Novel Electrochemical Biosensors for Clinical Assays

Biosensors, i.e., devices where biological molecules or bio(mimetic)structures are intimately coupled to a chemo/physical transducer for converting a biorecognition event into a measurable signal, have recently gained a wide (if not huge) academic and practical interest for the multitude of their applications in analysis, especially in the field of bioanalysis, medical diagnostics, and clinical assays. Indeed, thanks to their very simple use (permitting sometimes their application at home), the minimal sample pretreatment requirement, the higher selectivity, and sensitivity, biosensors are an essential tool in the detection and monitoring of a wide range of medical conditions from glycemia to Alzheimer’s disease as well as in the monitoring of drug responses. Soon, we expect that their importance and use in clinical diagnostics will expand rapidly so as to be of critical importance to public health in the coming years. This Special Issue would like to focus on recent research and development in the field of biosensors as analytical tools for clinical assays and medical diagnostics

A Crosstalk- and Interferent-Free Dual Electrode Amperometric Biosensor for the Simultaneous Determination of Choline and Phosphocholine

Choline (Ch) and phosphocholine (PCh) levels in tissues are associated to tissue growth and so to carcinogenesis. Till now, only highly sophisticated and expensive techniques like those based on NMR spectroscopy or GC/LC- high resolution mass spectrometry permitted Ch and PCh analysis but very few of them were capable of a simultaneous determination of these analytes. Thus, a never reported before amperometric biosensor for PCh analysis based on choline oxidase and alkaline phosphatase co-immobilized onto a Pt electrode by co-crosslinking has been developed. Coupling the developed biosensor with a parallel sensor but specific to Ch, a crosstalk-free dual electrode biosensor was also developed, permitting the simultaneous determination of Ch and PCh in flow injection analysis. This novel sensing device performed remarkably in terms of sensitivity, linear range, and limit of detection so to exceed in most cases the more complex analytical instrumentations. Further, electrode modification by overoxidized polypyrrole permitted the development of a fouling- and interferent-free dual electrode biosensor which appeared promising for the simultaneous determination of Ch and PCh in a real sample

Biomedical application of immobilized enzymes

Reports on chemical immobilization of proteins and enzymes first appeared in the 1960s. Since then, immobilized proteins and enzymes have been widely used in the processing of variety of products and increasingly used in the field of medicine. Here, we present a review of recent developments in immobilized enzyme use in medicine. Generally speaking, the use of immobilized enzyme in medicine can be divided into two major categories: biosensors and bioreactors. A brief overview of the evolution of the biosensor and bioreactor technology, of currently existing applications of immobilized enzymes, of problems that researchers encountered, and of possible future developments will be presented. © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 89: 979–990, 2000Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34503/1/2_ftp.pd

Electrochemical Sensors Based on Carbon Nanotubes

This review focuses on recent contributions in the development of the electrochemical sensors based on carbon nanotubes (CNTs). CNTs have unique mechanical and electronic properties, combined with chemical stability, and behave electrically as a metal or semiconductor, depending on their structure. For sensing applications, CNTs have many advantages such as small size with larger surface area, excellent electron transfer promoting ability when used as electrodes modifier in electrochemical reactions, and easy protein immobilization with retention of its activity for potential biosensors. CNTs play an important role in the performance of electrochemical biosensors, immunosensors, and DNA biosensors. Various methods have been developed for the design of sensors using CNTs in recent years. Herein we summarize the applications of CNTs in the construction of electrochemical sensors and biosensors along with other nanomaterials and conducting polymers

Elektrochemické biosenzory s prostorově oddělenou enzymatickou a detekční části pro selektivní analýzu v průtokovém uspořádání

This dissertation thesis presents the newly developed four highly reusable, stable as well as simple, and cost-effective electrochemical (bi)enzymatic biosensors for the selective and reliable determination of choline, acetylcholine, uric acid, and L-lactic acid in flow injection analysis. All biosensors are based on the concept of the spatial separation of the biorecognition part from detection one and amperometric monitoring of the enzymatically consumed oxygen via its four-electron reduction at the highly negative detection potential. In this way, the design of the biosensors includes an easily replaceable enzymatic mini-reactor(s) connected upstream to the flow cell that contains the appropriate silver amalgam-based transducer. The enzymatic mini-reactor based on choline oxidase, uricase, or lactate oxidase was used for choline, uric acid, or L-lactic acid biosensors, respectively. The acetylcholine bienzymatic biosensor includes the consequently connected choline oxidase- and acetylcholinesterase-based mini-reactors. The first part of this thesis focuses on the construction of two different silver amalgam-based electrodes. Specifically, this section discusses the fabrication of a silver solid amalgam electrode covered by mercury film operating in a wall-jet cell and also highlights the...Předpokládaná disertační práce pojednává o čtyřech nově vyvinutých, opakovaně použitelných, stabilních a zároveň jednoduchých a cenově výhodných elektrochemických (bi)enzymatických biosenzorech pro selektivní a spolehlivé stanovení cholinu, acetylcholinu, močové kyseliny a L-mléčné kyseliny. Všechny biosenzory jsou založeny na koncepci prostorového oddělení biorozpoznávací části od detekční a amperometrického monitorování enzymaticky spotřebovaného kyslíku prostřednictvím jeho čtyřelektronové redukce při vysoce záporném detekčním potenciálu. Konstrukce biosenzorů zahrnuje snadno vyměnitelný enzymatický/é mini-reaktor(y) připojený/é před průtokovou celou, která obsahuje příslušný převodník na bázi pevného stříbrného amalgámu. Enzymatické mini-reaktory založené na cholin oxidase, urikase nebo laktát oxidase byly použity pro biosenzory na cholin, močovou kyselinu nebo L-mléčnou kyselinu. Bienzymatický biosenzor na acetylcholin zahrnuje sériově propojené mini-reaktory na bázi acetylcholinesterasy a cholin oxidasy. První část této práce se zaměřuje na konstrukci dvou různých elektrod na bázi stříbrného amalgámu. Konkrétně tato část pojednává o výrobě stříbrné pevné amalgámové elektrody pokryté rtuťovým filmem pro wall-jet průtokovou celu a navíc, zdůrazňuje rozdíly mezi jejím využitím jako...Department of Analytical ChemistryKatedra analytické chemiePřírodovědecká fakultaFaculty of Scienc

Development of non-esterified fatty acid (NEFA) electrochemical biosensor for energy metabolism studies

PhD ThesisThere are many energy metabolism studies ongoing, including those for cardiovascular diseases and type-2-diabetes. With an increase in people being diagnosed with type-2-diabetes, there should be more ways to monitor not only the blood glucose levels but also the other biomarkers associated with type-2-diabetes. The metabolism biomarkers are essential in understanding the cause of diabetes early on. These biomarkers include: glucose, non-esterified fatty acid, lactate, urea, creatinine, glycosylated haemoglobin and cholesterol. Whilst glucose measurement has a clear role in type-2-diabetes management, the potential value of non-esterified fatty acid has not been explored or highlighted yet. The aim of this project is to develop an electrochemical biosensor for the non-esterified fatty acid in human blood, as non-esterified fatty acid can cause -cell loss in type-2-diabetes. Exploration of this biomarker would be a step forward in increasing research and patient understanding of the dynamic processes involved in establishing good metabolism control. The project uses the enzymes in commercial optical methods for non-esterified fatty acid detection. Oleic acid was used as the standard non-esterified fatty acid in this work. The electrochemical techniques employed are cyclic voltammetry, linear sweep voltammetry, chronoamperometry and electrochemical impedance spectroscopy. Enzyme electrodes were fabricated using the layer-by-layer immobilization of alternating polymer and enzyme combinations on carbon, cobalt phthalocyanine and single wall carbon nanotube screen printed electrodes. A chronoamperometric non-esterified fatty acid sensor was developed with the linear detection range of 0.10 mM to 0.90 mM oleic acid and with a sensitivity of 0.6562 A/mM oleic acid. This sensor was then further fabricated to detect non-esterified fatty acid concentrations in human plasma and serum samples. Commercial UV optical methods were used as method of validation of the blood sample concentrations. This work produced a platform for further non-esterified fatty acid detection studies.EPSRC

The Development of a Gamma-(γ)-Aminobutyric Acid (GABA) Biosensor and Characterisation of an L-Glutamate Biosensor for Neurochemical Analysis

The research presented in this thesis started off as a PhD project with the aim to develop and characterise an in vitro biosensor appropriate for in vivo detection and monitoring of gamma- (γ)-aminobutyric acid (GABA). The ambition was simultaneous monitoring of GABA and Lglutamate. It was also hoped that simultaneous D-serine monitoring would be performed using a newly developed and validated sensor for this co-agonist of the glutamatergic N-methyl Daspartate (NMDA) receptor. The development of a GABase-based biosensor was found to be too large an undertaking and consequently, the research plan converted to refinement of an Lglutamate biosensor. Some development work (pH and temperature studies) was also performed on the D-serine biosensor. Gamma-(γ)-aminobutyric acid is the major inhibitory neurotransmitter but has yet to receive wide examination in the scientific community. In contrast, L-glutamate, the major excitatory, neurotransmitter has not only experienced vast amounts of research but is also present in the public eye unlike GABA. As neurotransmitters are chemicals producing electrical stimulation in the brain, electrochemical techniques offer a unique insight into their operation and reactions. The development of a first generation GABA biosensor used an underlying L-glutamate biosensor as GABA is the precursor to L-glutamate. An appropriate enzyme unit activity for the GABase solution was the first barrier to be overcome. After this, the position of the GABase in the composite design was investigated. This experimentation didn’t garner any results that suggested a response would be produced. The active surface was examined to ensure that the production of hydrogen peroxide would be detected. An alternate reaction scheme was also investigated which didn’t produce any response either. This suggested the enzyme solution was, at least in part, at fault. Further exploration and refinement of the enzyme solution could potentially alleviate the issues encountered during this development work. The characterisation of the L-glutamate biosensor then became the priority. The optimal composite design was found to be: Pt/IrC – PoPD – (Sty – GluOx(100 U/mL) – BSA:GA(1.0:0.1 %) – PEI(1%))15 After the optimal design was found and had appropriate sensitivity (90.4 ± 2.0 nA∙cm−2∙μM−1) comparable to previously reported sensors, the in vitro characterisation was performed. This consisted of ensuring the biosensor would remain operational after implantation in the extracellular fluid i.e. under the chemical and physical parameters present in the brain. The shelf-life was found to be several weeks (28 days) and there was no recorded loss in sensitivity after repeated calibrations, or exposure to ex vivo rodent brain tissue. The sensor performed as desired under all physiologically relevant pH and temperature ranges. The interference was mitigated with the use of poly-ortho-phenylenediamine (PoPD) (interferent species were typically < 5% of the basal glutamate (10 μM) glutamate response) and reliable detection of L-glutamate was still observed. Preliminary in vivo characterisation performed in freely moving animals suggested the suitability of this sensor design for in vivo use. Expected signal changes were observed and a stable baseline over 16 days. Future work will include further in vivo characterisation and validation of this biosensor. Tentatively, the dual monitoring of L-glutamate and D-serine would be examined because of their co-agonist role at the NMDA receptor