Reagentless pH-stat for Microliter Fluid Specimens

• Published in: Analytical chemistry (Washington) Vol. 80; no. 11; pp. 4065 - 4069
• Main Authors: Kao, Linus T.-H, Hsu, Hung-Yi, Gratzl, Miklós
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.06.2008
• Subjects: Acetylcholinesterase - chemistry; Acetylcholinesterase - metabolism; Analytical chemistry; Animals; Applied sciences; Biochemistry; Buffers; Cattle; Chemical reactions; Chemistry; Electric Conductivity; Electrochemical methods; Enzyme kinetics; Exact sciences and technology; Global environmental pollution; Hydrogen; Hydrogen-Ion Concentration; Indicators and Reagents - chemistry; Microfluidic Analytical Techniques - methods; Nitrates - chemistry; Pollution; Potassium Compounds - chemistry; Reaction kinetics; Reproducibility of Results; Rotation; Substrate Specificity; Water; Chemical analysis; Enzyme; Electrochemical method; Esterases; Acetylcholinesterase; Carboxylic ester hydrolases; pH stat procedure; Electrolysis; Efficiency; Hydrolases; Environment; Alkalinity; Hydrogen ion; Hydroxyl; Monitoring
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac800161y

pH-stating is a common technique for monitoring kinetics of various biochemical reactions that involve the generation of hydrogen or hydroxyl ions. In this work, we describe a reagentless electrochemical micro-pH-stat where the titrant of acid or base is produced by water electrolysis on the rotating sample system (RSS) platform. RSS originated from the authors’ laboratory as a convective platform to support different analytical techniques in microliter-sized samples. As water electrolysis induces no volume change and the current that generates the reagent can be precisely measured even at low levels, very small samples in the microliter range become accessible for pH-stating: a reduction of more than an order of magnitude in specimen size relative to the most sensitive conventional methods. Nearly 100% current efficiency has been achieved with this system using a 250 µm Pt minidisc working electrode for electrolysis. The developed micro-pH-stat has been validated by the determination of the activity of erythrocyte acetylcholinesterase as a function of substrate concentration and pH. The optimal pH and activity profile obtained are in good agreement with those determined with standard techniques. The micro-pH-stat has the potential for applications for enzyme assays, reagentless pH control, acidity/alkalinity, and buffer capacity measurements in very small samples of biomedical and environmental origin.