetylcholine esterase (AChE) and choline oxidase (ChO) were co-immobilized onto modified Au electrode.

  • Fabricated electrochemical biosensor showed excellent detection limit and storage stability.

  • The developed sensor was tested in real serum samples and correlated with the standard HPLC method.

Abstract

Acetylcholinesterase (AChE) and choline oxidase (ChO) were co-immobilized on platinum nanoparticles (PtNPs) and metallic organic framework (MOF) modified Au electrode to construct an acetylcholine (ACh) biosensor. The morphology and structure of the nanoparticles modified working electrode were characterized by Scanning Electron Microscopy (SEM), Energy-dispersive X-ray spectroscopy (EDX), Cyclic voltammetry (CVs) and Electrochemical Impedance Spectroscopy (EIS) techniques. The prepared biosensor exhibit a wide linear range of 0.01–500 μM for acetylthiocholine chloride (ATCl) and a low detection limit of 0.01 μM. No interferences were observed for other possible compounds/substances present in the serum samples. For detection of ACh in clinical (serum) samples the results of the nanoparticle based biosensor were well correlate with the results obtained from HPLC method. The half-life of the working electrode was 4 months, when stored at 4 °C.

Introduction

Acetylcholine (ACh) is a kind of neurotransmitter which is present at neuromuscular junctions, at synapses in the ganglia of the visceral motor system, and at multiple sites within the central nervous system. Synthesis of ACh takes place in nerve terminals from acetyl coenzyme A and choline. This reaction is catalysed by choline acetyltransferase [1]. Altered levels of ACh are associated with dementia, hallucinations, memory loss and Alzheimer’s disease [2]. Different scientific groups have implemented various strategies for the estimation of ACh and its derivatives. These strategies include titrimetric estimation and electrochemical estimation using, conductrimetric, polarographic, amperometric and voltametric methods. Some scientific groups also demonstrated the spectroscopic, chromatographic and biological assay methods for the estimation of ACh, choline and its derivatives [3]. In the present work Platinum nanoparticles (PtNPs) and metallic organic frameworks (MOFs) are implemented for the amperometric detection of ACh.

Metallic nanoparticles (MNPs) exhibits high conductivity due to high fraction of surface atoms, and hence, can be utilized in various applications. MNPs specially, platinum nanoparticles (PtNPs) offer various advantages like coalescence, high stability, catalytic activity, enhanced corrosion resistance, which makes them a suitable candidate for biosensing applications [4]. PtNPs are already being utilized in the fabrication of biosensors. One such sensor is made for the detection of hydrogen peroxide (H2O2), where the synergistic electrocatalytic activity of carbon nanotube (CNT) and PtNPs enables the low potential detection of H2O2 with high sensitivity [5]. Another sensor employed glucose oxidase integrated with Pt-CNT-silicate matrix for more sensitive detection of glucose. This sensor is found to be more sensitive than the CNT based sensor [6].

MOFs fall under the category of highly porous materials. They offer unparalleled range of structural diversity, chemical and physical properties along with high degree of tenability. MOFs are extended crystalline structures. They are formed by the connection between clusters of cations or metal cations termed as nodes. The connections are provided by multi-topic organic molecules or ions termed as linker or strut [7]. MOF offers low cytotoxicity, which can be utilized for in vivo applications [8]. MOF can be tuned for the light absorption and emission properties of an analyte along with the specific affinity with the addition of various metal and organic building blocks. This property allows the sensor to circumvent interference from the undesired components during the analysis. The tenability of MOFs is also possible post-synthesis, which can be employed for specific molecular recognition [9]. For biosensing applications, this versatility opens up possibilities for cell or tissue-specific targeting. Due to reversible binding and fluorescence events, MOF also offers high degree of reusability [10]. The versatility of MOF makes them a potential candidate for biosensing application. The MOFs prepared by self-assembled organic linkers, metal ions or clusters are widely utilized as chemical sensors [11], [12], [13]. Luminescent MOF sensors have been widely explored for the sensitive detection of explosive chemicals [14], [15], oxygen [16], [17], various aromatic compounds [18] and amines [19], [20]. MOF has been employed for the detection of ascorbic acid [21] and glucose [22] using peroxidase-mimic MOF biosensors. MOF biosensors with nucleic acid probes have already been developed for the detection of proteins and DNA. One such sensor is fabricated to target HIV-1 U5 long terminal repeat sequence and thrombin by employing two-dimensional MOF N,N′-bis(2-hydroxyethyl) dithiooxamidatocopper (II) [Cu(H2dtoa) [23]. The method, described in the communication utilizes the changes in the impedance and value of current due to enzymatic reaction of ACh molecules present in the serum, to the enzymes (AChE-ChO) immobilized on the surface of PtNPs encapsulated MOF on working electrode.

Section snippets

Chemicals and reagents

AChE (EC 3.1.1.7, type VI-S; from electric eel; activity 200–600 U/mg solid) and ChO (EC 1.1.3.17, from Alcaligenes, sp. 10U/ mg solid) were bought from Sigma Aldrich Co.USA. Chemicals used in the process of PtNPs preparation are poly(vinylpyrrolidone) (PVP), which act as a stabilizer, dihydrogen hexachloroplatinate (H2PtCl6), and ethylene glycol (EG, serves the role of both solvent and reducing agent) procured from Sisco Research Laboratory, Mumbai. Chemicals used in the process of metallic

Characterization of various modification stages for the fabrication of working electrode

The various modification stages of the modified working electrode (AChE-ChO/PtNPs/MOF modified Au electrode) were characterized by SEM. Fig. 1A(a) of bare Au electrode shows the uniformly smooth surface. Fig. 1A(b) of MOF exhibit a high quality brick like morphology. Fig. 1A(c) shows spherical shape of PtNPs was clearly visible, suggesting substantial enhancement in the surface area of Au electrode after modification. When AChE and ChO were co-immobilized onto the PtNPs/MOF/Au electrode in Fig.

Conclusion

In the present study, a novel AChE-ChO/Pt/MOF/Au electrode was fabricated. The structural details of the fabricated electrode was analysed using SEM. The quantitative and qualitative characteristics were also examined using electrochemical techniques. Under the optimal conditions, a lower detection limit of 0.01 μM for ACh (S/N = 3) was obtained. The utilization of MOF and PtNPs in the development of a biosensor for ACh has prompted its progressed diagnostic execution by offering multiple

Acknowledgements

Dr. Utkarsh Jain received “Start-up Research Grant” by Science and Engineering Research Board (SERB) – Department of Science and Technology (DST), India (File No. YSS/2015/000023). The SERB – DST, India is highly acknowledged.

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