A highly sensitive label-free amperometric biosensor for norfloxacin detection based on chitosan-yttria nanocomposite

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Abstract

Here, non-invasive and label-free detection of trace-level of norfloxacin (NF) in human urine samples has been reported using the electrochemical technique. Nanostructured yttrium oxide (nY2O3) was synthesized at low-temperature using a one-step hydrothermal process. These nY2O3 were characterized by various methods including XRD, FT-IR, Raman spectroscopy, and TEM. A biosensing platform based on nY2O3 modified with chitosan (CH) was fabricated for the detection of NF. The nanocomposite film (CH-Y2O3/ITO) was characterized by FE-SEM, contact angle measurements, and electrochemical techniques. Further, fluoroquinolones antibodies (anti-FQ) were used to modify the CH-Y2O3/ITO electrode via covalent interaction. Non-specific sites were blocked by bovine serum albumin (BSA), those present on the anti-FQ/CH-Y2O3/ITO electrode surface. The response study of BSA/anti-FQ/CH-Y2O3/ITO bioelectrode towards NF detection revealed a wide range (1 pM-10 μM) with a lower detection limit of 3.87 pM using differential pulse voltammetry (DPV). The sensitivity obtained is as high as 10.14 μA μM−1 cm2 with a fast response time of ~10 min. Moreover, the diagnostic performance of the fabricated sensor was evaluated to detect NF in urine spiked sample. The recovery of NF from the spiked sample was observed from 90.5 to 101.1%, with a maximum relative standard deviation of 7.04. The obtained results of the fabricated bioelectrode (BSA/anti-FQ/CH-Y2O3/ITO) was validated with ELISA. The results were found better when compared with earlier described biosensors and commercially existing ELISA in terms of sensitivity and lower detection limit.

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

References (62)

  • M. Ghoneim et al.

    Determination of Norfloxacin by square-wave adsorptive voltammetry on a glassy carbon electrode

    J. Pharm. Biomed. Anal.

    (2001)
  • Y. Ni et al.

    Simultaneous determination of three fluoroquinolones by linear sweep stripping voltammetry with the aid of chemometrics

    Talanta

    (2006)
  • N. Prabhakar et al.

    Chitosan-iron oxide nanocomposite based electrochemical aptasensor for determination of malathion

    Anal. Chim. Acta

    (2016)
  • A. Kaushik et al.

    Chitosan–iron oxide nanobiocomposite based immunosensor for ochratoxin-a

    Electrochem. Commun.

    (2008)
  • R. Khan et al.

    Zinc oxide nanoparticles-chitosan composite film for cholesterol biosensor

    Anal. Chim. Acta

    (2008)
  • H. Guo et al.

    Preparation, characterization, and strong upconversion of monodisperse Y2O3: Er3+, Yb3+ microspheres

    Opt. Mater.

    (2009)
  • A. Ubaldini et al.

    Raman characterisation of powder of cubic RE2O3 (RE= Nd, Gd, Dy, Tm, and Lu), Sc2O3 and Y2O3

    J. Alloys Compd.

    (2008)
  • Y. Repelin et al.

    Vibrational spectroscopy of the C-form of yttrium sesquioxide

    J. Solid State Chem.

    (1995)
  • L. Lin et al.

    Synthesis of yttrium oxide nanoparticles via a facile microplasma-assisted process

    Chem. Eng. Sci.

    (2018)
  • A. Kaushik et al.

    Iron oxide nanoparticles–chitosan composite based glucose biosensor

    Biosens. Bioelectron.

    (2008)
  • T. Sato et al.

    Thermal transformation of yttrium hydroxides to yttrium oxides

    Thermochim. Acta

    (1988)
  • M. Aghazadeh et al.

    Low-temperature electrochemical synthesis and characterization of ultrafine Y (OH)3 and Y2O3 nanoparticles

    J. Rare Earths

    (2012)
  • B. Lakshminarasappa et al.

    Synthesis characterization and luminescence studies of 100 MeV Si8+ ion irradiated sol gel derived nanocrystalline Y2O3

    Nucl. Instrum. Methods Phys. Res., Sect. B

    (2014)
  • L.T.K. Giang et al.

    Preparation and characterization of yttrium hydroxide and oxide doped with rare earth ions (Eu3+, Tb3+) nano one-dimensional

    Phys. Procedia

    (2015)
  • S. Tiwari et al.

    L-cysteine capped lanthanum hydroxide nanostructures for non-invasive detection of oral cancer biomarker

    Biosens. Bioelectron.

    (2017)
  • P.K. Gupta et al.

    Improved electrochemical performance of metal doped zirconia nanoparticles for detection of Ochratoxin-A

    J. Electroanal. Chem.

    (2018)
  • A. Vasudev et al.

    Electrochemical immunosensor for label free epidermal growth factor receptor (EGFR) detection

    Biosens. Bioelectron.

    (2013)
  • D. Chauhan et al.

    Electrochemical immunosensor based on magnetite nanoparticles incorporated electrospun polyacrylonitrile nanofibers for vitamin-D3 detection

    Mater. Sci. Eng. C

    (2018)
  • A. Singh et al.

    Synthesis of Ag–Pt alloy nanoparticles in aqueous bovine serum albumin foam and their cytocompatibility against human gingival fibroblasts

    Colloids Surf. B: Biointerfaces

    (2009)
  • J. Singh et al.

    Preparation of sulfonated poly (ether–ether–ketone) functionalized ternary graphene/AuNPs/chitosan nanocomposite for efficient glucose biosensor

    Process Biochem.

    (2013)
  • R. Laxminarayan et al.

    Antibiotic resistance in India: drivers and opportunities for action

    PLoS Med.

    (2016)
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