Capillary electrophoresis with boron doped diamond electrode for amperometric detection of low molecular weight biological substrate

The research work in this thesis included the sensitive and selective separation of biological substance by capillary electrophoresis with a boron doped diamond electrode for amperometric detection. Chapter 1 introduced the capillary electrophoresis and electrochemical detection. It included the different modes of capillary electrophoresis, polyelectrolyte multilayers coating for open tubular capillary electrochromatography, different modes of electrochemical detection and carbon based electrodes. Chapter 2 showed the synthesized and electropolymerized N-acetyltyramine with a negatively charged sulfobutylether-β-cyclodextrin on a boron doped diamond (BDD) electrode followed by the electropolymerzation of pyrrole to form a stable and permselective film for selective dopamine detection. For comparison, a glassy carbon (GC) electrode with a combined electropolymerized permselective film of polytyramine and polypyrrole-1-propionic acid was used for selective detection of dopamine. The detection limit of dopamine was improved from 100 nM at a GC electrode to 5 nM at a BDD electrode. Chapter 3 showed field-amplified sample stacking using a fused silica capillary coated with gold nanoparticles embedded in poly(diallyldimethylammonium) chloride, which has been investigated for the electrophoretic separation of indoxyl sulphate, homovanillic acid and vanillylmandelic acid. The detection limit of the three analytes obtained by using a boron doped diamond electrode was around 75 nM, which was significantly below their normal physiological levels in biological fluids. This combined separation and detection scheme was applied to the direct analysis of these analytes and other interfereing chemicals including uric and ascorbic acids in urine samples without off-line sample treatment or preconcentration. Chapter 4 showed the selective detection of Pseudomonas Quinolone Signal, PQS for quorum sensing from its precursor HHQ, using a simply boron doped diamond electrode. Furthermore, by combining poly(diallyldimethylammonium) chloride modified fused silica capillary with a BDD electrode for amperometric detection, PQS was separated from HHQ and other analogues. The detection limit of PQS was as low as 65 nM. Different P. aeruginosa mutant strains were studied. Chapter 5 showed the separation of aminothiols by layer-by-layer coating of silica capillary with a boron doped diamond electrode. The capillary was layer-by-layer coated with the polycation poly(diallyldimethylammonium) chloride and negatively charged silica nanoparticles. All the aminothiols was separated and detected using a BDD electrode in an acidic electrolyte. It was a novel scheme for the separation and detection of glutathione reduced and oxidized forms, which is important for estimated overstressed level in the human system.

Biosensors using photonic crystal fibres

In this thesis, we present the unique properties of hollow-core photonic crystal fibres (HC-PCFs) for sensing applications in terms of viscosity detection and DNA sensing using a special poly(ethylene) glycol (PEGDA) hydrogel. The low loss HC-PCFs ensure a long interaction length between the sample and the optical signals. Thus in this thesis, we report the characterisation of filled HC-PCFs and the development of a selective filling process. For the first time, we report the investigation of a new viscometer device, and a new device for DNA sensing development, and also the chemical process for hydrogel growth was adapted to the fibres. By combining HC-PCFs with the hydrogel we enable 3D volumetric sample confinement within the HC-PCF, further increasing the interaction between the sample and the optical signal. However, the hydrogel has a large influence on the guidance properties of the HC-PCF and the HC-PCF has a strong influence on the growth process for the hydrogel itself. When we integrate the hydrogel and HC-PCFs we detect concentration levels as low as 400 nM of labelled DNA. However, using our technology for fluorescence detection we can achieve results two orders of magnitude better than those previously reported.

Design, fabrication and characterization of resonant waveguide grating based optical biosensors

The absence of rapid, low cost and highly sensitive biodetection platform has hindered the implementation of next generation cheap and early stage clinical or home based point-of-care diagnostics. Label-free optical biosensing with high sensitivity, throughput, compactness, and low cost, plays an important role to resolve these diagnostic challenges and pushes the detection limit down to single molecule. Optical nanostructures, specifically the resonant waveguide grating (RWG) and nano-ribbon cavity based biodetection are promising in this context. The main element of this dissertation is design, fabrication and characterization of RWG sensors for different spectral regions (e.g. visible, near infrared) for use in label-free optical biosensing and also to explore different RWG parameters to maximize sensitivity and increase detection accuracy. Design and fabrication of the waveguide embedded resonant nano-cavity are also studied. Multi-parametric analyses were done using customized optical simulator to understand the operational principle of these sensors and more important the relationship between the physical design parameters and sensor sensitivities. Silicon nitride (SixNy) is a useful waveguide material because of its wide transparency across the whole infrared, visible and part of UV spectrum, and comparatively higher refractive index than glass substrate. SixNy based RWGs on glass substrate are designed and fabricated applying both electron beam lithography and low cost nano-imprint lithography techniques. A Chromium hard mask aided nano-fabrication technique is developed for making very high aspect ratio optical nano-structure on glass substrate. An aspect ratio of 10 for very narrow (~60 nm wide) grating lines is achieved which is the highest presented so far. The fabricated RWG sensors are characterized for both bulk (183.3 nm/RIU) and surface sensitivity (0.21nm/nm-layer), and then used for successful detection of Immunoglobulin-G (IgG) antibodies and antigen (~1μg/ml) both in buffer and serum. Widely used optical biosensors like surface plasmon resonance and optical microcavities are limited in the separation of bulk response from the surface binding events which is crucial for ultralow biosensing application with thermal or other perturbations. A RWG based dual resonance approach is proposed and verified by controlled experiments for separating the response of bulk and surface sensitivity. The dual resonance approach gives sensitivity ratio of 9.4 whereas the competitive polarization based approach can offer only 2.5. The improved performance of the dual resonance approach would help reducing probability of false reading in precise bio-assay experiments where thermal variations are probable like portable diagnostics.

Continuous non-destructive monitoring of cell health using impedance based interdigitated electrode structured sensors

This article examines some preliminary tests which were performed in order to evaluate the best electrode configuration (width and spacing) for cell culture analyses. Biochips packaged with indium tin oxide (ITO) interdigitated electrodes (IDEs) were used to perform impedance measurements on A549 cells cultured on the surface of the biochip. Several tests were carried out using a 10 mM solution of Sodium Chloride (NaCl), cell medium and the cell culture itself to characterize some of the configurations already fabricated in the facilities at Tyndall National Institute.

Nanofabrication towards biophotonics

This thesis explores methods for fabrication of nanohole arrays, and their integration into a benchtop system for use as sensors or anti-counterfeit labels. Chapter 1 gives an introduction to plasmonics and more specifically nanohole arrays and how they have potential as label free sensors compared to the current biosensors on the market. Various fabrication methods are explored, including Focused Ion Beam, Electron Beam Lithography, Nanoimprint lithography, Template stripping and Phase Shift Lithography. Focused Ion Beam was chosen to fabricate the nanohole arrays due to its suitability for rapid prototyping and it’s relatively low cost. In chapter 2 the fabrication of nanohole arrays using FIB is described, and the samples characterised. The fabricated nanohole arrays are tested as bulk refractive index sensors, before a bioassay using whole molecule human IgG antibodies and antigen is developed and performed on the senor. In chapter 3 the fabricated sensors are integrated into a custom built system, capable of real time, multiplexed detection of biomolecules. Here, scFv antibodies of two biomolecules relevant to the detection of pancreatic cancer (C1q and C3) are attached to the nanohole arrays, and detection of their complementary proteins is demonstrated both in buffer (10 nM detection of C1q Ag) and human serum. Chapter 4 explores arrays of anisotropic (elliptical) nanoholes and shows how the shape anisotropy induces polarisation sensitive transmission spectra, in both simulations and fabricated arrays. The potential use of such samples as visible and NIR tag for anti-counterfeiting applications is demonstrated. Finally, chapter 5 gives a summary of the work completed and discusses potential future work in this area.

Development and optimisation of photonic crystal based nanosensors

This thesis involved the development of two Biosensors and their associated assays for the detection of diseases, namely IBR and BVD for veterinary use and C1q protein as a biomarker to pancreatic cancer for medical application, using Surface Plasmon Resonance (SPR) and nanoplasmonics. SPR techniques have been used by a number of groups, both in research [1-3] and commercially [4, 5] , as a diagnostic tool for the detection of various biomolecules, especially antibodies [6-8]. The biosensor market is an ever expanding field, with new technology and new companies rapidly emerging on the market, for both human [8] and veterinary applications [9, 10]. In Chapter 2, we discuss the development of a simultaneous IBR and BVD virus assay for the detection of antibodies in bovine serum on an SPR-2 platform. Pancreatic cancer is the most lethal cancer by organ site, partially due to the lack of a reliable molecular signature for diagnostic testing. C1q protein has been recently proposed as a biomarker within a panel for the detection of pancreatic cancer. The third chapter discusses the fabrication, assays and characterisation of nanoplasmonic arrays. We will talk about developing C1q scFv antibody assays, clone screening of the antibodies and subsequently moving the assays onto the nanoplasmonic array platform for static assays, as well as a custom hybrid benchtop system as a diagnostic method for the detection of pancreatic cancer. Finally, in chapter 4, we move on to Guided Mode Resonance (GMR) sensors, as a low-cost option for potential use in Point-of Care diagnostics. C1q and BVD assays used in the prior formats are transferred to this platform, to ascertain its usability as a cost effective, reliable sensor for diagnostic testing. We discuss the fabrication, characterisation and assay development, as well as their use in the benchtop hybrid system.