NANOMATERIAL BASED SENSORS FOR THE DETERMINATION OF BIOMOLECULES AND DRUGS

Abstract

Electrochemical investigation of biologically important compounds and drugs provide major challenges both from electro-mechanistic and analytical point of view. Electrochemistry at metal or carbon based electrode has emerged as an interesting area of analytical studies over the last few years, significantly changing the scope and sensitivity of electroanalytical methods. The field of nanoscience has blossomed over the last few decades, and the importance of nanotechnology has been increased due to the requirement of miniaturization in areas, such as computing, sensors, biomedical and many other applications. The advancements in these areas are depending largely due to the ability to synthesize nanoparticles of various materials, sizes and shapes, as well as to assemble them efficiently into complex architectures. Nanotechnology based electrochemical platform offers a promising tool for the attainment of multiple aims in biomolecular analysis. Nanomaterials prepared from metal, semiconductors and carbon or polymeric species have allured great attention due to their widespread applications in different areas of science. There has been a substantial progress in the construction of highly efficient nanomaterials based electrochemical sensors for the monitoring of biologically important molecules and pharmaceutical drugs. It is observed that the sensitive and selective detection of specific biomolecules and drugs is mandatory for elucidating the physiological processes as well as for early diagnosis and therapy of diseases. The recent upcoming of the new forms of nanomaterial/polymer composites have revolutionized the electrochemical research and brought many potential applications in nanoscience. Hybrid materials have enticed many researchers and opened a new dimension in the field of sensor fabrication due to their attractive electronic, optical, thermal and electro-catalytic properties over other conventional materials. These properties together with their nanometric size and high aspect ratio make them suitable for the electrochemical sensing of verities of organic compounds. Considering the significance of hybrid materials in the area of electrochemistry, in this thesis an attempt has been made to systematically utilize the different modification approaches employing nanomaterials, nanomaterial/polymer composites with a focus on the development of highly sensitive electrochemical sensors for the investigations of biomolecules and drugs. In a new perspective, aptamers which are small oligonucleic acids that specifically bind to the target molecules has also been explored. The advantages of aptamers are that they can be regenerated, highly stable to external factors, and do not require animal models for generation prepared through the SELEX (Systematic Evolution of Ligands by Exponential enrichment) process. The thesis is divided in six chapters. ii The first chapter of the thesis is “General Introduction” which presents a compendious review of the pertinent work and highlights the importance of electrochemical studies in biological system along with its application in diverse areas. This chapter has an overview of conventional electrodes, types of nanomaterial and modification of electrodes using nanomaterials. This chapter also deals with the illustration of the methodology employed in the present investigation comprising some theoretical aspects of voltammetric techniques. The second chapter of the thesis describes the application of gold nanoparticles decorated palladium for studying the electrochemical oxidation of dopamine which is one of the most important catecholamine that brain uses as the neurotransmitter as well as it is an important intermediate in the biosynthesis of other neurotransmitters of catecholamine family. Dopamine is also responsible for a variety of physiological functions like voluntary movement, ability to concentrate, feelings of pleasure, motivation and reward, gastrointestinal motility, pituitary hormone release, and higher cognitive processes. A stable layer of physisorbed gold nano particles at the surface of palladium has been used as a catalyst support. The modified sensor was characterized by field emission scanning electron microscopy (FE-SEM) and electrochemical impedance spectroscopy (EIS). The oxidation chemistry of dopamine has been investigated at bare and gold nanoparticle modified palladium sensor using cyclic and square wave voltammetry. The oxidation peak potential of dopamine shifted to lower values and peak current increased significantly, which is attributed to the electrocatalytic properties of nano gold modified palladium sensor. The peak potential of dopamine at pH 7.2 was 190 mV and 162 mV at bare and modified sensor respectively. The peak currents of dopamine were found to increase linearly with increase in the concentration of dopamine in the range 5–800 μM for bare and 0.5–1000 μM for nano gold modified palladium sensor respectively. The detection limit (3σ/b) and sensitivity were found to be 0.6 μM and 0.003 μA μM−1 for bare, 0.08 μM and 0.015 μA μM−1 for nano gold modified palladium respectively. The third chapter of the thesis deals with the single-walled carbon nanotube (SWCNT) embedded poly 1,5-diaminonapthalene (p-DAN) modified pyrolytic graphite sensor for the determination of sulfacetamide (SFA). SFA is a synthetic, highly potent antibacterial agent that is widely used for the treatment of numerous dermatological diseases. The surface morphology of the modified sensor has been characterized by FE-SEM, which revealed a good dispersion of the carbon nanotube in polymer matrix of 1,5-diaminonapthalene. SFA was determined using square wave voltammetry in phosphate buffer of pH 7.2, which acted as supporting electrolyte during analysis. The modified sensor has been found to an effective catalytic response towards iii the oxidation of SFA and excellent reproducibility and stability are observed. The peak current of SFA was found to be linearly dependent on the concentration of SFA in the concentration range 0.005–1.5 mM and detection limit and sensitivity of 0.11 μM (S/N3) and 23.977 μA mM1, respectively were observed. The analytical utility of the method has also been explored by determining SFA in various pharmacological dosage forms. The results obtained from the voltammetry have also been validated by comparing the results with those obtained from HPLC. The proposed method is sensitive, simple, rapid and reliable and is useful for the routine analysis of SFA in pharmaceutical laboratories. [Dopamine] [Sulfacetamide] In the fourth chapter of the thesis, the application of SWCNT for the modification of the surface of edge plane of pyrolytic graphite is presented. The electrochemical behavior of mometasone furoate (MF) has been studied at SWCNT modified pyrolytic graphite (MPG). The addition of cationic surfactant (cetyltrimethylammonium bromide, CTAB) was found to enhance the reduction current signal of MF, whereas, anionic (sodium dodecylsulfate, SDS) and non-ionic (Tween 60) surfactants exhibited opposite effect. Hence, detailed studies on the oxidation of mometasone are carried out in CTAB medium. A sensitive and selective electrochemical determination of MF by square wave voltammetry and cyclic voltammetry in phosphate buffer of pH 7.2 has been carried out in the presence of CTAB. The cathodic peak current showed a linear response for MF reduction in the concentration range 10–1000 M. The effective surface area of the modified sensor was found to be 0.225 cm2, The sensitivity and detection limit of MF were found to be 0.017 A M-1 and 1.23 M, respectively were observed. The reduction site in MF has also been established by the separation and characterization of the product of reduction by 1H NMR and FT-IR spectroscopic measurements and found to be carbonyl group at position 3. The developed method was successfully applied for the determination of MF in pharmaceutical preparations and in human urine. iv Tentative mechanism proposed for the reduction of MF The fifth chapter of the thesis illustrates incorporation of gold nanoparticles onto the p- DAN coated pyrolytic graphite for the quantification of cefpodoxime proxetil (CP), which is a semi-synthetic beta-lactum antibiotic belonging to the third generation of cephalosporin group. The modified sensor was characterized by X-ray photoelectron spectroscopy (XPS) and FESEM. The sensor exhibited an effective catalytic response towards oxidation of CP with excellent reproducibility and stability. The peak current of CP was found to be linear in the range of 0.1–12 M and detection limit and sensitivity of 39 nM (S/N3) and 4.621 A M1, respectively, were observed. The method was successfully applied for the determination of CP in pharmaceutical formulations and human urine samples. The common metabolites present in human urine such as uric acid, ascorbic acid, xanthine and hypoxanthine did not interfere in the determination. A comparison of the results obtained by using developed method with high performance liquid chromatography (HPLC) indicated a good agreement. The method is simple, sensitive, rapid and precise and is useful for the routine determination of CP in pharmaceutical dosages and biological samples. The last chapter of thesis (chapter six) describes the in vitro chloramphenicol detection in a Haemophilus influenza model using an aptamer-polymer based electrochemical biosensor. A sensitive and selective electrochemical biosensor for the determination of chloramphenicol (CAP) exploring its direct electron transfer processes in in vitro model and pharmaceutical samples. This biosensor exploits a selective binding of CAP with aptamer, immobilized onto the poly-(4-amino-3-hydroxynapthalene sulfonic acid) (p-AHNSA) modified edge plane pyrolytic graphite. The electrochemical reduction of CAP was observed in a well-defined peak. v A quartz crystal microbalance (QCM) study is performed to confirm the interaction between the polymer film and the aptamer. Cyclic voltammetry and square wave voltammetry were used to detect CAP. The in vitro CAP detection is performed using the bacterial strain of Haemophilus influenza. A significant accumulation of CAP by the drug sensitive Haemophilus influenza strain is observed for the first time in this study using a biosensor. Various parameters affecting the CAP detection in standard solution and in in vitro detection are optimized. The detection of CAP is linear in the range of 0.1–2500 nM with the detection limit and sensitivity of 0.02 nM and 0.102 A nM-1, respectively. CAP is also detected in the presence of other common antibiotics and proteins present in the real sample matrix, and negligible interference is observed. A representation of the biosensor fabrication and detection model used is presented.