Dual Lifetime Referencing Research Articles

Optical ammonia sensors based on fluorescent aza-BODIPY dyes\u2014\xa0a flexible toolbox

We present three types of optical ammonia sensors suitable for environmental, bioprocess, and reaction monitoring. A respective fluorescent BF2-chelated tetraarylazadipyrromethene dye (aza-BODIPYs) is physically entrapped in a polyurethane hydrogel (HydroMed D4) forming an emulsion system with vinyl-terminated polydimethylsiloxane (PDMS). The analyte-sensitive layer is covered by a hydrophobic membrane which excludes hydrophilic substances. Three different protection layers are tested, whereby the Teflon and the hydrophobic PES layers outperform a PDMS/TiO2 layer. Response times within their dynamic range of 15 s can be achieved, whereas the PDMS/TiO2-covered sensor requires at least 390 s. The three sensors entail the following concentration areas: first sensor 3 μg L−1–3 mg L−1 (LOD 0.23 μg L−1), second sensor 0.1–30 mg L−1 (LOD 28 μg L−1), and third sensor 3 mg L−1–1 g L−1 (LOD 0.51 mg L−1). Readout is performed with a commercially available phase fluorimeter combined with optical fibers. Dual-lifetime referencing (DLR) is used as referencing method and Egyptian blue acts as an inert reference material. No cross-sensitivity to pH changes can be detected.

Dual lifetime referencing enables pH-control for oxidoreductions in hydrogel-stabilized biphasic reaction systems.

pH-shifts are a serious challenge in cofactor dependent biocatalytic oxidoreductions. Therefore, a pH control strategy was developed for reaction systems, where the pH value is not directly measurable. Such a reaction system is the biphasic aqueous-organic reaction system, where the oxidoreduction of hydrophobic substrates in organic solvents is catalysed by hydrogel-immobilized enzymes, and enzyme-coupled cofactor regeneration is accomplished via formate dehydrogenase, leading to a pH-shift. Dual lifetime referencing (DLR), a fluorescence spectroscopic method, was applied for online-monitoring of the pH-value within the immobilizates during the reaction, allowing for a controlled dosage of formic acid. It could be shown that by applying trisodium 8-hydroxypyrene-1, 3, 6-trisulfonate as pH indicator and Ru(II) tris(4, 7-diphenyl-1, 10-phenantroline) (Ru[dpp]) as a reference luminophore the control of the pH-value in a macroscopic gel-bead-stabilized aqueous/organic two phase system in a range of pH 6.5 to 8.0 is possible. An experimental proof of concept could maintain a stable pH of 7.5 ± 0.15 during the reaction for at least 105 h. With these results, it could be shown that DLR is a powerful tool for pH-control within reaction systems with no direct access for conventional pH-measurement.

Development of microscopic time‐domain dual lifetime referencing luminescence detection for pH monitoring in microfluidic free‐flow isoelectric focusing

A lifetime‐based ratiometric microscale pH sensor system and a fluorescence microscopic setup was developed for the in‐line observation of pH in free‐flow isoelectric focusing based on the principle of time‐domain dual lifetime referencing (t‐DLR). The t‐DLR method has been developed for pH monitoring and various other applications in the fields of environmental monitoring and biotechnology. Here, we introduce the integration of pH sensor microstructures for t‐DLR in microfluidic channels and the application of the t‐DLR scheme for pH sensing in miniaturized electrophoretic procedures. The pH sensor was inkjet‐printed on glass in rows with a length of 10 mm, a height of 404 ± 18 nm, and a width of 371 ± 28 μm and integrated into a microfluidic chip generated by a laser cutting and lamination technique. It had a working range from pH 4 to 8 with a pKa of 6.10 ± 0.01 and fast response times under 500 ms. The sensor was used for the in‐line observation of the pH gradient during isoelectric focusing of the proteins β‐lactoglobulin A, conalbumin, and myoglobin and their identification by their pI. The obtained pIs were in good agreement with literature data demonstrating the applicability of the pH sensor in microfluidic continuous separations.

Dual lifetime referenced fluorometry for the determination of doxorubicin in urine

Dual lifetime referencing (DLR) is introduced as a rapid and self-referenced method for measuring the concentration of a fluorescent analyte in solution. The fluorescent cancer chemotherapeutic doxorubicin was chosen as a medically relevant analyte and blended with a reference dye (Ru(dpp)3) that displays overlapping excitation and emission spectra. The relative contributions of the short-lived (nanoseconds) fluorescent analyte and the long-lived (microseconds) reference dye define the observed lifetime. Measuring this lifetime by both frequency-domain DLR and time-domain DLR yields similar analytical ranges and limits of detection (0.4μM). To assess the matrix effect of medical samples, the standard addition method was employed to both modes of DLR. Urine was spiked with doxorubicin and recovery rates of ≥97% were obtained.

Dual-lifetime referencing (DLR): a powerful method for on-line measurement of internal pH in carrier-bound immobilized biocatalysts

BackgroundIndustrial-scale biocatalytic synthesis of fine chemicals occurs preferentially as continuous processes employing immobilized enzymes on insoluble porous carriers. Diffusional effects in these systems often create substrate and product concentration gradients between bulk liquid and the carrier. Moreover, some widely-used biotransformation processes induce changes in proton concentration. Unlike the bulk pH, which is usually controlled at a suitable value, the intraparticle pH of immobilized enzymes may deviate significantly from its activity and stability optima. The magnitude of the resulting pH gradient depends on the ratio of characteristic times for enzymatic reaction and on mass transfer (the latter is strongly influenced by geometrical features of the porous carrier). Design and selection of optimally performing enzyme immobilizates would therefore benefit largely from experimental studies of the intraparticle pH environment. Here, a simple and non-invasive method based on dual-lifetime referencing (DLR) for pH determination in immobilized enzymes is introduced. The technique is applicable to other systems in which particles are kept in suspension by agitation.ResultsThe DLR method employs fluorescein as pH-sensitive luminophore and Ru(II) tris(4,7-diphenyl-1,10-phenantroline), abbreviated Ru(dpp), as the reference luminophore. Luminescence intensities of the two luminophores are converted into an overall phase shift suitable for pH determination in the range 5.0-8.0. Sepabeads EC-EP were labeled by physically incorporating lipophilic variants of the two luminophores into their polymeric matrix. These beads were employed as carriers for immobilization of cephalosporin C amidase (a model enzyme of industrial relevance). The luminophores did not interfere with the enzyme immobilization characteristics. Analytical intraparticle pH determination was optimized for sensitivity, reproducibility and signal stability under conditions of continuous measurement. During hydrolysis of cephalosporin C by the immobilizate in a stirred reactor with bulk pH maintained at 8.0, the intraparticle pH dropped initially by about 1 pH unit and gradually returned to the bulk pH, reflecting the depletion of substrate from solution. These results support measurement of intraparticle pH as a potential analytical processing tool for proton-forming/consuming biotransformations catalyzed by carrier-bound immobilized enzymes.ConclusionsFluorescein and Ru(dpp) constitute a useful pair of luminophores in by DLR-based intraparticle pH monitoring. The pH range accessible by the chosen DLR system overlaps favorably with the pH ranges at which enzymes are optimally active and stable. DLR removes the restriction of working with static immobilized enzyme particles, enabling suspensions of particles to be characterized also. The pH gradient developed between particle and bulk liquid during reaction steady state is an important carrier selection parameter for enzyme immobilization and optimization of biocatalytic conversion processes. Determination of this parameter was rendered possible by the presented DLR method.

Dual lifetime referenced trace ammonia sensors

Dual lifetime referencing is applied to trace ammonia sensors. Two schemes are presented to achieve luminescence lifetime based measurements in the frequency domain: the first is based on a resonance energy transfer (RET) system with eosin ethylester as indicator applying a coumarin derivative as donor. The second consists of a resonance energy transfer cascade with a coumarin derivative, sulforhodamine B and the absorption dye bromophenol blue as ammonia-sensitive dye. The large Stokes shift due to the energy transfer and the resulting spectral properties of the sensing layers enable dual lifetime referencing using an Ir(III)-coumarin complex for both ammonia detection systems. The sensing systems are applicable for the monitoring of ammonia in the range of either 1–5000μg/l or 50–50,000μg/l, respectively.

Luminescence decay time encoding of magnetic micro spheres for multiplexed analysis

Magnetic microspheres are optically encoded by doping with three luminescent dyes. The combination of a fluorophore with a nanosecond decay profile and two phosphorescent Ruthenium metal ligand complexes with a microsecond decay profile generates a characteristic signature described by three features: bead brightness, luminescent decay time and dual lifetime referencing (DLR). The beads are identified by time resolved imaging in the microsecond range. A series of fluorophores is tested and the interference of the resulting luminescent code in the red and green label detection channels is investigated. A detailed staining procedure is worked out to increase the staining efficiency of the dyes with hydrophilic character into the lipophilic polystyrene microspheres. A mathematical model is established to calculate the dye amounts that are needed for staining a bead family with a specific feature set. Nineteen bead families were prepared representing the grid points in the three planes of a cube referring to the three features. The coefficient of variation over all bead families is 7%, 1.4% and 1.6% for bead brightness, luminescence decay time and DLR, respectively. The combination of these features and the bead size as additional feature enables the creation of 840 distinguishable bead families.

Dual lifetime referenced optical sensor membrane for the determination of copper(II) ions

A sensor membrane has been developed for the determination of copper(II) ions that displays excellent performance due to internal referencing of luminescence intensities. The applied sensing scheme (dual lifetime referencing) makes use of the indicator lucifer yellow (LY) and an inert reference luminophore (a ruthenium complex entrapped in polyacrylonitrile beads). Both are contained in a hydrogel matrix. The copper-dependent fluorescence intensity change of LY can be converted in either a phase shift or time dependent parameter. The sensing membrane is capable of determining copper(II) with an outstanding high selectivity over a dynamic range between 1 and 1000 μM in neutral or weekly acidic conditions. The advantages of the referencing method over intensity based measurements was demonstrated by the measurement of turbid solutions. The scheme was also applied to two-dimensional measurements in the time domain. Sensor integrated microtiterplates were imaged with a CCD camera gated with square pulses in the microsecond range.

Dual lifetime referencing as applied to a chloride optical sensor.

A membrane with an optical response to chloride has been developed that contains two luminophores that display two largely different decay times. The first luminophore (the "reference") is a chloride-insensitive ruthenium metal-ligand complex possessing a decay time in the microsecond range. The second luminophore is the short-lived chloride-quenchable fluorescent probe lucigenin. Both are contained in a hydrogel matrix and are excited by a blue LED emitting sinusoidally modulated light. Under these conditions, the chloride-dependent fluorescence intensity of lucigenin can be converted in an analyte-dependent fluorescence phase shift that depends on the ratio of the two luminescence intensities and can be measured at modulation frequencies of typically 45 kHz. The dynamic range of this sensor can be adjusted by either varying the ratio of the two luminophores or selecting a particular optical filter combination.

Optical sensor for seawater salinity.

An optical sensor for the measurement of salinity in seawater has been developed. It is based on a chloride-quenchable fluorescent probe (lucigenin) immobilized on a Nafion film. Two approaches for measuring salinity via chloride concentration are presented. In the first, a change in salinity corresponds to a change in the fluorescence intensity of lucigenin. In the second, the fluorescence intensity information is converted into a phase angle information by adding an inert phosphorescent reference luminophore (a ruthenium complex entrapped in poly(acrylonitrile) beads). Under these conditions, the chloride-dependent fluorescence intensity of lucigenin can be converted into a chloride-dependent fluorescence phase shift which serves as the analytical information. This scheme is referred to as dual lifetime referencing (DLR). The sensor was used to determine the salinity in seawater and brackish water of the North Sea.