Fabrication of polypyrrole based biosensors for trace metal analysis

Traditionally, trace metal analysis are performed with spectroscopic methods, such as atomic absorption spectroscopy (AAS), inductively coupled plasma atomic emission spectrometry (ICP-AES), and inductively coupled plasma mass spectrometry (ICP-MS). These techniques require expensive instrumentation which are not readily accessible and not easily amenable for in-field use. A possible solution for overcoming these limitations can be realized through availability of sensitive and reliable biosensors, which can reliably detect metal ions at trace and ultra-trace concentrations. In this dissertation, various approaches used for the fabrication of highly sensitive metal biosensors are reported. In the first approach, different configurations of glucose biosensors were constructed by immobilization of glucose oxidase (GOx) into conducting polypyrrole films and their analytical performances were demonstrated for amperometric and potentiometric detection of glucose. The resulting GOx biosensors were subsequently employed for metal ion detection based on the inhibition of the activity of the entrapped GOx by the trace metals. The possibility of achieving further improvement in sensitivity and detection limit by co-entrapment of various redox mediators or gold nanoparticles with the GOx into the polypyrrole (PPy) were also investigated for potentiometric and amperometric metal biosensors. In the second approach, the development of a novel strategy for construction of a highly sensitive and selective copper biosensor was achieved by entrapment of Gly-Gly-His tripeptide into conducting polypyrrole film. The analytical applications of both PPy-GOx and PPy-Gly-Gly-His biosensors were successfully demonstrated for reliable determination of metal ions in a number of different samples. Overall, the thesis has been structured into 8 chapters, starting with a general introduction in chapter 1. The discussion in this chapter focuses on the sources and effects of trace metals, methods of analysis and aims of the study. Chapter 2 focuses on the review of the advances and recent developments of biosensors in the last two decades, notably the construction of enzyme, whole-cell and chelating protein biosensors for detection of trace metal ions. The principles of the operation of different configurations of trace metal biosensors are described. Considerations for choosing the desired biomolecule for achieving both sensitive and selective biosensing of trace metals are discussed. Chapter 3 reports the inhibitive potentiometric detection of trace metals with ultra-thin polypyrrole glucose oxidase biosensor. The first part of this Chapter describes the development of a potentiometric glucose biosensor for glucose detection. The biosensor was developed by immobilization of GOx into polypyrrole films by galvanostatic polymerization. Electrochemical and morphological characterization of the resulting ultra-thin PPy-GOx film formed in the absence of supporting electrolyte provided a better understanding of its improved potentiometric response for glucose and was useful in ascertaining whether further improvement can be achieved prior to its utilization for indirect metal analysis. This study demonstrated that the morphology and electrocatalytic activity of PPy-GOx film were significantly dependent on electrodeposition parameters. Evidences from the electron micrographs and chronopotentiograms show that, for higher GOx concentrations, the amount of the enzyme entrapped into the PPy film increased and resulted in more conductive and sensitive glucose biosensor. The observed morphology by SEM also revealed variations in the apparent activity which correlated with the changes in the conductivity of the PPy-GOx film. The observed morphologies revealed details of the GOx tertiary structure that influence its biosensing properties. The resulting PPy-GOx biosensor enabled detection of a minimum glucose concentration of 6.0 µM and a linear concentration range of 6.0 µM to 40 mM with a sensitivity of 77 mV decade-1. This performance was far more effective for glucose detection than those reported in most studies. Another advantage of the biosensor is that it can be used for at least ten consecutive measurements without any significant loss in the sensitivity. The second part of Chapter 3 describes the successful utilization of the PPy-GOx potentiometric biosensor for the inhibitive detection of Cu2+, Hg2+, Cd2+ and Pb2+ ions. The analysis of the nature of the metal inhibition of the immobilized GOx in the PPy-GOx biosensor was achieved by Dixon and Cornish-Bowden plots. The shapes of the curves (sigmoidal, parabolic and linear) obtained for the inhibitors suggest that the inhibition by the metal ions may not be exclusively directed at the essential -SH group, but involve additional binding sites of the enzyme. More specifically, the plots suggest that the inhibition is competitive for Cd2+, while being non-competitive inhibition for other metal ions. The use of an ultra-thin (55 nm thick) PPy-GOx film enabled inhibitive potentiometric detection of metals down to minimum detectable concentrations of 0.08 μM Cu2+, 0.03 μM Hg2+, 0.02 μM Pb2+ and 0.04 μM Cd2+. In addition, good linear concentration ranges were achieved for Cu2+ (0.08-16 μM), Hg2+ (0.03-5 μM), Pb2+ (0.10-15 μM) and Cd2+ (0.04-62 μM). Unfortunately, the response time (100 s) achieved with this PPy-GOx biosensor was too long and further improvement is still necessary. Chapter 4 describes the inhibitive amperometric detection of trace metals with ultra-thin polypyrrole-glucose biosensor. In the first part, another strategy was developed for the fabrication of a novel bilayer amperometric glucose biosensor modified with permselective layer for exclusion of unwanted organic interferences during glucose detection. In addition to its ability to remove interferences, the inclusion of the outer layer enabled better retention of GOx in the inner layer and led to improvement in sensitivity. This was achieved by galvanostatic entrapment of GOx in the PPy film as inner layer, followed by the formation of an outer conducting PPy-Cl layer. Optimum conditions for formation of the inner layer include a 300 U mL-1 GOx, 0.2 M Py and a charge of 25 mC cm2; while the outer layer was formed from 0.15 Py, 0.1 M potassium chloride and a charge of 3.6 mC cm2. With this configuration, the bilayer biosensor successfully rejected 75% of 1.5 mM ascorbic acid, causing less than 18 % suppression of the current response of the PPy-GOx monolayer biosensor. The bilayer biosensor exhibited very good reproducibility (1.9% rsd, n=10), high stability (more than 2 months), fast response time (3-5 s), wide linear range (0.5–24 mM), low Km (8.4 mM), high Imax (77 µAcm-2 ), low detection limit (27 µM), minimum detectable concentration (0.5 mM) and good sensitivity (3.5 µA cm-2 mM-1). The successful application of the bilayer biosensor to the determination of glucose in non-alcoholic beverages was also demonstrated. The second part of Chapter 4 exploits the developed amperometric glucose biosensor for the determination of mercury, copper, lead and cadmium. The data obtained show that Hg2+, Cu2+, Pb2+ and Cd2+ ions inhibited GOx activity, resulting in decreases in glucose responses. The linear ranges obtained for Hg2+ and Cu2+ were 0.48-3.3 µM and 1.5-11 µM, respectively, with corresponding minimum detectable concentrations of 0.48 and 1.5 µM, respectively. The response for Cd2+ demonstrated linearity between 4.0 and 26 µM, while the Pb2+ response was linear from 1.6 to 9.0 µM. The minimum detectable concentrations were 1.6 and 4.0 µM for Pb2+ and Cd2+, respectively. The analysis of Dixon and Cornish-Bowden plots indicates that Hg2+ and Cu2+ demonstrated non-competitive inhibition, but was mixed inhibition for Pb2+, while competitive inhibition was observed for Cd2+. The low inhibition constant, Ki values obtained in this study indicates that the 55 nm PPy-GOx film employed exhibits higher inhibition than thicker films. Application of the inhibitive amperometric biosensor to tap water samples confirms the possibility of using the bilayer PPy-GOx biosensor for rapid screening of metal ions in real samples. The biosensor demonstrated a fast response time of 20 s which is much lower than the 100 s reported in Chapter 4 for inhibitive potentiometric biosensor for trace metals. Chapter 5 explores the co-entrapment of mediators with GOx into PPy films by a single step electroplymerization process for improvement of amperometric glucose response and metal detection described in Chapter 4. The five mediators considered were potassium hexacynoferrocynide (K3[Fe(CN)6]), methylene blue (MB), bromophenol blue (BB), methyl viologen (MV) and gold nanoparticles (AuNPs). The co-entrapment of these mediators with GOx for amperometric detection of glucose resulted in considerable improvement in sensitivity, response time, linear concentration range and Nernstian behaviour beyond those achieved in Chapter 4. The best improvement in glucose amperometric response was obtained with a PPy-GOx-[Fe(CN)6]4- electrode, requiring only the addition of 1 mM of [Fe(CN)6]4- into a monomer solution which contained 0.15 M Py and 150 U mL-1 GOx. The biosensor gave a fast response time of 3 s and has a very wide linear range from 1.2-50 mM with a minimum detectable concentration of 0.25 mM, detection limit (17 µM) and a sensitivity of 7.1 µA cm-2 mM-1. The resulting electron mediated biosensor from Chapter 5 was applied for inhibitive amperometric detection of Hg2+, Cu2+, Cd2+ and Pb2+ ions based on the suppression of the glucose response of the PPy-GOx-[Fe(CN)6]4- biosensor.  (...)