Effect of yeast pretreatment on the characteristics of yeast-modified electrodes as mediated amperometric biosensors for lactic acid
Carbon paste electrode modified with baker' and wine yeast Saccharomyces cerevisiae (a source of flavocytochrome b(2)) were investigated as amperometric biosensors for L-lactic acid. Before immobilization on the electrode surface, yeast cells were pretreated with various electrolytes, alcohols and weak organic acids. Electrode responses to L-lactic acid were tested in the presence of various mediators (potassium ferricyanide, phenazine methosulfate, 2,6-dichlorophenolindophenol sodium salt hydrate, 1,2-naphthoquinone-4-sulfonic acid sodium salt). The highest (144+/-7 nA per 0.2 mM L-lactic acid) and the most stable responses were obtained after yeast pretreatment with 30% ethanol using potassium ferricyanide as a mediator. Different electrode sensitivities with mediator phenazine methosulphate probably reflected diverse changes in yeast membrane (and/or cell wall).
Available from: Arto Heiskanen
- "Ferricyanide    and redox hydrogels  have been employed in applications with prokaryotic cells. Upon chemical treatment of cells, communication with intracellular redox enzymes has been demonstrated using ferricyanide as electron transfer mediator in prokaryotic cells  and in eukaryotic cells  . Single quinone mediators, which due to their lipophilicity are able to penetrate the cellular plasma membrane, have been utilized with prokaryotic   as well as eukaryotic    cells. "
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ABSTRACT: This work describes a mediated amperometric method for simultaneous real-time probing of the NAD(P)H availability in two different phenotypes, fermentative and respiratory, of the phosphoglucose isomerase deletion mutant strain of S. cerevisiae, EBY44 [ENY.WA-1A pgi1-1D::URA3], and its parental strain, ENY.WA-1A. The developed method is based on multichannel detection using microelectrode arrays. Its versatility was demonstrated by using four microelectrode arrays for simultaneously monitoring the NAD(P)H availability of both geno- and phenotypes under the influence of two different carbon sources, glucose and fructose, as well as the cytosolic and mitochondrial inhibitor and uncoupler, dicoumarol. The obtained results indicate that the method is capable of accurately and reproducibly (overall relative standard error of mean 3.2%) mapping the real-time responses of the cells with different genotype-phenotype combinations. The ENY.WA cells showed the same response to glucose and fructose when dicoumarol was used; fermentative cells indicated the presence of cytosolic inhibition and respiratory cells a net effect of mitochondrial uncoupling. EBY44 cells showed cytosolic inhibition with the exception of respiratory cells when fructose was used as carbon source.
Bioelectrochemistry (Amsterdam, Netherlands) 04/2009; 76(1-2):180-8. DOI:10.1016/j.bioelechem.2009.02.015 · 4.17 Impact Factor
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ABSTRACT: In general, L-lactate respiration is difficult to detect in living yeast cells due to the small activity of L-lactate oxidizing enzymes within the mitochondria. Genetically modified cells of methylotrophic yeast Hansenula polymorpha overproducing L-lactate:cytochrome c-oxidoreductase (EC 184.108.40.206, also known as flavocytochrome b(2), FC b(2)) were physically immobilized by means of a dialysis membrane onto various types of electrode materials in order to investigate the possibility of electrochemically detecting L-lactate respiration. It could be shown that in the case of genetically modified Hansenula polymorpha cells in contrast to cells from the parental strain, enhanced L-lactate-dependent respiration could be detected. Due to overproduction of FC b(2) the O(2) reduction current is decreased upon addition of L-lactate to the electrolyte solution. The electron transfer pathway in the L-lactate-dependent respiration process involves a cascade over three redox proteins, FC b(2), cytochrome c and Complex-IV, starting with L-lactate oxidation and ending with oxygen reduction. By means of selective inhibition of Complex IV with CN(-), lactate respiration could be proven for causing the decrease in the O(2) reduction.
Bioelectrochemistry (Amsterdam, Netherlands) 05/2009; 76(1-2):175-9. DOI:10.1016/j.bioelechem.2009.04.002 · 4.17 Impact Factor
Available from: Marina Nisnevitch
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ABSTRACT: Biosensors are bioanalytical devices which transform a biorecognition response into a measurable physical signal. Although biosensors are a novel achievement of bioanalytical chemistry, they are not only a subject of intensive research, but also a real commercial product (Kissinger, 2005). The estimated world analytical market is about $20 billion per year of which 30 % is in the health care field. The biosensors market is expected to grow from $6.72 billion in 2009 to $14.42 billion in 2016 (http://www.marketresearch.com, Analytical Review of World Biosensors Market).
Although up to now IUPAC has not accepted an official definition of the term biosensor, its electrochemical representative is defined as “a self-contained integrated device, which is capable of providing specifc quantitative or semi-quantitative analytical information using a biological recognition element (biochemical receptor) which is retained in direct spatial contact with an electrochemical transduction element’’ (Thevenot et al., 2001). Generally, the biosensor is a hybrid device containing two functional parts: a bioelement (an immobilized biologically active material) and a physical transducer. As bioelements can be used pieces of tissue, microbial cells, organelles, natural biomembranes or liposomes, receptors, enzymes, antibodies and antigens, abzymes, nucleic acids and other biomolecules and even biomimetics which imitate structural and functional features of the natural analogue. The bioelement is a recognition unit providing selective binding or biochemical/metabolic conversion of the analyte that results in changes of physical or physico-chemical characteristics of the transducer (Scheller et al., 1991; Schmidt & Karube, 1998; Gonchar et al., 2002; Nakamura & Karube, 2003; Sharma et al., 2003; Investigations on Sensor Systems and Technologies, 2006). The bioelement in such constructions is usually prepared in immobilized form and often covered with an outer membrane (or placed between two membranes in a sandwich manner), which either prevents the penetration of interfering substances into a sensitive bioselective layer and transducer surface, or creates a diffusion barrier for the analyte. Such membrane structures increase the stability of the biorecognizing element, enhance its selectivity and provide the diffusion limitations for biochemical reactions. Electrochemical, optical, piezoelectric, thermoelectric, transistor, acoustic and other elements are used as transducers in biosensor systems. Electrochemical (amperometric, potentiometric, conductometric) and optical (surface plasmon resonance) devices are the most exploited transducers in commercially available biosensors (Commercial Biosensors, 1998).
Basically, biosensors can be regarded as information transducers in which the energy of biospecific interactions is transformed into information about the nature and concentration of an analyte in the sample. The most essential advantages of biosensors are excellent chemical selectivity and high sensitivity, possibility of miniaturization and compatibility with computers. Their drawbacks are limited stability and a rather complicated procedure for preparation of the biologically active material.
The enzyme biosensors are the most widespread devices (Zhao & Jiang, 2010); many of them are produced commercially. Enzyme biosensors are characterized by their high selectivity. They also provide fast output due to high activity and high local enzyme concentration in a sensitive layer. The drawbacks of enzyme biosensors are insufficient stability and the high price of purified enzymes. The cell sensors, especially microbial ones, have been actively developed only in recent years (Shimomura-Shimizu & Karube, 2010a, 2010b; Su et al., 2011). Cell biosensors have a range of considerable advantages when compared to their enzyme analogues: availability of cells, low price and simple procedure of cell isolation, possibility to use long metabolic chains, avoiding purification of enzymes and coenzymes, advanced opportunity for metabolic engineering, integrity of the cell response (important in assaying total toxicity and mutagenic action of environmental pollutants), possibility to retain viability of sensoring cells and even to provide their propagation, and, in some cases, higher stability of cell elements compared to enzyme ones. The main drawbacks of microbial biosensors are a rather low signal rate due to a lower concentration of enzymes involved in cellular response, as well as low selectivity of cell output (e.g. in the case of microbial O2 electrode sensors due to a broad substrate specificity of cellular respiration).
These drawbacks are not absolute, taking into account recent progress in genetic engineering and the possibility to over-express the key analytical enzyme in the cell (Gonchar et al., 2002).
The most biosensors have been created for clinical diagnostics (D’Orazio, 2003; Song et al., 2006; Belluzo et al., 2008). They exploit enzymes as biocatalytic recognition elements and immunoreagents and DNA fragments as affinity tools for biorecognition of the target analytes (metabolites, antigens, antibodies, nucleic acids) coupled to electrochemical and optical modes of transduction. For simultaneous detection of multiple analytes, microarray technique is developed for automated clinical diagnostics (Seidel & Niessner, 2008). For continuous monitoring of living processes, reagentless implantable biosensors have been developed (Wilson & Ammam, 2007).
Biosensors are regarded as very promising tools for cancer clinical testing (Rasooly & Jacobson, 2006; Wang, 2006). New genomic and proteomic approaches are being used for revealing cancer biomarkers related with genetic features, changes in gene expression, protein profiles and post-translational modifications of proteins.
Recent progress in nanobiotechnology allows using nanomolecular approaches for clinical diagnostic procedures (Salata, 2004; Jain, 2007). The most important applications are foreseen in the areas of biomarker monitoring, cancer diagnosis, and detection of infectious microorganisms. Analytical nanobiotechnology uses different nanoscaled materials (gold and magnetic nanoparticles, nanoprobes, quantum dots as labels, DNA nanotags) for molecular detection (Baptista et al., 2008; Medintz et al., 2008; Sekhon & Kamboj, 2010). The use of nanomaterials in biosensors has allowed the introduction of many new signal transduction technologies into biosensorics and improvement of bioanalytical parameters of the nanosensors - selectivity, response time, miniaturization of the biorecognition unit (Jianrong et al., 2004; Murphy, 2006).
Biosensors - Emerging Materials and Applications, 07/2011; , ISBN: 978-953-307-328-6
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