NANOMATERIALS MODIFIED PAPER BASED BIOSENSORS FOR CANCER DETECTION

Abstract

Nanostructured materials have recently aroused much interest as immobilization matrices for biosensor applications. This is because these materials exhibit interesting properties such as a large surface-to-volume ratio, high surface reaction activity, high catalytic efficiency and strong adsorption ability that make them potential candidate materials to play a catalytic role for the fabrication of a biosensor. The nanomaterials based biosensors have been found to show improved sensitivity, detection limit and stability. Besides this, large surface area of a nanomaterial provides an improved loading of biomolecules with desired orientation. For biosensing applications, a wide range of conducting substrates such as glassy carbon, indium tin oxide (ITO), and gold coated glass substrate are currently being used. However, the rigidity, brittleness and cost limit their applications towards the development of a wearable, flexible, cost effective and disposable point-of-care device. Conducting paper based biosensors have recently been attracting considerable attention because of their light weight, flexibility, portability, high sensitivity, fast response time and disposability. Moreover, coupling of a conducting paper based device (e.g. electrodes and transistors) with biological systems provides an efficient platform for the conduction of both electronic and ionic charge carriers that play a major role for communication with a desired biomolecule. Many methods including inkjet printing, screen printing, spin coating can be utilized to modify a paper to make it conducting. These methods, however, require skilled personal, costly conducting ink (gold, silver and graphite) paste, equipments, and are time-consuming. vi Conducting polymers have been considered as a promising candidate to make paper conducting due to delocalization of π electrons since they are known to facilitate rapid electron transfer, mechanical flexibility and solution processablity. Doping of a conducting polymer has been found to enhance its electronic, optical, physical, chemical and electrochemical properties. Among the various conducting polymers, poly(3,4ethylenedioxythiophene): poly(styrenesulfonate) (PEDOT:PSS) has been considered to be a potential candidate for development of a conducting paper due to its homogeneous entrapment in/on a paper using simple dip coating method. Moreover, the conductivity of PEDOT:PSS modified paper can be significantly enhanced and tuned by treatment with a desired solvent. Further, incorporation of a nanomaterial may modulate the performance of a conducting paper in terms of electrochemical kinetics, signal stability and sensitivity. Recently, nanomaterials such as reduced graphene oxide (RGO), carbon nanotubes (CNTs), polymeric (PEDOT:PSS) electrospun nanofibers have recently aroused much interest in electrochemical sensors. This is due to their large surface area, enhanced biomolecule loading and excellent electrochemical properties. Therefore incorporation of nanomaterials in the PEDOT:PSS coated paper may further improve electrochemical performance of the desired paper sensor. Cancer is currently a serious concern and a medical threat to the contemporary world. According to a recent report, there have been 8.2 million cancer deaths, 14.1 million new cancer cases and 32.6 million people are living with cancer necessitating its early detection. Carcinoembryonic antigen (CEA) has been found to be an important biomarker for the diagnosis and routine monitoring of cancer due to its association with colon, lung, ovarian and breast cancer that are responsible for more than half of all cancer deaths each year. The human CEA gene is clustered on vii chromosome 19q and it comprises of 29 genes. CEA (a glycoprotein) comprises of ~ 60% carbohydrate having molecular mass of ~180-200 KDa, is one of the most widely used cancer biomarker. It plays an important role in early monitoring, screening and disease recurrence. In this context, determination of CEA in blood serum has been proposed for clinical diagnosis and monitoring of cancer. The aim of this research is to develop a substitute for conventional electrode (ITO, gold and glassy carbon), that have been found to have limitations in applications for fabrication of smart point-of-care devices and their application in biosensing for CEA detection. We have fabricated PEDOT:PSS based conducting paper as electrode for CEA biomarker detection. Efforts have been made to improve the conductivity of PEDOT:PSS coated paper by using different solvents like ethylene glycol and formic acid. Further, incorporation of nanomaterials such as reduced graphene oxide, carbon nanotubes, PEDOT:PSS electrospun nanofibers modulate the paper electrode performance in terms of electrochemical kinetics and biosensing characteristics. The response of the paper electrode is validated using CEA concentration of serum samples of cancer patients obtained via immunoassay technique. The thesis comprises of six Chapters as discussed below: Chapter 1 on Introduction and Literature Survey highlights the detailed description of nanomaterials based conducting paper including their fabrication strategies, characteristics and potential applications. Beside this, a brief discussion on cancer, conventional techniques used for cancer biomarker detection and scope of biosensors in cancer detection. The attempts have been made to discuss limitations of conventional electrodes used in biosensing application and give a detailed literature review on paper based electrochemical biosensors. viii Chapter 2 on Materials and characterization techniques describes the various materials used for the fabrication of paper based biosensors for CEA detection using electrochemical technique. Further, the analytical techniques such as four points probe conductivity measurement, X-Ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), energy dispersive X-Ray spectroscopy (EDX), transmission electron microscopy (TEM), and electrochemical techniques used for characterization of the conducting paper, nanomaterial modified conducting paper and immuno-electrodes have been discussed in details. Attempts have also been made to describe the procedures and protocols used to immobilize antibodies and to estimate the various parameters related to the characteristics of paper based biosensors. Chapter 3 on Reduced graphene oxide modified conducting paper sensor for cancer detection deals with results of the studies relating to the fabrication of a paper based sensor comprising of poly (3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) and reduced graphene oxide (RGO) nanocomposite. The effect of various solvents like methanol, ethylene glycol and H2SO4 on the electrical conductivity of PEDOT:PSS coated Whatman paper has been investigated. The conductivity of this solution processed conducting paper significantly is found to increase by 2 orders of magnitude on being treated with ethylene glycol. Further, incorporation of RGO into the solution processed conducting paper results in improved electrochemical performance and signal stability. The fabricated electrodes have been used for the detection of the cancer biomarker, CEA. This low cost, flexible and environment friendly conducting paper based biosensor has been utilized for cancer biomarker (CEA) detection, that reveals high sensitivity of 25.8 μAng-1mLcm-2 in the linear detection range of 2-8 ngmL-1 with a good storage ix stability (21 days). The response of paper electrode has been validated using CEA concentration of serum sample of cancer patient. This paper electrode can be decomposed by simple incineration and has immense potential as a smart medical diagnostic kit or a point-of-care biosensor. In order to further improve the performance of conducting paper, formic acid and CNT have been used as a suitable solvent and dopant, respectively. Compared to RGO, metallic impurities present in CNT have been found to improve the electrochemistry of CNT. Chapter 4 on Carbon nanotube modified conducting paper sensor for cancer detection used nanocomposite of PEDOT:PSS and carbon nanotubes (CNT) to fabricate conducting paper (CNT/CP) via dip coating. It is found that conductivity of this paper increases by 2 orders of magnitude on being treated with formic acid (CNT/FA@CP) due to removal of the non-conducting molecule PSS from electrode surface. This fabricated paper is flexible, electrochemical active, and it can be easily disposed off by simple incineration. This smart conducting platform has been used for conjugation of the anti-carcinoembronic antigen (anti-CEA) protein for quantitative estimation of CEA. The PEDOT:PSS-CNT based electrochemical paper immunosensor exhibits sensitivity of 7.8 μAng-1mLcm-2 with improved linear detection range of 2-15 ngmL-1 and feasibility of paper electrode have been validated with CEA concentration in serum samples of cancer patient. It has been observed that incorporation of carbon nanotubes improved heterogenous electron transfer rate constant (5 times) and linear detection range with respect to PEDOT:PSS-RGO based conducting paper. CEA biomarker released in serum of healthy person is < 3 ngmL-1 with a cut-off value 5 ngmL-1 and for cancer patient it is reported to be a maximal of 20 ngmL-1. x Chapter 5 on PEDOT:PSS/PVA nanofiber decorated conducting paper sensor for cancer detection pertains to results of studies relating to the fabrication of a flexible, cost-effective, lightweight, label free and environment friendly electrochemical sensor. For this purpose, nanofibers of PEDOT:PSS/PVA have been grown on a conducting paper platform using electrospinning technique. The deposition of electrospun nanofibers (EsNf) is found to result in improved mechanical strength, large surface area, enhanced biomolecule loading and electrochemical characteristics. This platform provides improved charge transfer between electrode and solution resulting in higher sensitivity towards the electrochemical detection of cancer biomarker (CEA). The results of the amperometric response studies indicate that the paper electrode (BSA/anti-CEA/PEDO:PSS/PVA-EsNf/CP) can be used to estimate CEA in the range, 0.2 to 25 ngmL-1, has high sensitivity of 14.2 μAng-1mLcm-2 and shelf life of 22 days. This paper sensor covers the entire physiological range of CEA secreted in serum sample (< 3 to 20 ngmL-1) with improved lower detection limit. This modified conducting paper electrode is a promising alternative over expensive conventional electrodes (ITO, gold and glassy carbon) for fabrication of smart point of care devices. Chapter 6 on Summary and future prospects contains brief summary of the studies related to nanomaterial modified paper based biosensor for cancer detection. This Chapter also highlights the future prospects of nanomaterials modified conducting paper sensor for detection of other biomolecule including cancer biomarker. The work described in this thesis is summarized in the form of following publications: xi 1. S. Kumar, P. Rai, J.G. Sharma, A. Sharma and B.D. Malhotra, PEDOT:PSS/PVA-nanofibers-decorated conducting paper for cancer diagnostics, Advanced Materials Technologies, 2016. (doi: 10.1002/admt.201600056). 2. S. Kumar, M. Willander, J.G. Sharma and B.D. Malhotra, A solution processed carbon nanotube modified conducting paper sensor for cancer detection, Journal of Material Chemistry B, 2015, 3, 9305–9314. (Impact factor 4.9) 3. S. Kumar, S. Kumar, S. Srivastava, B.K. Yadav, S.H. Lee, J.G. Sharma, D.C. Doval and B.D. Malhotra, Reduced graphene oxide modified smart conducting paper for Cancer biosensor, Biosensors and Bioelectronics, 2015, 73, 114- 122. (Impact factor 7.5).