Ratiometric porphyrin-based layers and nanoparticles for measuring oxygen in biosamples
Introduction
The last decades have witnessed widespread interest in optical chemical sensing mainly due to its sensitivity and ease of use [1], [2]. Most of the approaches are based on the application of smart materials whose optical properties are sensitive to target analytes. In addition, miniaturization of sensing systems has opened new possibilities to measure and monitor analyte concentrations. The homogeneous deposition of the indicator in thin polymer layers permeable for the analyte is a common method for sensor preparation [3]; nevertheless some approaches, in which indicators were co-polymerized together with the polymers, were also tested [4], [5]. Noticeably, monitoring of biologically relevant parameters such as pH, carbon dioxide and oxygen or biomolecules is of interest in many fields of science and technology. Especially the measurement of oxygen is relevant in numerous areas, such as automobile industry [6], marine research [7], biotechnology [8] and medicine [9]. The balance between oxygen supply and oxygen consumption is of paramount importance for tissues [10] as well as microorganisms [11]. The interest in determining the content of oxygen (pO2) has spurred the development of oxygen sensing techniques starting from the first amperometric sensors [12] through fiber optic devices [13] into the field of fluorescent nanosensors [14]. Oxygen electrodes or fiber optic-based devices are useful to measure global pO2, but they are invasive and can only measure oxygen at the tip of the electrode one point at a time. This makes it difficult to measure the oxygen distribution and to evaluate oxygen heterogeneities in ischemic tissues or microorganisms.
Several nanoparticles stained with dyes sensitive to oxygen have been developed during the last years. In these studies a wide variety of porphyrin-based oxygen-sensitive dyes as well as polymers was used: Polyacrylamide and poly(dodecyl methacrylate) was applied for the preparation of so-called “PEBBLEs” [15]. Also styrene was used to obtain nanoparticles sensitive to oxygen [16], [17]. Another interesting example is the application of liposomes as scaffold for nanosensors [18]. Recently, the preparation and characterization of poly(styrene-block-vinylpyrrolidone) nanoparticles has been presented [19].
In this work we describe the preparation of ratiometric polystyrene-based oxygen-sensitive layers and nanosensors. Layers and nanosensors are characterized and used to measure oxygen concentration in biological samples.
Section snippets
Materials and chemicals
The phosphorescent oxygen probe platinum(II)meso-tetra(pentafluorophenyl)porphine (PtTFPP) was purchased from Frontier Scientific (Logan, UT, USA) while N,N′-bis(1-hexylheptyl)perylene-3,4:9,10-bis-(dicarboximide) (S13) and sodium dodecyl sulfate (SDS) were bought from Fluka (Buchs, Switzerland). 4,4′-Azobis(4-cyanovaleric acid) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Styrene (Aldrich, Schnelldorf, Germany) and divinylbenzene (Aldrich) were purified immediately before use by
Phosphorescence quenching of PtTFPP and S13 loaded ratiometric nanoparticles and layers
As described in Section 2 nanobeads were prepared by microemulsion polymerization of styrene. Both, indicator and reference dye were included during the polymerization process. We choose polystyrene as polymer matrix because the lipophilic dyes can be easily incorporated. Besides, the formation of the nanoparticles is simple to control, and the surface of the particles can be easily functionalized. To confer oxygen sensitivity to the nanobeads we entrapped
Conclusions
A ratiometric oxygen sensor was developed by combining an indicator for oxygen and an inert reference dye. The dyes were incorporated in polymer layers and nanoparticles. The sensor response was assessed in aqueous solution as well as in yeast culture. Both sensor systems responded to changes in the partial pressure of oxygen. Porphyrin phosphorescence was quenched by oxygen whereas fluorescence of the reference dye remained essentially unaffected. Our experiments showed that sensor layers are
Acknowledgements
This work was supported by the Marie Curie Fellowship within the EU Projects MTKD-CT-2005-029554, entitled “Sensor Nanoparticles for Ions and Biomolecules” and “Nanomaterials for Applications in Sensors, Catalysis and Emerging Technologies” (MRTN-CT-2006-033873). This support is gratefully acknowledged. We are grateful to Frank Steiniger and Dr. Sandor Nietzsche (Centre for Electron Microscopy, Universitätsklinikum Jena) for their help with the AFM measurements.
Piotr J. Cywinski studied Physics and Chemistry at the Technical University of Lodz, Poland. Currently he is a postdoctoral researcher in the Institute of Physical Chemistry at the Friedrich-Schiller-University Jena. His research is focused on the design and development of novel luminescent nanosensors to be used for the intracellular imaging.
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Piotr J. Cywinski studied Physics and Chemistry at the Technical University of Lodz, Poland. Currently he is a postdoctoral researcher in the Institute of Physical Chemistry at the Friedrich-Schiller-University Jena. His research is focused on the design and development of novel luminescent nanosensors to be used for the intracellular imaging.
Artur J. Moro was born in the village of Cascais, close to Lisbon, Portugal. He made is diploma degree in Applied Chemistry with an Organic Chemistry specialization at the Faculty of Sciences and Technology of the Universidade Nova de Lisboa. Since 2006, he has been working as a PhD student, as part of a project within the Framework Program 6 of the European Union Marie Curie Actions.
Dr. Sarmiza Elena Stanca obtained her PhD from the Babes-Bolyai University of Cluj Napoca, Romania, where she developed electrochemical biosensors. Currently, she works as Marie Curie fellow at the Institute of Physiology II at the Friedrich-Schiller-University Jena. Her main interests are fluorescent nanosensors and their application in physiology.
Dr. Christoph Biskup studied Medicine and Chemistry in Cologne, Germany. Currently, he runs the laser scanning microscope laboratory at the Institute of Physiology II at the Friedrich-Schiller-University Jena. His research is focused on the development of fluorescence techniques that can be used to quantify cellular parameters and elucidate cellular regulatory networks. Developments include apart from sensors novel multidimensional fluorescence imaging techniques.
Gerhard J. Mohr received his Ph.D. in Chemistry (1996) at Karl-Franzens University Graz. Then he moved to the Centre for Chemical Sensors at ETH Zurich where he developed chemosensors based on reversible chemical reactions. Since 2001 he works at the Institute of Physical Chemistry in Jena as a Marie Curie and Heisenberg fellow, focusing on fluorescent sensor nanoparticles.