Leah Tolosa

Optical assay for glucose based on the luminescnence decay time of the long wavelength dye Cy5

An optical assay for glucose is described based on the luminescence decay time of a long wavelength dye (Cy5) which can be excited with currently available red laser diodes. Concanavalin A was covalently labeled with Cy5 which served as the donor in an assay based on fluorescence resonance energy transfer (FRET). The acceptor was Malachite Green which was covalently linked to insulin which served as a carrier protein. To provide binding affinity for ConA Malachite Green insulin was also covalently labeled with maltose (MIMG). Binding of Cy5ConA to MIMG resulted in a decreased intensity and decay time of Cy5 as observed by time-correlated single photon counting. Glucose was detected by competitive displacement of MIMG from Cy5ConA, resulting in increased intensity and decay time. This glucose assay has several features which can result in practical real world assays for glucose. The long absorption wavelength of Cy5 allows excitation with red laser diodes, which can be readily pulsed or amplitude-modulated for time-domain or frequency-domain decay time measurements. Additionally, decay times can be measured through skin using long wavelength excitation and emission, suggesting the possibility of an implanted glucose sensor. And finally, the assay affinity and reversibility can in principle be adjusted by controlling the extent and type of sugar labeling of the carrier protein.

Directional Surface Plasmon-Coupled Emission from a 3 nm Green Fluorescent Protein Monolayer

High‐sensitivity detection schemes are of great interest for a number of applications. Unfortunately, such schemes are usually high‐cost. We demonstrate a low‐cost approach to a high‐sensitivity detection scheme based on surface plasmon‐coupled emission (SPCE). The SPCE of a monomolecular layer of green fluorescent protein (GFP) is reported here. The protein was electrostatically attached to a thin, SiO2‐protected silver film deposited on a quartz substrate. The visible, directional emission of GFP was observed at a sharp, well‐defined angle of 47.5° from the normal to the coupling prism, and the spectrum corresponded to that of GFP. The SPCE resulting from the reverse Kretschmann configuration showed a 12‐fold enhancement over the free space fluorescence. The directional emission was 97% p‐polarized. The directionality and high polarization can be coupled with the intrinsic spectral resolution of SPCE to be used in the design miniaturized spectrofluorometers. The observation of SPCE in the visible region of the spectrum from a monolayer of protein opens up new possibilities in protein‐based sensing.

Dual optical sensor for oxygen and temperature based on the combination of time domain and frequency domain techniques

In measuring specific conditions in the real world, there are many situations where both the oxygen concentration and the temperature have to be determined simultaneously. Here we describe a dual optical sensor for oxygen and temperature that can be adapted for different applications. The measurement principle of this sensor is based on the luminescence decay times of the oxygen-sensitive ruthenium complex tris-4,7-diphenyl-1,10-phenanthroline ruthenium(III) [Rudpp] and the temperature-sensitive europium complex tris(dibenzoylmethane) mono(5-amino-1,10-phenanthroline)europium(III) [Eudatp]. The excitation and emission spectra of the two luminophores overlap significantly and cannot be discriminated in the conventional way using band pass filters or other optical components. However, by applying both the frequency and time domain techniques, we can separate the signals from the individual decay time of the complexes. The europium complex is entrapped in a poly(methyl methacrylate) (PMMA) layer and the ruthenium complex is physically adsorbed on silica gel and incorporated in a silicone layer. The two layers are attached to each other by a double sided silicone based tape. The europium sensing film was found to be temperature-sensitive between 10 and 70°C and the ruthenium oxygen-sensitive layer can reliably measure between 0 and 21% oxygen.

Microsecond dynamics of biological macromolecules.

Publisher Summary This chapter discusses that fluorescence spectroscopy is widely used to study the nanosecond timescale dynamics of biological macromolecules. The spectral observables are sensitive to nanosecond dynamics because emission also occurs on the nanosecond time scale. While nanosecond biopolymers dynamics are important, these rapid processes reflect mostly local fluorophore motions and its interactions with the immediate environment. However, biological macromolecules also display structural changes on the microsecond time scale. Processes that occur on the microsecond time scale include domain flexing in proteins and lateral diffusion in membranes. It is also likely that nucleic acid junctions and structured RNAs display microsecond motions. It discusses that fluorescence is now capable of detecting microsecond dynamics. This change in time scale is made possible by the development of metal-ligand complexes (MLCs), which display decay times ranging from 10 nsec to more than 10/μsec. The MLCs display several spectral characteristics that make them useful probes, including high photostability, a large Stokes shift, and polarized emission. The chapter presents data and simulations showing the possibility of measuring protein domain flexing, lateral diffusion in membranes, and microsecond rotational correlation times. Fluorescence is no longer trapped on the nanosecond time scale, and can be used to quantify dynamic processes from nanoseconds to microseconds to milliseconds because the lanthanides display millisecond decay times.

Development and application of an excitation ratiometric optical pH sensor for bioprocess monitoring

The development of a fluorescent excitation ratiometric pH sensor (AHQ‐PEG) using a novel allylhydroxyquinolinium (AHQ) derivative copolymerized with polyethylene glycol dimethacrylate (PEG) is described. The AHQ‐PEG sensor film is shown to be suitable for real‐time, noninvasive, continuous, online pH monitoring of bioprocesses. Optical ratiometric measurements are generally more reliable, robust, inexpensive, and insensitive to experimental errors such as fluctuations in the source intensity and fluorophore photobleaching. The sensor AHQ‐PEG in deionized water was shown to exhibit two excitation maxima at 375 and 425 nm with a single emission peak at 520 nm. Excitation spectra of AHQ‐PEG show a decrease in emission at the 360 nm excitation and an increase at the 420 nm excitation with increasing pH. Accordingly, the ratio of emission at 420:360 nm excitation showed a maximum change between pH 5 and 8 with an apparent pKa of 6.40. The low pKa value is suitable for monitoring the fermentation of most industrially important microorganisms. Additionally, the AHQ‐PEG sensor was shown to have minimal sensitivity to ionic strength and temperature. Because AHQ is covalently attached to PEG, the film shows no probe leaching and is sterilizable by steam and alcohol. It shows rapid (∼2 min) and reversible response to pH over many cycles without any photobleaching. Subsequently, the AHQ‐PEG sensor film was tested for its suitability in monitoring the pH of S. cereviseae (yeast) fermentation. The observed pH using AHQ‐PEG film is in agreement with a conventional glass pH electrode. However, unlike the glass electrode, the present sensor is easily adaptable to noninvasive monitoring of sterilized, closed bioprocess environments without the awkward wire connections that electrodes require. In addition, the AHQ‐PEG sensor is easily miniaturized to fit in microwell plates and microbioreactors for high‐throughput cell culture applications.

On the Possibility of Real-Time Monitoring of Glucose in Cell Culture by Microdialysis Using a Fluorescent Glucose Binding Protein Sensor

Although glucose sensors with millimolar sensitivity are still the norm, there is now a developing interest in glucose sensors with micromolar sensitivity for applications in minimally invasive sampling techniques such as fast microdialysis and extraction of interstitial fluid by iontophoresis and laser poration. In this regard, the glucose binding protein (GBP) with a binding constant for glucose in the micromolar range is of particular relevance. GBP is one of the soluble binding proteins found in the periplasmic space of Gram‐negative bacteria. Because of its hinge‐like tertiary structure, glucose binding induces a large conformational change, which can be used for glucose sensing by attaching a polarity sensitive fluorescent probe to a site on the protein that is allosterically responsive to glucose binding. Correspondingly, the resulting optical biosensor has micromolar sensitivity to glucose. Because binding is reversible, the biosensor is reusable and can be stored at 4 °C for 6 months without losing its sensitivity. In this paper, we show the feasibility of using the GBP biosensor to monitor glucose in microdialysis. The effect of perfusion rate, bulk glucose concentration and temperature on microdialysis efficiency was determined. Additionally, the glucose concentrations in mammalian cell culture were monitored to demonstrate the applicability of this sensor in complex and dynamic processes over a period of time. As the sensor is sensitive to micromolar glucose, high dialysis efficiency is not required when the bulk glucose concentration is within the millimolar physiological range. Thus, a perfusion rate of 10 μL/min or faster can be used, resulting in delay times of 1 min or less.

On the design of low-cost fluorescent protein biosensors.

There is a large body of knowledge on proteins and their ligands that is available to the sensor researcher for the successful design of fluorescent biosensors. Chemically synthesized receptors rarely match the sensitivity and selectivity of proteins.Additionally, proteins are easily produced and manipulated through recombinant protein techniques. Although limitations exist in the prediction of signal response of proteins labeled with fluorescent probes, thoughtful experimentation can lead to useful, highly responsive fluorescent protein assays. Conversion of these assays into sensor devices may require additional manipulation of the fluorescence properties of the labeled proteins. We have shown that this can be achieved by a second fluorophore serving as a reference for ratiometric measurements. The choice of reference is contingent on the low-cost, miniaturized design of the device. Accordingly, the reference fluorophore is excitable with the same LED as the signal transducing probe and has a fluorescence decay lifetime that is orders of magnitude longer.Alternating illumination with intensity modulated light at two frequencies allows for ratiometric sensing without the need for bulky filter wheels while collecting the signals over a wide range of emission wavelengths. The result is a simple optoelectronics design that is cost-effective and small enough to be portable.In summary, the process of designing protein-based fluorescent biosensors for practical applications requires the systematic collaboration of a cross-disciplinary group of molecular biologists, chemists and engineers.

Comparing the performance of the optical glucose assay based on glucose binding protein with high-performance anion-exchange chromatography with pulsed electrochemical detection: efforts to design a low-cost point-of-care glucose sensor.

Background: The glucose binding protein (GBP) is one of many soluble binding proteins found in the periplasmic space of gram-negative bacteria. These proteins are responsible for chemotactic responses and active transport of chemical species across the membrane. Upon ligand binding, binding proteins undergo a large conformational change, which is the basis for converting these proteins into optical biosensors. Methods: The GBP biosensor was prepared by attaching a polarity-sensitive fluorescent probe to a single cysteine mutation at a site on the protein that is allosterically responsive to glucose binding. The fluorescence response of the resulting sensor was validated against high-performance anion-exchange chromatography (HPAEC) with pulsed electrochemical detection. Finally, a simple fluorescence reader was built using a lifetime-assisted ratiometric technique. Results: The GBP assay has a linear range of quantification of 0.100–2.00 μM and a sensitivity of 0.164 μM−1 under the specified experimental conditions. The comparison between GBP and HPAEC readings for nine blind samples indicates that there is no statistical difference between the analytical results of the two methods at the 95% confidence level. Although the methods of fluorescence detection are based on different principles, the response of the homemade device to glucose concentrations was comparable to the response of the larger and more expensive tabletop fluorescence spectrophotometer. Conclusions: A glucose binding protein labeled with a polarity-sensitive probe can be used for measuring micromolar amounts of glucose. Using a lifetime-assisted ratiometric technique, a low-cost GBP-based micromolar glucose monitor could be built.

Polarization-Based Sensing with a Self-Referenced Sample

We describe a new method of fluorescence sensing based on fluorescence polarization. The sensor consists of two compartments, both of which contain the sensing fluorophore. One side of the sensor contains a constant concentration of analyte, and the other contains the unknown concentration. Emission from both sides is observed through polarizers, with the polarization from the sample being rotated 90° from that of the reference. Changes in the fluorescence intensity of the sample result in changes in the measured polarization for the combined emission. We show that this approach can be used to measure glucose and calcium using fluorophores which show analyte-dependent intensity changes, and no change in the spectral shape. Only a single fluorophore is required, this being the sensing fluorophore in both sides of the sensor. We also show that polarization sensing of glucose and calcium can be performed with visual detection of the polarization. In this case the only electronic component is the light source. These simple schemes can be used with a variety of analytes. The only requirement is a change in fluorescence intensity in response to the analyte.

Glucose monitoring in neonates: need for accurate and non-invasive methods.

Neonatal hypoglycaemia can lead to devastating consequences. Thus, constant, accurate and safe glucose monitoring is imperative in neonatal care. However, point-of-care (POC) devices for glucose testing currently used for neonates were originally designed for adults and do not address issues specific to neonates. This review will address currently available monitoring options and describe new methodologies for non-invasive glucose monitoring in newborns.