Development of a screen-printed cholesterol biosensor: Comparing the performance of gold and platinum as the working electrode material and fabrication using a self-assembly approach

Gold (Au) and platinum (Pt) were used as the working electrode material to detect cholesterol in solution through enzymatically generated hydrogen peroxide (H₂O₂). Both gold and platinum were capable of detecting cholesterol through the electrochemical oxidation of H₂O₂, and could be used as the working electrode material. By comparison, however, Au was preferable over Pt in terms of higher response current and better sensitivity. Therefore, Au was chosen as the working electrode material for the fabrication of a thick-film screen-printed cholesterol biosensor consisting of three electrodes on an alumina substrate (working: Au, reference: Ag/AgCl, and counter: Au). The immobilization of the enzyme cholesterol oxidase (ChOx, E.C. 1.1.3.6) on the Au working electrode was achieved using a self-assembly approach. A thiol, 3-mercaptopropionic acid (MPA), was self-assembled onto the gold working electrode forming a thin organic layer that served as the anchor for the enzyme immobilization. 1-Ethyl-3(3-dimethylamino propyl)carbodiimide methiodide (EDC) was then used to immobilize the enzyme ChOx covalently on the gold working electrode through the carbodiimide coupling between the carboxyl (–COOH) groups of the self-assembled MPA layer and the amino (–NH₂) groups of the enzyme. Electrochemical measurements showed that this biosensor responded well to cholesterol, confirming that the self-assembly immobilization method was effective. The reproducibility, the interference, and the storage stability of the biosensor were studied and assessed.     

Proteins from extremophiles as stable tools for advanced biotechnological applications of high social interest

Extremophiles are micro-organisms adapted to survive in ecological niches defined as 'extreme' for humans and characterized by the presence of adverse environmental conditions, such as high or low temperatures, extreme values of pH, high salt concentrations or high pressure. Biomolecules isolated from extremophiles possess extraordinary properties and, in particular, proteins isolated from extremophiles represent unique biomolecules that function under severe conditions, comparable to those prevailing in various industrial processes. In this article, we will review some examples of recent applications of thermophilic proteins for the development of a new class of fluorescence non-consuming substrate biosensors for monitoring the levels of two analytes of high social interest, such as glucose and sodium.     

Self-powered photoelectrochemical biosensing platform based on Au NPs@ZnO nanorods array

Photoanodes, which are used in photoelectrochemical (PEC) water splitting, have been shown to be applicable in the construction of a PEC biosensing platform. This was realized by replacing water oxidization with oxidation of an appropriate test molecule. Here, we have demonstrated the feasibility of adopting photoanodes consisting of zinc oxide nanorods arrays decorated with plasmonic gold nanoparticles (Au NPs@ZnO NRs) for the self-powered PEC bioanalysis of glutathione (GSH) in phosphate-buffered saline (PBS) at an applied bias potential of 0 V vs. Ag/AgCl. This heterostructure exhibited enhanced PEC properties because of the introduction of the Au/ZnO interface. Under visible light illumination, hot electrons from surface-plasmon resonance (SPR) at the Au NP surface were injected into the adjacent ZnO and subsequently driven to the photocathode. Under ultraviolet (UV) light illumination, the photogenerated electrons in ZnO tended to transfer to the fluorine-doped tin oxide due to the step-wise energy band structure and the upward energy band bending at the ZnO/ electrolyte interface. These results indicate that plasmonic metal/semiconductor heterostructure photoanodes have great potential for self-powered PEC bioanalysis applications and extended field of other photovoltaic beacons.

Open image in new window

     

A novel microbial BOD biosensor developed by the immobilization of P. Syringae in micro‐cellular polymers

BACKGROUND: A biochemical oxygen demand (BOD) sensor, based on an immobilized Pseudomonas syringae in highly porous micro‐cellular polymer (MCP) in combination with a dissolved oxygen electrode, has been developed for the analysis of biodegradable organic compounds in aqueous samples. Microorganisms were immobilized in a molded MCP disk and a wastewater sample was injected into the biocomposite disk by a flow injection system. Dissolved oxygen (DO) changes as a measure of soluble BOD was read with a DO probe placed into a flow cell carrying biocatalytically activated disk. RESULTS: Optimal response of the MCP BOD sensor was obtained at pH 6.8 and 25 °C with a typical response time of 3–5 min for a 2 mm thick molded polymeric disk. The sensor showed detection linearity over the range 5–100 mg L^?1 BOD₅ (r² > 0.99) at a flow rate of 0.6 mL min^?1. The repeatability and reproducibility of the sensor response were found to be 3.08% and 7.77%, respectively. BOD values produced with this biosensor for various municipal and industrial wastewaters correlated well with those determined by the conventional 5‐day BOD test. CONCLUSION: This new biosensor was different from present amperometric BOD biosensor configurations in which the biocatalyst (microbial/enzymatic) is placed between cellulose and Teflon membranes installed on a DO probe. The use of a molded MCP disk coniainng microbial activity offers better stability and lifetime for commercial use in environmental monitoring. Copyright © 2008 Society of Chemical Industry     

Grating coupled optical waveguide interferometer for label-free biosensing

A phase shifting optical waveguide interferometer is demonstrated for label-free evanescent wave biosensing in aqueous solutions. In the proposed configuration, the reference and measurement arms of the interferometer are combined inside an integrated optical Ta₂O₅ waveguide using an ion implanted grating. Biomolecules adsorbing on the waveguide surface shift the phase in the measurement arm, which is modulated at the same time by a periodically relaxing liquid crystal phase modulator. It is demonstrated that by analyzing the periodic intensity response at the end facet of the waveguide, the phase shifts in the measurement arm can be monitored in real-time with a precision of 10⁻⁴ rad. The high phase resolution allows the detection of surface adsorbed molecule densities below 1 pg/mm² without using any labeling or on-chip referencing. The instrument performance is demonstrated by monitoring in situ protein adsorption and affinity binding of low molecular weight pure biotin to an immobilized avidin layer.     

Nanocrystalline-diamond thin films with high pH and penicillin sensitivity prepared on a capacitive Si-SiO2 structure

A capacitive field-effect EDIS (electrolyte-diamond-insulator-semiconductor) sensor with improved pH and penicillin sensitivity has been realised using a nanocrystalline-diamond (NCD) film as sensitive gate material. The NCD growth process on SiO₂ as well as an additional surface treatment in oxidising medium have been optimised to provide high pH-sensitive, non-porous O-terminated films without damage of the underlying SiO₂ layer. The surface morphology of O-terminated NCD thin films and the layer structure of EDIS sensors have been studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) methods. To establish the relative coverage of the surface functional groups generated by the oxidation of NCD surfaces, X-ray photoelectron spectroscopy analysis was carried out. The hydrophilicity of NCD thin films has been studied by water contact-angle measurements. A nearly Nernstian pH sensitivity of 54-57 mV/pH has been observed for O-terminated NCD films treated in an oxidising boiling mixture for 80 min and in oxygen plasma. The high pH-sensitive properties of O-terminated NCD have been used to develop an EDIS-based penicillin biosensor. A freshly prepared penicillin biosensor possesses a high sensitivity of 85 mV/decade in the concentration range of 0.1-2.5 mM penicillin G. The lower detection limit is 5 μM.     

Quick and reliable estimation of BOD load of beverage industrial wastewater by developing BOD biosensor

An amperometric biosensor for determination of biochemical oxygen demand in wastewater has been developed to overcome the time consuming monitoring procedures. The performance and stability of the immobilized membrane have been investigated at 37 °C and pH 6.8. Immobilized microbial membranes maintain their stability and activity after intermittent use for 400 cycles when stored at 4 °C in sodium phosphate buffer pH 6.8. The response time of the BOD sensor was only 90 min, being independent of the concentration, and the lower detection limit was 1 mg/l. The obtained BOD values showed correlation with that of the conventional method for BOD determination (BOD5) with a deviation of ±10%. Moreover, the sensor exhibits good repeatability (3.39–4.45%) and reproducibility (1.85–2.25%). Software was added to upgrade this sensor and to make it a promising candidate for online monitoring.     

A novel hydrogen peroxide biosensor based on Au-graphene-HRP-chitosan biocomposites

Graphene was prepared successfully by introducing -SO₃⁻ to separate the individual sheets. TEM, EDS and Raman spectroscopy were utilized to characterize the morphology and composition of graphene oxide and graphene. To construct the H₂O₂ biosensor, graphene and horseradish peroxidase (HRP) were co-immobilized into biocompatible polymer chitosan (CS), then a glassy carbon electrode (GCE) was modified by the biocomposite, followed by electrodeposition of Au nanoparticles on the surface to fabricate Au/graphene/HRP/CS/GCE. Cyclic voltammetry demonstrated that the direct electron transfer of HRP was realized, and the biosensor had an excellent performance in terms of electrocatalytic reduction towards H₂O₂. The biosensor showed high sensitivity and fast response upon the addition of H₂O₂, under the conditions of pH 6.5, potential −0.3 V. The time to reach the stable-state current was less than 3 s, and the linear range to H₂O₂ was from 5 × 10⁻⁶ M to 5.13 × 10⁻³ M with a detection limit of 1.7 × 10⁻⁶ M (S/N = 3). Moreover, the biosensor exhibited good reproducibility and long-term stability.     

Measurement of hydrogen peroxide in an advanced oxidation process using an automated biosensor

A hydrogen peroxide biosensor was used to monitor hydrogen peroxide concentrations in a UV/hydrogen peroxide immobilised Fenton advanced oxidation process (AOP). The biosensor is based on gas phase monitoring and thus is more resistant to fouling from the liquid phase constituents of industrial processes. The biosensor is supplied with catalase continually, therefore overcoming any problems with enzyme degradation, which would occur in an immobilised enzyme biosensor. The biosensors response was linear within the experimental range 30-400mg H(2)O(2)l(-1) with a R(2) correlation of 0.99. The hydrogen peroxide monitor was used to monitor residual peroxide in an AOP, operated with a step overload of hydrogen peroxide, with correlation factors of 0.96-0.99 compared to offline hydrogen peroxide determinations by UV spectroscopy. Sparging the sample with nitrogen was found to be effective in reducing the interference from dissolved gases produced with the AOP itself. It is proposed that this biosensor could be used to improve the effectiveness of AOPs via hydrogen peroxide control.