title: Proton-sensing transistor systems for detecting ion leakage from plasma membranes under chemical stimuli.
abstract: The membrane integrity of live cells is routinely evaluated for cytotoxicity induced by chemical or physical stimuli. Recent progress in bioengineering means that high-quality toxicity validation is required. Here, we report a pH-sensitive transistor system developed for the continuous monitoring of ion leakage from cell membranes upon challenge by toxic compounds. Temporal changes in pH were generated with high reproducibility via periodic flushing of HepG2 cells on a gate insulator of a proton-sensitive field-effect transistor with isotonic buffer solutions with/without NH4Cl. The pH transients at the point of NH4Cl addition/withdrawal originated from the free permeation of NH3across the semi-permeable plasma membranes, and the proton sponge effect produced by the ammonia equilibrium. Irreversible attenuation of the pH transient was observed when the cells were subjected to a membrane-toxic reagent. Experiments and simulations proved that the decrease in the pH transient was proportional to the area of the ion-permeable pores on the damaged plasma membranes. The pH signal was correlated with the degree of hemolysis produced by the model reagents. The pH assay was sensitive to the formation of molecularly sized pores that were otherwise not measurable via detection of the leakage of hemoglobin, because the hydrodynamic radius of hemoglobin was greater than 3.1nm in the hemolysis assay. The pH transient was not disturbed by inherent ion-transporter activity. The ISFET assay was applied to a wide variety of cell types. The system presented here is fast, sensitive, practical and scalable, and will be useful for validating cytotoxins and nanomaterials.
  
  
  
  The plasma membrane toxicity and hemolysis are widely and routinely evaluated in biomaterials science and biomedical engineering. Despite the recent development of a variety of methods/materials for efficient gene/drug delivery systems to the cytosol, the methodologies for safety validation remain unchanged in many years while leaving some major issues such as sensitivity, accuracy, and fast response. The paper describes a new way of measuring the plasma membrane leakage in real time upon challenge by toxic reagents using a solid-state transistor that is sensitive to proton as the smallest indicator. Our system was reliable and was correlated to the results from hemolysis assay with advanced features in sensitivity, fast response, and wide applicability to chemical species. The downsizing and integration features of semiconductor fabrication technologies may realize cytotoxicity assays at the single-cell level in multi-parallel.
authors.affiliation: Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU), 2-3-10 Kanda-Surugadai, Chiyoda, Tokyo 101-0062 Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Imaizumi
authors.affiliation: Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU), 2-3-10 Kanda-Surugadai, Chiyoda, Tokyo 101-0062 Japan. Electronic address: goda.bsr@tmd.ac.jp.
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Goda
authors.affiliation: Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU), 2-3-10 Kanda-Surugadai, Chiyoda, Tokyo 101-0062 Japan.
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Schaffhauser
authors.affiliation: Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8563, Japan.
authors.forename: Jun-Ichi
authors.initials: JI
authors.lastname: Okada
authors.affiliation: Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU), 2-3-10 Kanda-Surugadai, Chiyoda, Tokyo 101-0062 Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Matsumoto
authors.affiliation: Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU), 2-3-10 Kanda-Surugadai, Chiyoda, Tokyo 101-0062 Japan. Electronic address: miyahara.bsr@tmd.ac.jp.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Miyahara
mesh_terms: D000641:Ammonia / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007477:Ions; D011522:Protons; D012756:Sheep; D013997:Time Factors; D014173:Transistors, Electronic
chemical_list: D007477:Ions; D011522:Protons; D000641:Ammonia
keywords: Cell membrane; Hemolysis; Ion-sensitive field-effect transistor; Potentiometry; Proton-sponge effect