Stefan Nagl

Dual Fluorescence Sensor for Trace Oxygen and Temperature with Unmatched Range and Sensitivity

An optical dual sensor for oxygen and temperature is presented that is highly oxygen sensitive and covers a broad temperature range. Dual sensing is based on luminescence lifetime measurements. The novel sensor contains two luminescent compounds incorporated into polymer films. The temperature-sensitive dye (ruthenium tris-1,10-phenanthroline) has a highly temperature-dependent luminescence and is incorporated in poly(acrylonitrile) to avoid cross-sensitivity to oxygen. Fullerene C70 was used as the oxygen-sensitive probe owing to its strong thermally activated delayed fluorescence at elevated temperatures that is extremely oxygen sensitive. The cross-sensitivity of C70 to temperature is accounted for by means of the temperature sensor. C70 is incorporated into a highly oxygen-permeable polymer, either ethyl cellulose or organosilica. The two luminescent probes have different emission spectra and decay times, and their emissions can be discriminated using both parameters. Spatially resolved sensing is achieved by means of fluorescence lifetime imaging. The response times of the sensor to oxygen are short. The dual sensor exhibits a temperature operation range between at least 0 and 120 degrees C, and detection limits for oxygen in the ppbv range, operating for oxygen concentrations up to at least 50 ppmv. These ranges outperform all dual oxygen and temperature sensors reported so far. The dual sensor presented in this study is especially appropriate for measurements under extreme conditions such as high temperatures and ultralow oxygen levels. This dual sensor is a key step forward in a number of scientifically or commercially important applications including food packaging, for monitoring of hyperthermophilic microorganisms, in space technology, and safety and security applications in terms of detection of oxygen leaks.

Multiplex bacterial growth monitoring in 24-well microplates using a dual optical sensor for dissolved oxygen and pH.

Non‐invasive, simultaneous optical monitoring of oxygen and pH during bacterial cultivation in 24‐well microplates is presented using an integrated dual sensor for dissolved oxygen and pH values. The dual sensor is based on oxygen‐sensitive organosilica microparticles and pH‐sensitive microbeads from a polymethacrylate derivative embedded into a polyurethane hydrogel. The readout is based on a phase‐domain fluorescence lifetime‐based method referred to as modified frequency domain dual lifetime referencing using a commercially available detector system for 24‐well microplates. The sensor was used for monitoring the growth of Pseudomonas putida bacterial cultures. The method is suitable for parallelized, miniaturized bioprocessing, and cell‐based high‐throughput screening applications. Biotechnol. Bioeng. 2008;100: 430–438.

Method for simultaneous luminescence sensing of two species using optical probes of different decay time, and its application to an enzymatic reaction at varying temperature

AbstractChemical sensing, imaging and microscopy based on the use of fluorescent probes has so far been limited almost exclusively to the detection of a single parameter at a time. We present a scheme that can overcome this limitation by enabling optical sensing of two parameter simultaneously and even at identical excitation and emission wavelengths of two probes provided (a) their decay times are different enough to enable two time windows to be recorded, and (b) the emission of the shorter-lived probe decays to below the detectable limit while that of the other still can be measured. We refer to this new scheme as the dual lifetime determination (DLD) method and show that it can be widely varied by appropriate choice of probes and experimental settings. DLD is demonstrated to work by sensing oxygen and temperature independently from each other by making use of two probes, one for oxygen (a platinum porphyrin dissolved in polystyrene), and one for temperature [a europium complex dissolved in poly(vinyl methylketone)]. DLD was applied to monitor the consumption of oxygen in the glucose oxidase-catalyzed oxidation of glucose at varying temperatures. The scheme is expected to have further applications in cellular assays and biophysical imaging. FigurePrinciple behind the dual lifetime determination (DLD) method

Progress in microchip enantioseparations

Advances in microfluidic chips for chiral separations from 2003 to early 2009 are discussed. Microchip‐based separation techniques promise higher speed, throughput, portability, less sample and reagent consumption, better environmental compatibility, reduced cost and the prospect of system integration. Microchip electrophoresis is the most promising technique for miniaturized enantioseparations and has been performed with a variety of designs and analytes, however, other formats such as microchip electrochromatography are also gaining in popularity. Microchip fabrication, chemistry and detection issues are critically discussed and highlighted. Integration of enantioseparation techniques into multifunctional microchips are currently a rapidly advancing area of research and methods are discussed that may eventually enable enantioseparations to be the part of a holistic chemical microchip.

Protein–protein interaction analysis in single microfluidic droplets using FRET and fluorescence lifetime detection

Herein, we demonstrate the feasibility of a protein-protein interaction analysis and reaction progress monitoring in microfluidic droplets using FRET and microscopic fluorescence lifetime measurements. The fabrication of microdroplet chips using soft- and photolithographic techniques is demonstrated and the resulting chips reliably generate microdroplets of 630 pL and 6.71 nL at frequencies of 7.9 and 0.75 Hz, respectively. They were used for detection of protein-protein interactions in microdroplets using a model system of Alexa Fluor 488 labelled biotinylated BSA, Alexa Fluor 594 labelled streptavidin and unlabelled chicken egg white avidin. These microchips could be used for quantitative detection of avidin and streptavidin in microdroplets in direct and competitive assay formats with nanomolar detection limits, corresponding to attomole protein amounts. Four droplets were found to be sufficient for analytical determination. Fluorescence intensity ratio and fluorescence lifetime measurements were performed and compared for microdroplet FRET determination. A competitive on-chip binding assay for determination of unlabelled avidin using fluorescence lifetime detection could be performed within 135 s only.

Monitoring On‐Chip Pictet–Spengler Reactions by Integrated Analytical Separation and Label‐Free Time‐Resolved Fluorescence

High-throughput screening for optimal reaction conditions and the search for efficient catalysts is of eminent importance in the development of chemical processes and for expanding the spectrum of synthetic methodologies in chemistry. In this context we report a novel approach for a microfluidic chemical laboratory integrating organic synthesis, separation and time-resolved fluorescence detection on a single microchip. The feasibility of our integrated laboratory is demonstrated by monitoring the formation of tetrahydroisoquinoline derivatives by Pictet-Spengler condensation. After on-chip reaction the products and residual starting material were separated enantioselectively on the same chip. On-chip deep UV laser-induced fluorescence detection with time-correlated single photon counting was applied for compound assignment. The system was utilized to screen reaction conditions and various substrates for Pictet-Spengler reactions on-chip. Finally, the microlab was successfully applied to investigate enantioselective reactions using BINOL-based phosphoric acids as organocatalysts.

Microfluidic platforms employing integrated fluorescent or luminescent chemical sensors: a review of methods, scope and applications

Herein we critically review microfluidic platforms that contain integrated fluorescent or luminescent chemical sensor assemblies. These were employed in particular for miniaturized oxygen and pH sensing. Microchips with optical temperature sensing capability are also covered since these share many concepts and applications. Other analytes and derived parameters from the above analytes are found in some sensing approaches in microfluidics.After an introduction, the work is structured into three main chapters dealing with the fabrication and microintegration of these sensors, readout and detection strategies, and applications of these microsystems. The fabrication is discussed with a focus on soft lithography-based approaches in polydimethylsiloxane (PDMS) or PDMS and glass hybrid devices that form the majority of work so far. Alternative approaches, particularly using glass or quartz as the main chip material are also covered. Detection techniques employed to date are the subject of the next chapter, where simple intensity as well as lifetime- or wavelength-referenced schemes are presented and the utility of image-based sensing on the microscale is discussed.Lastly, exciting applications of these microfluidic chips are highlighted. Luminescent oxygen and pH sensing has been of particular interest in the field of microbioreactors but other areas are also of interest, particularly chemical reactors and electrophoresis. Optical temperature sensing is discussed and its use in fundamental studies as well as in enzyme reactors. Integrated microsystems with biosensing capabilities and some for monitoring of metal ions and other analytes are also presented.

Microfluidic chips for chirality exploration

Microfluidic chips applied to the investigation of chirality allow reaction, separation and analysis of minuscule amounts of enantiomeric molecules. Chiral chip technology is employed in fields as diverse as pharmaceutical high throughput screening and deep space exploration missions.

Rapid Isoelectric Point Determination in a Miniaturized Preparative Separation Using Jet-Dispensed Optical pH Sensors and Micro Free-Flow Electrophoresis

Herein, the fabrication, characterization, calibration, and application of integrated microfluidic platforms for fast isoelectric point (pI) determinations via free-flow electrophoresis with integrated inkjet-printed fluorescent pH sensor microstructures are presented. These devices allow one to determine the pI of a biomolecule from a sample mixture with moderately good precision and without addition of markers in typically less than 10 s total separation and analysis time. Polyhydroxyethyl methacrylate (pHEMA) hydrogels were covalently coupled with fluorescein and hydroxypyrene trisulfonic acid (HPTS)-based pH probes. These were piezoelectrically jet-dispensed onto acrylate-modified glass as pH sensor microarrays with a diameter of 300-600 μm and thicknesses of 0.4-2.4 μm with high spatial accuracy. Microchip fabrication and integration of these pH sensor arrays was realized by multistep liquid-phase photolithography from oligoethylene glycol precursors resulting in glass-based microfluidic free-flow isoelectric focusing (μFFIEF) chips with integrated pH observation capabilities. The microchips were characterized with regard to pH sensitivity, response times, photo-, and flow stability. Depending on the sensor matrix, they allowed IEF within a pH range of roughly 5.5-10.5 with good sensitivity and fast response times. These microchips were used for FFIEF of small molecule markers and several protein mixtures with simultaneous monitoring of local pH. This allowed the determination of their pI via multispectral imaging of protein and pH sensor fluorescence without addition of external markers. Obtained pIs were generally in good agreement with known data, demonstrating the applicability of the method for pI determination in micropreparative procedures within a time frame of a few seconds only.

Micro flow reactor chips with integrated luminescent chemosensors for spatially resolved on-line chemical reaction monitoring

Real-time chemical reaction monitoring in microfluidic environments is demonstrated using luminescent chemical sensors integrated in PDMS/glass-based microscale reactors. A fabrication procedure is presented that allows for straightforward integration of thin polymer layers with optical sensing functionality in microchannels of glass-PDMS chips of only 150 μm width and of 10 to 35 μm height. Sensor layers consisting of polystyrene and an oxygen-sensitive platinum porphyrin probe with film thicknesses of about 0.5 to 4 μm were generated by combining spin coating and abrasion techniques. Optimal coating procedures were developed and evaluated. The chip-integrated sensor layers were calibrated and investigated with respect to stability, reproducibility and response times. These microchips allowed observation of dissolved oxygen concentration in the range of 0 to over 40 mg L(-1) with a detection limit of 368 μg L(-1). The sensor layers were then used for observation of a model reaction, the oxidation of sulphite to sulphate in a microfluidic chemical reactor and could observe sulphite concentrations of less than 200 μM. Real-time on-line monitoring of this chemical reaction was realized at a fluorescence microscope setup with 405 nm LED excitation and CCD camera detection.