Dopamine biosensor based on surface functionalized nanostructured nickel oxide platform
Introduction
Dopamine is known to be a significant catecholamine neurotransmitter that shows important role in the functions of central and peripheral nervous system, renal and hormonal systems of human and other mammals (Heien et al., 2005, Wightman et al., 1988). It is produced by decarboxylation of 3,4-dihydroxy phenylalanine and operated as a precursor in the synthesis of neurotransmitters epinephrine and norepinephrine. Dopamine also acts as a neuromodulator in brain circuitry and responsible for several physiological conditions such as mood, behavior, memory, attention and movement (Robinson et al., 2003). Abnormal metabolism and concentration of dopamine in body can lead to neurological diseases like Parkinson's disease, schizophrenia, epilepsy, senile dementia and attention deficit hyperactivity disorder (ADHD) (Cao et al., 2008, Huffman and Venton, 2009, Mo and Ogorevc, 2001, Robinson et al., 2003, Wightman et al., 1988). Moreover in emergency condition, dopamine is infused to the patients showing the symptoms of myocardial infraction, hypertension, bronchial asthma and during acute heart surgery (Beitollahi et al., 2008). Due to such physiological and pathophysiological effects, it is essential to develop a quantitative method to accurately estimate dopamine for diagnosis and continuous monitoring of neurological disorders.
The currently available analytical methods for dopamine determination are high performance liquid chromatography (Carrera et al., 2007, Muzzi et al., 2008), UV spectrometry (Barreto et al., 2008), capillary electrophoresis (Kang et al., 2005, Li et al., 2010, Thabano et al., 2009), liquid chromatography-electrospray tandem mass spectrometry (El-Beqqali et al., 2007), flow injection analysis with spectrophotometric detection (Deftereos et al., 1993), coulometric (Myers et al., 1998), fluorescence (Chen et al., 2011, Khattar and Mathur, 2013, Nikolelis et al., 2004) and electrochemical detection (Chen and Peng, 2003, Matos et al., 2000, Mecker and Martin, 2008, Umasankar and Chen, 2008, Zhang et al., 2013). Apart from electrochemical detection, most of the procedures are complex, cumbersome, expensive, time consuming and requires lots of sample. Due to electrochemical activity, dopamine can be efficiently detected by applying appropriate potential across the electrodes (Njagi et al., 2010). With this in view, enzyme biosensor combined with electrochemical detection may be advantageous owing to rapid detection, simplicity and ease in miniaturization. Further, for reliable and rapid determination of dopamine and to increase sensitivity and selectivity of the biomedical devices, enzyme can be proficiently used as biorecognition element. Tyrosinase or polyphenol oxidase (EC 1.14.18.1) is binuclear copper containing enzyme that catalyzes oxidation of phenolic compounds in two steps to their respective o-quinones, which is further reduced at appropriate redox potential to form original phenols again (Tembe et al., 2008, Zhou et al., 2007). o-quinones are electroactive species and can be reduced at moderately negative potential (Tembe et al., 2006, Zhou et al., 2007) which is helpful in the prevention of the polymerization of phenols and interferences from oxidizable species (Koile and Johnson, 1979). Dopamine is a catechol like phenolic substance, can be detected with enhanced specificity using tyrosinase enzyme molecules through direct reduction of bio-catalytically liberated o-dopaquinone species (Njagi et al., 2010, Tsai and Chiu, 2007, Zhou et al., 2007).
For suitable immobilization of tyrosinase enzyme molecules, several materials such as graphite (Nistor et al., 1999), conducting polymer (Arslan et al., 2005, Rajesh and Kaneto, K., 2004), nafion membrane (Furbee Jr et al., 1993), carbon paste (Caruso et al., 1999), hydrogel (Daigle and Leech, 1997) and biopolymers (Liu et al., 2005) have been used as matrices to improve stability of the enzyme in sensor probe. Most of these matrices used to develop tyrosinase based system suffer from complexity and reduced sensitivity (Tembe et al., 2008, Tembe et al., 2006). In this context, nanostructured metal oxides can be effectively used as matrices for biomolecule immobilization due to presence of fascinating electrochemical and optical properties, catalytic effects and efficient charge transfer abilities from biomolecules to a particular substrate (Doong and Shih, 2010, Thanh and Green, 2010). Nanostructured metal oxides have attracted plentiful attention of the researchers owing to their adsorption capabilities, effective surface area for biomolecule immobilization with desired orientation and better conformation that leads to high biological activity of the immobilized bio-sensing molecules (Caruso, 2001, Solanki et al., 2011). Among the various nanostructured metal oxides, nickel oxide nanoparticles have recently been used in bio-sensing application (Li et al., 2008) due to the presence of high electro-catalytic activity, fast electron transport property, high surface energy, chemical stability, low cost and biocompatibility (Ali et al., 2013, Kavitha and Yuvaraj, 2011, Salimi et al., 2007). In addition the existence of variable oxidation states in nickel oxide nanoparticles helps in easy mobility of the electrons (Mohan et al., 2011). Beside this, high isoelectric point (IEP 10.8) of NiO nanoparticles make it suitable for tyrosinase enzyme (IEP 4.7) immobilization. The NiO nanoparticles can be synthesized through sol–gel, reverse micelle, microwave irradiation and laser induced fragmentation method (Justin et al., 2010, Singh et al., 2011). In sol–gel method NiO nanoparticles are generally prepared by using non-ionic, anionic and cationic surfactants to control pore volume, surface area, crystallite size and organization of NiO nanocrystals (Justin et al., 2010). Among three types of surfactants, the anionic surfactant shows finest result in respect of higher surface redox activity, lower crystallite size, porous morphology and higher surface area (Justin et al., 2010). These properties are possibly most suitable for biomolecule immobilization and electrochemical measurement. Hence in our experiment sodium dodecyl sulphate (SDS) has been used as anionic surfactant during synthesis of NiO nanoparticles.
The electrochemical detection of dopamine is greatly affected by the presence of co-existing interfering substances ascorbic acid and uric acid because of similarity in oxidation potential of dopamine close to these interfering species (Liu et al., 2013, Zhang et al., 2005). Dopamine requires higher oxidation potential (between 0.5 and 0.7 V) which includes oxidation range of many other electrochemically active species (Njagi et al., 2010). To avoid the effect of interferents, tyrosinase based system has been used in selective and specific detection of dopamine and the estimation of dopamine has been monitored through direct reduction of o-dopaquinone species at relatively lower potential (~−0.15 V).
In the present study, sol–gel method with anionic surfactant SDS has been used to synthesize NiO nanoparticles (NPs) and tyrosinase enzyme has been immobilized on synthesized NiO NPs by physio-adsorption technique. The NiO NPs-tyrosinase conjugates, characterized by different methods, was then deposited on indium tin oxide (ITO) coated polyethylene terephthalate (PET) substrate by solution casting method. These enzyme strips were used for biosensor measurements and several optimization steps were performed to characterize the dopamine biosensor in synthetic samples, with interferents and in real sample.
Section snippets
Materials and reagents
Tyrosinase (EC 1.14.18.1 from Agaricus bisporus with activity of 1000 U/mg of solid), dopamine hydrochloride (C8H11NO2·HCl), Poly (vinyl alcohol) (PVA, MW 130000), ITO coated PET substrate and sodium hydroxide pellets (NaOH) were procured from Sigma–Aldrich (USA). Nickel nitrate hexahydrate [Ni (NO3)2·6H2O] and sodium dodecyl sulphate (NaC12H25SO4) were purchased from Alfa Aesar (UK). The stock solution of tyrosinase (1 mg/mL) was freshly prepared in phosphate buffer (50 mM, pH 6.5). All solutions
Nanoparticle characterization
The NiO NPs were characterized using various techniques to ascertain their composition, homogeneity and sizes.
Conclusions
In summary, nickel oxide NPs have been synthesized by sol–gel method and successfully functionalized with tyrosinase enzyme molecules to prepare a simple, easy to fabricate, relatively specific, highly reusable and sensitive bio-sensing platform for the detection of neurotransmitter dopamine. The electrochemical response studies of the fabricated electrode showed improved sensitivity (0.0602 µA/µM) for dopamine detection in wide linear detection range (2–100 µM) with fast response time of 45 s due
Acknowledgments
The work was supported by a research grant from Department of Biotechnology, Government of India (Grant No. BT/PR7190/PID/6/719/2012). The author Appan Roychoudhury is a recipient of Junior Research Fellowship from Indian Institute of Technology Delhi.
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