Analytische Chemie
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In recent years, the demand for reliable, versatile, fluorescent pH and oxygen sensors has increased rapidly in many biomedical applications since these analytes are important indicators of cell function or certain diseases. Therefore, sensor particles are needed that are small enough to penetrate cells, non-toxic, and allow for close-up optical monitoring. When developing such sensor systems, one must consider the pH and oxygen range detectable by the sensor dye and the matrix material of the used carrier particles. Here, we present the development of pH- and oxygen-responsive polymeric beads functionalized with fluorescent dyad molecules that consist of an analyte-responsive fluorophore and an analyte-inert dye.
Optical pH sensors utilizing colorimetric or fluorescent indicator dyes are highly promising in many biomedical and life science applications where electrochemical sensors fail. For instance, optical sensors are not prone to electrical interferences, they are noninvasive and enable remote measurements. Moreover, fluorescence detection is very fast, highly sensitive, and provides several readout parameters ideal for multiplexing with nanometer resolution using simple, inexpensive, and miniaturizable instrumentation. Here, we present the design of a dyad sensor molecule, consisting of an analyte-responsive and an analyte inert reference fluorophore.
pH presents one of the most important analytes in the life and material sciences, indicating, e.g., diseases and corrosion processes. This includes the optical monitoring of pH in living cells for studying cellular internalization pathways, such as phagocytosis, endocytosis, and receptor mediated internalization with the aid of molecular and nanoscale fluorescent sensors. Nanoparticle (NP)-based sensors, that are labelled or stained with a multitude of sensor dyes, have several advantages as compared to conventional molecular probes like enhanced brightness, i.e., amplified signals, ease of designing ratiometric systems by combining analyte-sensitive and inert reference dyes, and increased photostability. Moreover, this can enable the use of hydrophobic dyes in aqueous environments. Versatile templates and carriers for the fabrication of nanosensors by staining and/or labelling with different fluorophores and sensor molecules are surface-functionalized particles like silica (SiO2) and polystyrene (PS) particles. Here we present a platform of blue-red-green fluorescent pH nanosensors for the measurement of acidic, neutral, and basic pH utilizing both types of matrices and two spectrally distinguishable sensor dyes with an integrated reference dye and demonstrate its applicability for cellular studies.
Fluorescence imaging microscopy is an essential tool in biomedical research. Meanwhile, various fluorescent probes are available for the staining of cells, cell membranes, and organelles. Though, to monitor intracellular processes and dysfunctions, probes that respond to ubiquitous chemical parameters determining the cellular function such as pH, pO2, and Ca2+ are required. This review is focused on the progress in the design, fabrication, and application of photoluminescent nanoprobes for sensing and imaging of pH in living cells. The advantages of using nanoprobes carrying fluorescent pHindicators compared to single molecule probes are discussed as well as their limitations due to the mostly lysosomal uptake by cells. Particular attention is paid to ratiometric dual wavelength nanosensors that enable intrinsic referenced measurements. Referencing and proper calibration procedures are basic prerequisites to carry out reliable quantitative pH determinations in complex samples such as living cells. A variety of examples will be presented that highlight the diverseness of nanocarrier materials (polymers, micelles, silica, quantum dots, carbon dots, gold, photon upconversion nanocrystals, or bacteriophages), fluorescent pH indicators for the weak acidic range, and referenced sensing mechanisms, that have been applied intracellularly up to now.