Development of an integrated electrochemical system for in vitro yeast viability testing.

This work describes the development and testing of a microfabricated sensor for rapid cell growth monitoring, especially focused on yeast quality assessment for wine applications. The device consists of a NMOS ISFET sensor with Si(3)N(4) gate, able to indirectly monitor extracellular metabolism through pH variation of the medium, and a solid-state reference electrode implemented with PVC membranes doped with lipophilic salts (tetrabutylammonium-tetrabutylborate (TBA-TBB) and Potassium tetrakis(4-chlorphenyl)borate (KTClpB)). The use of a solid state reference electrode enables the implementation of a large number of cell assays in parallel, without the need of external conventional reference electrodes. Microbial growth testing has been performed both in standard culture conditions and on chip at different concentrations of ethanol in order to carry out a commonly used screening of wine yeast strains. Cell growth tests can be performed in few hours, providing a fast, sensitive and low cost analysis with respect to the conventional procedures.

[1]  T. Arakawa,et al.  Yeast-derived recombinant human insulin-like growth factor I: Production, purification, and structural characterization , 1990, Journal of protein chemistry.

[2]  Xiangqin Cui,et al.  Sensors and Actuators B , 2003 .

[3]  Isao Karube,et al.  An integrated three-electrode system with a micromachined liquid-junction Ag/AgCl reference electrode , 1999 .

[4]  J. Pietenpol,et al.  Cell-cycle dysregulation and anticancer therapy. , 2003, Trends in pharmacological sciences.

[5]  K Asami,et al.  Dielectric analysis of yeast cell growth. , 1995, Biochimica et biophysica acta.

[6]  Johan Bobacka,et al.  Solid‐Contact Reference Electrodes Based on Lipophilic Salts , 2009 .

[7]  Jae Ho Shin,et al.  Solvent-Processible Polymer Membrane-Based Liquid Junction-Free Reference Electrode , 1998 .

[8]  H. Müller,et al.  The proton extrusion of growing yeast cultures as an on‐line parameter in fermentation processes: Ammonia assimilation and proton extrusion are correlated by an 1:1 stoichiometry in nitrogen‐limited fed‐batch fermentations , 1990 .

[9]  Michael J. Schöning,et al.  New concepts of miniaturised reference electrodes in silicon technology for potentiometric sensor systems , 2004 .

[10]  Piet Bergveld,et al.  Thirty years of ISFETOLOGY ☆: What happened in the past 30 years and what may happen in the next 30 years , 2003 .

[11]  J. Simon,et al.  Yeast as a model system for anticancer drug discovery , 2004, Nature Reviews Cancer.

[12]  P. Piper.  The heat shock and ethanol stress responses of yeast exhibit extensive similarity and functional overlap. , 1995, FEMS microbiology letters.

[13]  Y. Takahara,et al.  On-Line Monitoring of Cell Concentrations during Yeast Cultivation by Dielectric Measurements , 1991 .

[14]  P. Bühlmann,et al.  The phase-boundary potential model. , 2004, Talanta.

[15]  Maurizio Valle,et al.  Bioelectrochemical signal monitoring of in-vitro cultured cells by means of an automated microsystem based on solid state sensor-array. , 2003, Biosensors & bioelectronics.

[16]  Hyo il Jung,et al.  An impedimetric biosensor for real-time monitoring of bacterial growth in a microbial fermentor , 2009 .

[17]  ScienceDirect,et al.  Toxicology and Applied Pharmacology , 1959, Nature.

[18]  Hongkai Wu,et al.  Fabrication of a microfluidic Ag/AgCl reference electrode and its application for portable and disposable electrochemical microchips , 2010, Electrophoresis.

[19]  Dermot Diamond,et al.  Development of miniature all-solid-state potentiometric sensing system , 2010 .

[20]  J. Worth Estes,et al.  Trends in pharmacological sciences in 1784 , 1984 .

[21]  A. van den Berg,et al.  Monitoring of yeast cell concentration using a micromachined impedance sensor , 2005, The 13th International Conference on Solid-State Sensors, Actuators and Microsystems, 2005. Digest of Technical Papers. TRANSDUCERS '05..

[22]  Chen Jianrong,et al.  Ion sensitive field effect transducer-based biosensors. , 2003, Biotechnology advances.

[23]  Ralf Ehret,et al.  Online monitoring of cell metabolism for studying pharmacodynamic effects. , 2007, Toxicology and applied pharmacology.

[24]  Lindsey J. Leach,et al.  Genetic Dissection of Ethanol Tolerance in the Budding Yeast Saccharomyces cerevisiae , 2007, Genetics.

[25]  A. Bhunia,et al.  Microscale electronic detection of bacterial metabolism , 2002 .

[26]  Katsuhide Fujita,et al.  The genome-wide screening of yeast deletion mutants to identify the genes required for tolerance to ethanol and other alcohols. , 2006, FEMS yeast research.

[27]  G. Fleet.  Wine Microbiology and Biotechnology , 1993 .

[28]  Timothy D. Strong,et al.  Solid-state ion sensors with a liquid junction-free polymer membrane-based reference electrode for blood analysis , 2000 .

[29]  J. W. Parce,et al.  Biosensors based on the energy metabolism of living cells: the physical chemistry and cell biology of extracellular acidification. , 1992, Biosensors & bioelectronics.

[30]  Characterisation of porous ceramic plugs for use in electrochemical sensors , 2002 .

[31]  L. Lorenzelli,et al.  Development and characterization of a multiparametric microsensor for yeast cell growth monitoring , 2009 .

[32]  Robert Koncki,et al.  Screen-printed reference electrodes for potentiometric measurements , 2004 .

[33]  P Silley,et al.  Impedance microbiology--a rapid change for microbiologists. , 1996, The Journal of applied bacteriology.

[34]  Marcel Ottens,et al.  Lab‐scale fermentation tests of microchip with integrated electrochemical sensors for pH, temperature, dissolved oxygen and viable biomass concentration , 2008, Biotechnology and bioengineering.

[35]  F Gòdia,et al.  On-line monitoring of yeast cell growth by impedance spectroscopy. , 2005, Journal of biotechnology.

[36]  G. Stewart,et al.  A study of ethanol tolerance in yeast. , 1990, Critical reviews in biotechnology.

[37]  H. Nam,et al.  Potentiometric properties of ion-selective electrode membranes based on segmented polyether urethane matrices. , 1997, Analytical chemistry.