Fabrication of novel nanobiosensors for detection of β-lactam antibiotics with the aids of nanomaterials

The β-lactam antibiotics are potent and inexpensive molecules that have been in use for decades, but remained one of the most widely prescribed in modern medicine. Inappropriate use of this class of drug partly due to popularity has resulted in the emergence of resistance strain, and β-lactam antibiotics have finally found its way into the human food chain. The need to monitor the unintended ingestion of β-lactam antibiotics by humans has resulted in an increasing demand for accurate determination of this class of antibiotics in food of animal origin and pharmaceutical products. The conventional methods available for determination of β-lactam antibiotics are fraught with several drawbacks, such as extensive sample preparation and clean-up procedures, and high detection limits. Sensitive and reliable biosensors are needed to address the drawbacks associated with the conventional methods. The aim of this thesis is to fabricate novel nanobiosensors for penicillin detection in milk and antibiotics by exploring the use of various nanomaterials, such as carbon nanotubes and gold nanowires array for achieving performance enhancement. This study has employed two approaches to achieve this goal. The first approach employed the entrapment of penicillinase within conducting polypyrrole films, while the second approach used chemical crosslinking of penicillinase with glutaraldehyde and bovine serum albumin. Both approaches utilized multi-walled carbon nanotubes and gold nanowires array to improve the analytical performance of the nanobiosensors. The results of this study are presented in eight chapters which are summarised separately below. Chapter 1 provides the background to the project, as well as a comprehensive literature review of the past and recent developments in the detection of β-lactam antibiotics. It also discusses the importance of β-lactam antibiotics and provides an introduction to the theory and general principles of biosensors. Chapter 2 focuses on the fabrication of a polypyrrole (PPy)-based penicillinase potentiometric biosensor based on the use of an ultra-thin film grown from a supporting electrolyte-free pyrrole monomer solution. The biosensor was developed by physical entrapment of penicillinase (P’Nase) within the PPy film by galvanostatic polymerization. Cyclic voltammetry and electrochemical impedance spectroscopy confirmed successful entrapment of P’Nase within the PPy matrix. Chronopotentiometric measurements demonstrate that the incorporation of P’Nase into the PPy film increases the difficulty in film formation, while also decreasing the stabilization potential due to increased film conductivity. The ultra-thin PPy-P’Nase-Pen G biosensor detected penicillin concentration as low as 15 μM and achieved a sensitivity of 78 mV/decade, a detection limit of 0.31 µM and a linear range of 117 – 552 µM. The PPy-P’Nase-Pen G electrode was successfully used for detection of penicillin in spiked milk samples with an excellent average recovery of 99.3 ± 3.8 %. The achieved analytical performances of the biosensor were more effective than some of the earlier reported PPy-based penicillin potentiometric biosensors. The performance of the biosensor developed in chapter 2 was further improved by co-entrapment of multi-walled carbon nanotubes (MWCNTs) with penicillinase within the PPy matrix. The study is described in Chapter 3, where it was demonstrated that the successful co-entrapment of MWCNTs significantly enhanced the penicillin potentiometric response obtained with the resulting biosensor. The successful incorporation of MWCNTs within the PPy film was confirmed by cyclic voltammetry and EIS. The EIS plots exhibits a decrease in the charge transfer resistance after the entrapment of MWCNTs within the matrix and indicated an improvement in the film conductivity. The presence of MWCNTs almost doubled the sensitivity obtained in chapter 2. The PPy-P’Nase-PenG-MWCNTs nanobiosensor also achieved a lower minimum detectable concentration of 5 µM and lower detection limit of 0.18 µM, a sensitivity of 108 mV/decade, and a wider linear range of 98 - 500 µM. The PPy-P’Nase-PenG-MWCNTs nanobiosensor demonstrates a reasonable stability and enabled reliable quantification of penicillin in milk samples and amoxicillin tablets, achieving an excellent recovery of 101.7 ± 2.3 % for milk samples and 101 ± 1 % for amoxicillin tablets. Chapter 4 investigates the use of crosslinking with glutaraldehyde (GLA) and bovine serum albumin (BSA) for immobilization of MWCNTs and P’Nase on a platinum electrode for potentiometric detection of penicillin. The successful integration of MWCNTs in the P’Nase- GLA-BSA matrix led to a better performance of the electrode. The resulting Pt-GLA-BSA-P’Nase-MWCNTs nanobiosensor achieved a sensitivity of 85 mV/decade for penicillin detection and a linear range of 50 - 645 µM, which is wider than achieved in the previous chapters. Also, the nanobiosensor achieved a detection limit of 0.10 µM and a lower minimum detectable concentration of 2.5 µM, which is half of the value, obtained in chapters 2 and 3. The successful application of the nanobiosensor to the determination of penicillin in milk and amoxicillin was also demonstrated. The data obtained for the recovery study for this nanobiosensor show that an average percentage recovery of 99.0 ± 0.8 % was achieved for milk samples and 106.3 ± 1.6 % for amoxicillin tablets. The Pt-GLA-BSA-P’Nase-MWCNTs nanobiosensor recovered 519 ± 7 mg to 544 ± 4 mg penicillin from the tablets with a dosage of 500 mg amoxicillin/tablet, resulting in a percentage recovery range of 104.0 ± 1.4 – 109.0 ± 0.7. On the other hand, analysis of penicillin in the milk samples gave a percentage range of 94.7 ± 0.4 – 104.0 ± 2.0. Chapter 5 investigates the use of a chemical crosslinking method for integration of P’Nase with gold nanowires array (AuNWA) as a support. The AuNWA were electrochemically grown into the pores of an anodic aluminium oxide (AAO) template by a two-step anodization method. Field emission scanning electron microscopy (FESEM) of the AAO and AuNWA revealed a highly ordered morphology and upright AuNWA with a diameter of 80 nm and a length of 16 µm. The use of the AuNWA-GLA-BSA-P’Nase nanobiosensor achieved a sensitivity of 91 mV/decade, a linear range of 59 – 240 µM, a detection limit of 0.15 µM and a minimum detectable concentration of 5 µM. The utilization of the AuNWA-GLA-BSA-P’Nase nanobiosensor gave excellent recovery of 103.4 ± 2.6 % for the detection of penicillin in milk and 104 ± 2 % for the detection of penicillin in amoxicillin samples. Chapter 6 investigates the use of AuNWA for further improvement of the achieved sensitivity and linear concentration range by co-integration of MWCNTs and P’Nase with AuNWA. This approach used involves the crosslinking of P’Nase and MWCNTs with GLA and BSA on an AuNWA support. The resulting AuNWA-GLA-BSA-MWCNTs nanobiosensor gave an improved sensitivity of 94 mV/decade and a wide linear range of 99 - 698 µM, which is comparable to that obtained in Chapter 4. However, a higher minimum detectable concentration of 5 µM and a detection limit of 0.22 µM were achieved. The biosensor was successfully applied to the determination of penicillin in milk and amoxicillin tablets with slightly elevated average percentage recovery of 108.3 ± 2.2 % for milk analysis and 107 ± 4 % average recovery for amoxicillin. Analysis of the results shows that the use of AuNWA for the fabrication of the biosensor by this approach is beneficial for improving the sensitivity and achieving a wide linear range. Chapter 7 explores the use of PPy for co-entrapment of MWCNTs and AuNWA for achieving further improvement for potentiometric detection of penicillin. In this approach, P’Nase and MWCNTs were co-entrapped within PPy film onto a highly ordered AuNWA for biosensing of penicillin. An inner layer of a thin film of PPy was first grown on the AuNWA to provide a uniform surface for the deposition of the outer layer in which P’Nase is immobilized. Optimum conditions for the formation of inner layer include a 0.05 M Py, polymerization time of 90 seconds and a current density of 0.9 mA/cm2. The outer layer was formed with 0.2 M pyrrole, 60 U/mL penicillinase, 0.01 M Pen G, 0.1 M KCl, 0.5 g/L MWCNTs, a current density of 0.9 mA/cm2 and a polymerization period of 120 s. The resulting AuNWA-PPy-Cl/PPy-P'Nase-MWCNTs nanobiosensor enabled the detection of as low as 5 µM penicillin and achieved a detection limit of 0.15 µM and exhibited a sensitivity of 57 mV/decade and a linear range of 50 - 521 µM. The influence of AuNWA is manifested in the significant increase in conductivity as the biosensor constructed on AuNWA is twice as sensitive as the one assembled on conventional gold electrode using the same configuration. Although, the AuNWA-PPy-Cl/PPy-P'Nase-MWCNTs bilayer nanobiosensor is not as sensitive as those reported in previous chapters, it enabled accurate determination of penicillin in amoxicillin tablets under optimised conditions achieving an average percentage recovery of 100.5 ± 1.4 %. The analytical performances of all the biosensors fabricated and reported in the previous chapters are summarised, compared and reviewed in Chapter 8. This chapter also gives recommendation for future modification of the techniques employed in this study for better performance.