A highly sensitive label-free amperometric biosensor for norfloxacin detection based on chitosan-yttria nanocomposite
Introduction
The advent of β-lactam antibiotics for the treatment of numerous communicable diseases in humans as well as in animals marked the beginning of the era of antimicrobial therapy in 1940s. Antibiotics are mainly used because of their specific activity to counter gram-positive and gram-negative bacteria. Globally, India ranks first, followed by China and the USA, in consuming antibiotics [1]. This overuse of drugs leads to antibiotic resistance in bacteria, which became a challenging problem in many areas like health centers, hospitals, and in societies because of an overall increase in the number of patients and the associated cost of treatment [2,3]. In future, the phenomena of antibiotic resistance will be more challenging. The inability to diagnose bacterial infections is the prime restraining factor, which leads to inappropriate use of antibiotics and a decrease in the survival rate during septic conditions. Therefore, there is a need to monitor the usage and release of these drugs into the environment through the human body.
Norfloxacin (NF) is a member of fluoroquinolone (FQ) antibiotics of the third generation and with chemical name 1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(piperazine-1-yl) quinolone-3-carboxylic acid. It is the first choice to treat various infectious illnesses caused by E. coli, Campylobacter, Salmonella shigella, and V. colera [4,5]. The properties like the 6th positioned fluorine atom in NF offers amplified potency to counter gram-negative bacteria, and the position of piperazine moiety at 7th place are the reasons for the anti-pseudomonal action of NF [6]. It's well known that extensive use of antibiotics will eventually lead to more and more organisms becoming resistant [7]. The various non-target toxic effects of NF have also been reported in the earlier works published [8]. As far as human health is concerned, the detection of NF in clinical and biological samples is of utmost importance because of its potentially toxic effects. NF has been detected by sequential analytical methods like fluorometry [9], spectrophotometry [10], and high-performance liquid chromatography (HPLC) [[11], [12], [13], [14], [15], [16], [17]]. Though HPLC has been extensively used owing to its selectivity, susceptibility, and capability to diminish interferences, but it is time-taking, requires wide solvent-usage and high-class devices, with a high maintenance cost which limits its usage. Electrochemical detection of an analyte based on nanomaterial modified electrodes is a precise method in analytical chemistry for the determination of biomolecules and drugs [18,19]. In the previously reported literature, very few studies have involved the electrochemical method for the determination of NF [20,21]. New methods for the detection of NF are therefore needed to develop, with having high speed, specificity, robustness, low-cost, and easy to handle instrumentation.
Biosensors have an outstanding performance capability such as high accuracy and precision, low cost, quick response, user-friendly operation, reliability, relatively compact size, and continuous real-time assessment [22]. Nanoparticles (NPs) are currently used to enhance the overall quantitative performance of electrochemical biosensors for biological and chemical detection [23]. Due to the exciting nanoscale morphology, non-toxicity, and catalytic properties, nanosized metal oxides (NMOs) have attracted significant interest among the various types of nanomaterials developed as immobilizing matrices for the development of biosensors. Optimized rare earth metal oxide NPs could be useful for nanobiosensors/sensors, bioprobes, drug discovery, genetic analysis, medical diagnostics, flow cytometry, and high-throughput screening [24]. They can easily be used for non-invasive, nondestructive, and real-time in vivo diagnosis of numerous ailments. Among them, the nanostructured yttrium oxide (nY2O3) has recently awakened much interest as biological immobilization matrix due to its excellent properties such as its high dielectric constant, chemical inertness, thermal stability, high surface-to-volume ratio, extremely rapid mobility of ions, charge transfer capability, biocompatibility, wide bandgap and interesting electrochemical properties making it a suitable biosensing material [25,26]. The thin film of yttrium oxide become strongly conductive due to the low dielectric constant (13) value, which make it a probable aspirant for application towards the biosensors development [27]. Besides this, the nY2O3 is a desirable inorganic metal oxide consisting of yttrium and oxygen elements. The oxygen moieties in nY2O3 help in the functionalization and covalent immobilization of antibodies. However, the major problem of the agglomeration of nY2O3 onto a specific matrix containing biological molecules has led to limited biosensing applications. This issue can be overcome by modifying nY2O3 with chitosan (CH), biopolymer matrix to develop a nanocomposite film so that they are adequate for biosensor application [[28], [29], [30], [31]]. CH has an excellent ability to form films, biocompatible and biodegradable polymer, and is widely adopted in the nanocomposite fabrication for immobilization of biomolecules by affixing covalently to their amino/hydroxyl groups [[32], [33], [34], [35]].
In this article, a rare earth metal oxide, nY2O3 has been synthesized using a one-step hydrothermal synthesis method to develop a susceptible and highly sensitive biosensing platform for the detection of NF. It is found that the CH-Y2O3 composite is not yet reported in the literature for electrochemical devices. This fabricated BSA/anti-FQ/CH-Y2O3/ITO bioelectrode exhibited higher sensitivity and low detection limit (3.87 pM), when compared with previously described immunosensors and also commercially available enzyme-linked immunosorbent assay (ELISA) towards the detection of the NF.
Section snippets
Reagents and materials
High purity yttrium nitrate [Y(NO3)3.6H2O], 1-(3-(dimethylamino)-propyl)-3-ethylcarbodiimide hydrochloride (EDC), acetonitrile, bovine serum albumin (BSA) were procured from Sigma Aldrich. Potassium hydroxide, acetone, hydrogen peroxide (H2O2), and ethanol obtained from Fisher Scientific. Chitosan (medium molecular weight: 190–310 kDa, viscosity: 298 mPa s, deacetylation degree: 90.12%, structural composition: (C6H11NO4)n, Sodium hydroxide pellets (NaOH), norfloxacin antigen (NF), sodium
Structural characterizations
XRD was performed to study the phase, crystallinity, and structure of the nY2O3. Fig. 1(a) shows the obtained XRD plot for a 2θ angle ranging from 10 to 80°. The characteristic peaks were obtained at 29.2°, 48.5°, and 57.6° marked corresponding to (222), (440) and (622) planes. Few minor peaks were also found at 20.5° (211), 33.8° (400), 35.9° (411), 39.8° (332), 43.5° (431), 53.2° (611), and 59.0° (631). It was observed that these peaks of diffraction precisely matched with the JCPDS No.
Conclusions
BSA/anti-FQ/CH-Y2O3/ITO bioelectrode has been successfully prepared for the development of norfloxacin biosensor. nY2O3 have been synthesized via a one-step hydrothermal process at low-temperature and were confirmed by using XRD, Raman, TEM, and FTIR. The CH-Y2O3 nanocomposite was prepared by the addition of nY2O3 (1 mg mL−1) into CH in acetic acid (1%) solution. The CH-Y2O3/ITO thin films were fabricated via the drop-casting of 30 μL of CH-Y2O3 suspension on ITO surface and used for
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgment
The authors thank to AIRF, JNU, for their characterization facilities. PRS thanks to the Government of India for financial supports for this work through joint Indo-Russia project (Department of Bitechnology (DBT) DBT/IC-2/Indo-Russia/2017-19/02 and Ministry of Science and Education of Russian Federation, agreement № 14.613.21.0061 by 17.07.2017, unique identification number of the project RFMEFI61317X0061). GBVSL is grateful to Department of Science and Technology (DST), New Delhi, India for
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