Fabricating an Amperometric Cholesterol Biosensor by a Covalent Linkage between Poly(3-thiopheneacetic acid) and Cholesterol Oxidase

In this study, use of the covalent enzyme immobilization method was proposed to attach cholesterol oxidase (ChO) on a conducting polymer, poly(3-thiopheneacetic acid), [poly(3-TPAA)]. Three red-orange poly(3-TPAA) films, named electrodes A, B and C, were electropolymerized on a platinum electrode by applying a constant current of 1.5 mA, for 5, 20 and 100 s, respectively. Further, 1-ethyl-3-(3-dimethylamiopropyl)carbodiimide hydrochloride (EDC · HCl) and N-hydroxysuccinimide (NHS) were used to activate the free carboxylic groups of the conducting polymer. Afterwards, the amino groups of the cholesterol oxidase were linked on the activated groups to form peptide bonds. The best sensitivity obtained for electrode B is 4.49 mA M−1 cm−2, with a linear concentration ranging from 0 to 8 mM, which is suitable for the analysis of cholesterol in humans. The response time (t95) is between 70 and 90 s and the limit of detection is 0.42 mM, based on the signal to noise ratio equal to 3. The interference of species such as ascorbic acid and uric acid increased to 5.2 and 10.3% of the original current response, respectively, based on the current response of cholesterol (100%). With respect to the long-term stability, the sensing response retains 88% of the original current after 13 days

Cholesterol biosensing with a polydopamine-modified nanostructured platinum electrode prepared by oblique angle physical vacuum deposition

This paper reports a novel cholesterol biosensor based on nanostructured platinum (Pt) thin films prepared by Magnetron Sputtering (MS) in an oblique angle (OAD) configuration. Pt thin films were deposited onto a gold screen-printed electrode and characterized using Rutherford Back Scattering (RBS), Scanning Electron Microscopy (SEM), X-ray Diffraction (XRD), Cyclic Voltammetry (CV), X-ray Photo-electron Spectroscopy (XPS), Atomic Force Microscopy (AFM) and wetting analysis. Our results confirmed that the film is highly porous and formed by tilted nanocolumns, with an inclination of around 40° and a total thickness of 280 nm. XRD and CV analysis confirmed the polycrystalline nature of the Pt thin film. Cholesterol oxidase (ChOx) was covalently immobilized using a bioinspired polymer, polydopamine (PDA), via Schiff base formation and Michael-type addition. After being immobilized, ChOx displayed apparent activation energy of 34.09 kJ mol−1 and Michaelis constant (KM) values of 34.09 kJ mol−1 and 3.65 mM, respectively, confirming the high affinity between ChOx and cholesterol and the excellent ability of the PDA film for immobilizing biological material without degradation. Under optimized working conditions the developed biosensor presented a sensitivity of 14.3 mA M−1cm−2 (R2:0.999) with a linear range up to 0.5 mM and a limit of detection of 10.5 μM (S/N = 3). Furthermore, the biosensor exhibited a fast response (<8 s), good anti-interference properties and high stability after relatively long-term storage (2 months).Ministerio de Economía y Competitividad IPT-2012-0961-300000, MAT2013-40852-

Fabrication and Optimization of ChE/ChO/HRP-AuNPs/c-MWCNTs Based Silver Electrode for Determining Total Cholesterol in Serum

The developed method used three enzymes comprised of cholesterol esterase, cholesterol oxidase, and peroxidase for fabrication of amperometric biosensor in order to determine total cholesterol in serum samples. Gold nanoparticles (AuNPs) and carboxylated multiwall carbon nanotubes (cMWCNTs) were used to design core of working electrode, having covalently immobilized ChO, ChE, and HRP. Polyacrylamide layer was finally coated on working electrode in order to prevent enzyme leaching. Chemically synthesised Au nanoparticles were subjected to transmission electron microscopy (TEM) for analysing the shape and size of the particles. Working electrode was subjected to FTIR and XRD. The combined action of AuNP and c-MWCNT showed enhancement in electrocatalytic activity at a very low potential of 0.27 V. The pH 7, temperature 40°C, and response time of 20 seconds, respectively, were observed. The biosensor shows a broad linear range from 0.5 mg/dL to 250 mg/dL (0.01 mM–5.83 mM) with minimum detection limit being 0.5 mg/dL (0.01 mM). The biosensor showed reusability of more than 45 times and was stable for 60 days. The biosensor was successfully tested for determining total cholesterol in serum samples amperometrically with no significant interference by serum components

Quantum dot encoded magnetic beads for multiplexed fluorescence biosensing

In recent years, the use of encoded beads has received considerable attention due to their potential for measuring multiple analytes in solution.(1-4) This can be achieved without the need for knowledge of their spatial position, as in the case of microarray technology. Encoded bead technology also relies on the solution kinetics rather than diffusion to a fixed surface as in the case of microarray technology, offering the possibility of developing rapid high throughput screening methods. This thesis describes the production, characterisation and application of quantum dot encoded beads prepared using layer-by-layer assembly of different colour quantum dots around a magnetic bead. To achieve this, two different strategies were used to make “coloured” barcodes. The first strategy used thiol chemistry to immobilise quantum dots in a layer-by-layer assembly onto magnetic beads whereas the second strategy uses the interaction between quantum dot-biotin and quantum dot-streptavidin conjugates to create constructs on the magnetic bead surface. The development of both of these immobilisation strategies was characterisation using X-ray photoelectron spectroscopy and fluorescence spectroscopy of immobilised quantum dot structures onto a plain glass substrate. After the preparation of encoded beads, they were characterised using single bead fluorescence spectroscopy. It was found that attempts to prepare barcodes by layer-by-layer assembly of CdSe/ZnS quantum dots using thiol chemistry onto magnetic beads did not comply with the necessary barcode characteristics i.e., different colour coded beads could not be distinguished from each other. However, the encoded beads prepared using layer-by-layer assembly of quantum dot-biotin and quantum dot-streptavidin conjugates onto streptavidin coated magnetic beads gave distinct multicolour coded bead spectra. These barcodes were characterised in terms of different spectral responses, stability at raised temperatures, stability in biotin solutions, and long-term stability after storage. Encoded beads prepared using layer-by-layer assembly of quantum dot-biotin and quantum dot-streptavidin conjugates onto streptavidin coated magnetic beads were then used to develop multiplexed immunoassays. Four different barcodes were prepared and used to perform model multiplexed immunoassays. The barcodes were identified upon the basis of different spectral response measured using single bead fluorescence spectroscopy. Finally, a quantitative immunoassay for human IgG was performed using these barcodes, which showed that different concentrations of human IgG can be determined in solution

Carbon Nanotubes for Electronics and Energy

Ever since their discovery, carbon nanotubes have been touted as a new material for the future and a correspondingly lengthy list of possible applications are often cited in the literature. This excitement for carbon nanotubes is a result of their richly varying physical, electronic and optical properties, where it is possible to have single, double and multiple carbon walls with each wall potentially being either semiconducting or metallic and possessing unique optical transitions covering the ultraviolet to infrared spectral range. However, to date the realization of many of the proposed applications has been hindered by exactly the characteristic that made carbon nanotubes so attractive in the first place, namely the inherent inhomogeneity and varying properties of as-prepared or grown material. In order to become a true advanced material of the future, methods to prepare carbon nanotubes with defined length, wall number, diameter, electronic and optical property are necessary. Additionally, such methods to sort carbon nanotubes must afford high purity levels, be amenable to large-scale preparation and be compatible with subsequent integration into device architectures. In this work these issues are addressed with the use of gel based sorting techniques, which with the use of an automated gel permeation system allows for the routine preparation of milligram quantities of metallic and semiconducting carbon nanotubes, chirality pure single walled carbon nanotubes and even double walled carbon nanotubes sorted by their outer-wall electronic type. Having developed techniques to prepare large quantities, methodologies to control the order and orientation of this 1 D nanomaterial on the macro scale are developed. Inks of carbon nanotubes with liquid crystal concentrations and aligned films thereof are developed and this newfound control over the electronic and structural property opened the door for energy related applications. For example the use of thin films as the transparent electrodes in silicon:carbon nanotube solar cells or as the light harvesting layer in combination with fullerenes with the goal of creating an all carbon solar cell. Likewise on the few nanotube level the unique optical transitions of different nanotube chiralities are used in the fabrication of nanoscale photosensitive elements