Microchamber arrays with an integrated long luminescence lifetime pH sensor
- 420 Downloads
A pH probe with a microsecond luminescence lifetime was obtained via covalent coupling of 6-carboxynaphthofluorescein (CNF) moieties to ruthenium-tris-(1,10-phenanthroline)2+. The probe was covalently attached to amino-modified poly-(2-hydroxyethyl)methacrylate (pHEMA) and showed a pH-dependent FRET with luminescence lifetimes of 681 to 1260 ns and a working range from ca. pH 6.5 to 9.0 with a pKa of 7.79 ± 0.14. The pH sensor matrix was integrated via spin coating as ca. 1- to 2-μm-thick layer into “CytoCapture” cell culture dishes of 6 mm in diameter. These contained a microcavity array of square-shaped regions of 40 μm length and width and 15 μm depth that was homogeneously coated with the pH sensor matrix. The sensor layer showed fast response times in both directions. A microscopic setup was developed that enabled imaging of the pH inside the microchamber arrays over many hours. As a proof of principle, we monitored the pH of Escherichia coli cell cultures grown in the microchamber arrays. The integrated sensor matrix allowed pH monitoring spatially resolved in every microchamber, and the differences in cell growth between individual chambers could be resolved and quantified.
KeywordsLong-lifetime luminescent pH sensor FRET-based pH imaging Polymeric microcavity array Escherichia coli cell growth Bioprocess miniaturization
Financial support of this work by the German Research Foundation (DFG, NA 947/1-2) and the German Ministry of Education and Research (BMBF, V4KMU10/126) is gratefully acknowledged.
Compliance with ethical standards
Conflict of interest
Elisabeth Poehler, Simon A. Pfeiffer, and Stefan Nagl declare they have no competing interests. Marc Herm and Michael Gaebler are active for Synthon Chemicals and plan to make the pH probe molecule described herein (Ru(CNF)3) commercially available. Benedikt Busse is associated with zell-kontakt GmbH that offer “CytoCapture” microchamber arrays for sale.
- 17.Hanson GT, McAnaney TB, Park ES, Rendell MEP, Yarbrough DK, Chu S, Xi L, Boxer SG, Montrose MH, Remington SJ (2002) Green fluorescent protein variants as ratiometric dual emission pH sensors. 1. Structural characterization and preliminary application. Biochemistry 41(52):15477–15488CrossRefGoogle Scholar
- 21.Schreml S, Meier RJ, Kirschbaum M, Kong SC, Gehmert S, Felthaus O, Küchler S, Sharpe JR, Wöltje K, Weiß KT, Albert M, Seidl U, Schröder J, Morsczeck C, Prantl L, Duschl C, Pedersen SF, Gosau M, Berneburg M, Wolfbeis OS, Landthaler M, Babilas P (2014) Luminescent dual sensors reveal extracellular pH-gradients and hypoxia on chronic wounds that disrupt epidermal repair. Theranostics 4(7):721–735CrossRefGoogle Scholar
- 31.Leventis N, Rawashdeh AM, Elder IA, Yang J, Dass A, Sotiriou-Leventis C (2004) Synthesis and characterization of Ru(II)tris(1,10-phenanthroline)-electron acceptor dyads incorporating the 4-benzoyl-N-methylpyridinium cation or N-benzyl-N′-methyl viologen. Improving the dynamic range, sensitivity, and response time of sol–gel-based optical oxygen sensors. Chem Mater 16(8):1493–1506CrossRefGoogle Scholar