Barrier tissue protects the body against external factors by restricting the passage of molecules. The gastrointestinal epithelium is an example of barrier tissue with the primary purpose of allowing the passage of ions and nutrients, while restricting the passage of pathogens and toxins. It is well known that the loss of barrier function can be instigated by a decrease in extracellular calcium levels, leading to changes in protein conformation and an increase in paracellular transport. In this study, ethylene glycol-bis(beta-aminoethyl ether)- N, N, N', N'-tetra acetic acid (EGTA), a calcium chelator, was used to disrupt the gastrointestinal epithelial barrier. The effect of EGTA on barrier tissue was monitored by a novel label-free method based on an organic electrochemical transistor (OECT) integrated with living cells and validated against conventional methods for measuring barrier tissue integrity. We demonstrate that the OECT can detect breaches in barrier tissue upon exposure to EGTA with the same sensitivity as existing methods but with increased temporal resolution. Due to the potential of low cost processing techniques and the flexibility in design associated with organic electronics, the OECT has great potential for high-throughput, disposable sensing and diagnostics.
References
[1]
Boulenc, X.; Marti, E.; Joyeux, H.; Roques, C.; Berger, Y.; Fabre, G. Importance of the paracellular pathway for the transport of a new bisphosphonate using the human Caco-2 monolayers model. Biochem. Pharmacol. 1993, 46, 1591–1600.
[2]
Farquhar, M.G.; Palade, G.E. Junctional complexes in various epithelia. J. Cell Biol. 1963, 17, 375–412, doi:10.1083/jcb.17.2.375.
[3]
Gaillard, J.L.; Finlay, B.B. Effect of cell polarization and differentiation on entry of listeria monocytogenes into the enterocyte-like Caco-2 cell line. Infec. Immunity 1996, 64, 1299–1308.
[4]
Anderson, J.M.; Balda, M.S.; Fanning, A.S. The structure and regulation of tight junctions. Curr. Opin. Cell Biol. 1993, 5, 772–778, doi:10.1016/0955-0674(93)90024-K.
[5]
Anderson, J.M. Molecular structure of tight junctions and their role in epithelial transport. News Physiol. Sci. 2001, 16, 126–130.
[6]
Anderson, J.M.; van Itallie, C.M. Tight junctions: Closing in on the seal. Curr. Biol. 1999, 9, R922–R924, doi:10.1016/S0960-9822(00)80105-0.
[7]
Guttman, J.A.; Finlay, B.B. Tight junctions as targets of infectious agents. Biochim. Biophys. Acta 2009, 1788, 832–841, doi:10.1016/j.bbamem.2008.10.028.
[8]
Colegio, O.R.; van Itallie, C.M.; McCrea, H.J.; Rahner, C.; Anderson, J.M. Claudins create charge-selective channels in the paracellular pathway between epithelial cells. Am. J. Physiol. Cell Physiol. 2002, 283, C142–C147.
[9]
Matter, K.; Balda, M.S. Occludin and the functions of tight junctions. Int. Rev. Cytol. 1999, 186, 117–146, doi:10.1016/S0074-7696(08)61052-9.
[10]
Anderson, J.M.; Stevenson, B.R.; Goodenough, D.A.; Mooseker, M.S. Molecular characterization of zo-1, a peripheral membrane-protein of the tight junction. J. Cell Biol. 1986, 103, A71, doi:10.1083/jcb.103.1.71.
[11]
Fanning, A.S.; van Itallie, C.M.; Anderson, J.M. Zonula occludens-1 and-2 regulate apical cell structure and the zonula adherens cytoskeleton in polarized epithelia. Mol. Biol. Cell 2012, 23, 577–590, doi:10.1091/mbc.E11-09-0791.
[12]
Baum, B.; Georgiou, M. Dynamics of adherens junctions in epithelial establishment, maintenance, and remodeling. J. Cell Biol. 2011, 192, 907–917, doi:10.1083/jcb.201009141.
Angst, B.D.; Marcozzi, C.; Magee, A.I. The cadherin superfamily: Diversity in form and function. J. Cell Sci. 2001, 114, 629–641.
[15]
Nagar, B.; Overduin, M.; Ikura, M.; Rini, J.M. Structural basis of calcium-induced e-cadherin rigidification and dimerization. Nature 1996, 380, 360–364.
[16]
Balda, M.S.; Gonzalez-Mariscal, L.; Contreras, R.G.; Macias-Silva, M.; Torres-Marquez, M.E.; Garcia-Sainz, J.A.; Cereijido, M. Assembly and sealing of tight junctions: Possible participation of G-proteins, phospholipase C, protein kinase C and calmodulin. J. Membrane Biol. 1991, 122, 193–202, doi:10.1007/BF01871420.
[17]
Sambuy, Y.; De Angelis, I.; Ranaldi, G.; Scarino, M.L.; Stammati, A.; Zucco, F. The Caco-2 cell line as a model of the intestinal barrier: Influence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell Biol. Toxicol. 2005, 21, 1–26, doi:10.1007/s10565-005-0085-6.
[18]
Artursson, P.; Karlsson, J. Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells. Biochem. Biophys. Res. Commun. 1991, 175, 880–885.
[19]
Artursson, P.; Magnusson, C. Epithelial transport of drugs in cell culture. II: Effect of extracellular calcium concentration on the paracellular transport of drugs of different lipophilicities across monolayers of intestinal epithelial (Caco-2) cells. J. Pharm. Sci. 1990, 79, 595–600.
[20]
Ivanov, A.I.; Nusrat, A.; Parkos, C.A. Endocytosis of epithelial apical junctional proteins by a clathrin-mediated pathway into a unique storage compartment. Mol. Biol. Cell 2004, 15, 176–188.
[21]
Artursson, P. Epithelial transport of drugs in cell culture. I: A model for studying the passive diffusion of drugs over intestinal absorptive (Caco-2) cells. J. Pharm. Sci. 1990, 79, 476–482.
[22]
Raiman, J.; Tormalehto, S.; Yritys, K.; Junginger, H.E.; Monkkonen, J. Effects of various absorption enhancers on transport of clodronate through Caco-2 cells. Int. J. Pharm. 2003, 261, 129–136.
[23]
Collares-Buzato, C.B.; McEwan, G.T.; Jepson, M.A.; Simmons, N.L.; Hirst, B.H. Paracellular barrier and junctional protein distribution depend on basolateral extracellular Ca2+ in cultured epithelia. Biochim. Biophys. Acta 1994, 1222, 147–158.
[24]
Jimison, L.H.; Tria, S.A.; Khodagholy, D.; Gurfinkel, M.; Lanzarini, E.; Hama, A.; Malliaras, G.G.; Owens, R.M. Measurement of barrier tissue integrity with an organic electrochemical transistor. Adv. Mater. 2012, 24, 5919–5923.
[25]
Tria, S.A.; Jimison, L.H.; Hama, A.; Bongo, M.; Owens, R.M. Validation of the organic electrochemical transistor for in vitro toxicology. BBA-Gen. Subjects, 2012. Available online: http://dx.doi.org/10.1016/j.bbagen.2012.12.003..
[26]
Weber, C.R.; Shen, L.; Wu, L.; Wang, Y.; Turner, J.R. Occludin is required for tumor necrosis factor (TNF)-mediated regulation of tight junction (TJ) barrier function. Gastroenterology 2011, 140, S64.
[27]
DeFranco, J.A.; Schmidt, B.S.; Lipson, M.; Malliaras, G.G. Photolithographic patterning of organic electronic materials. Org. Electron. 2006, 7, 22–28.
[28]
Khodagholy, D.; Gurfinkel, M.; Stavrinidou, E.; Leleux, P.; Herve, T.; Sanaur, S.; Malliaras, G.G. High speed and high density organic electrochemical transistor arrays. Appl. Phys. Lett. 2011, 99, 163304:1–163304:3.
[29]
Bernards, D.A.; Malliaras, G.G.; Toombes, G.E.S.; Gruner, S.M. Gating of an organic transistor through a bilayer lipid membrane with ion channels. Appl. Phys. Lett. 2006, 89, 053505:1–053505:3.
[30]
Bernards, D.A.; Malliaras, G.G. Steady-state and transient behavior of organic electrochemical transistors. Adv. Funct. Mater. 2007, 17, 3538–3544.
[31]
White, H.S.; Kittlesen, G.P.; Wrighton, M.S. Chemical derivatization of an array of three gold microelectrodes with polypyrrole: Fabrication of a molecule-based transistor. J. Am. Chem. Soc. 1984, 106, 5375–5377.
[32]
Moyes, S.M.; Morris, J.F.; Carr, K.E. Roles of pre-treatment time and junctional proteins in Caco-2 cell microparticle uptake. Int. J. Pharm. 2011, 407, 21–30.
[33]
Balda, M.S.; Whitney, J.A.; Flores, C.; Gonzalez, S.; Cereijido, M.; Matter, K. Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein. J. Cell Biol. 1996, 134, 1031–1049.
[34]
Armitage, W.J.; Juss, B.K.; Easty, D.L. Response of epithelial (mdck) cell junctions to calcium removal and osmotic stress is influenced by temperature. Cryobiology 1994, 31, 453–460.
[35]
Miyoshi, J.; Takai, Y. Molecular perspective on tight-junction assembly and epithelial polarity. Adv. Drug Deliv. Rev. 2005, 57, 815–855.
[36]
Sheth, P.; Samak, G.; Shull, J.A.; Seth, A.; Rao, R. Protein phosphatase 2A plays a role in hydrogen peroxide-induced disruption of tight junctions in Caco-2 cell monolayers. Biochem. J. 2009, 421, 59–70.
[37]
Rothen-Rutishauser, B.; Riesen, F.K.; Braun, A.; Gunthert, M.; Wunderli-Allenspach, H. Dynamics of tight and adherens junctions under egta treatment. J. Membrane Biol. 2002, 188, 151–162.
[38]
Ma, T.Y.; Tran, D.; Hoa, N.; Nguyen, D.; Merryfield, M.; Tarnawski, A. Mechanism of extracellular calcium regulation of intestinal epithelial tight junction permeability: Role of cytoskeletal involvement. Microsc. Res. Technique 2000, 51, 156–168.
[39]
Balda, M.S.; Whitney, J.A.; Flores, C.; Gonzalez, S.; Cereijido, M.; Matter, K. Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein. J. Cell Biol. 1996, 134, 1031–1049.
[40]
Van Itallie, C.M.; Fanning, A.S.; Bridges, A.; Anderson, J.M. Zo-1 stabilizes the tight junction solute barrier through coupling to the perijunctional cytoskeleton. Mol. Biol. Cell 2009, 20, 3930–3940.
