Axel Duerkop

Detection of nanomolar concentrations of copper(II) with a Tb-quinoline-2-one probe using luminescence quenching or luminescence decay time.

We present a time-resolved (gated) luminescence-based method for determination of Cu2+ ions in microtiterplate format in the nanomolar concentration range using the novel long-lived terbium-[1-methyl-4-hydroxy-3-(N-2-ethyl-5-aminothiadiazolyl-)-carbamoyl-quinoline-2-one] (TbL) complex. The probe works best in Tb:L = 1:2 stoichiometry at neutral pH. The dynamic range is from 10 to 300 nmol L(-1) of Cu2+ and the limit of detection is 4.3 nmol L(-1). This is the lowest limit of detection achieved so far for luminescent lanthanide-based probes for copper. It is shown that gating can efficiently suppress intense, short decaying background fluorescence e.g. that of Rhodamine 6G. The assay can be performed by measurement of luminescence decay time, as well. Stern-Volmer studies indicate that static quenching dominates over dynamic quenching. TbL2 was tested for the effect of some relevant interferents and the assay was applied to the determination of copper in tap water samples. The results achieved were in good agreement with those of a reference method.

SDS-PAGE of proteins using a chameleon-type of fluorescent prestain.

A new prestaining method for protein SDS-PAGE was developed using the fluorogenic amino-reactive label Py-1. This resulted in one of the fastest, most sensitive, and environmentally friendly protocols available. It is mainly due to the unique optical properties of Py-1, which is blue and virtually nonfluorescent but turns to red and becomes much more strongly fluorescent once it is conjugated to the amino group of a protein. Staining times of 30 min are adequate to visualize subnanogram quantities of proteins because pre-electrophoretic labeling Py-1 does not require the time-consuming steps of washing or fixation of gels. LODs as low as 16 pg of protein are found which is better than the best (commercial) poststains and comparable to the best (commercial) prestains. In addition, prestaining requires marginal amounts of staining solution. The change in electrophoretic mobility and band broadening is at a low level because Py-1 causes a mass shift of 288 Da per bound molecule only. By virtue of the small mass shift it causes, this stain is compatible with mass spectrometric protein analysis even though it acts as a covalent label.

Time-Resolved Fluorescence-Based Assay for the Determination of Alkaline Phosphatase Activity and Application to the Screening of Its Inhibitors

A single-step end point method is presented for determination of the activity of the enzyme alkaline phosphatase (ALP) using the effect of enhancement of fluorescence of the easily accessible europium(III)-tetracycline 3:1 complex (Eu3TC). Its luminescence, peaking at 616 nm if excited at 405 nm, is enhanced by a factor of 2.5 in the presence of phosphate. Phenyl phosphate was used as a substrate that is enzymatically hydrolyzed to form phenol and phosphate. The latter coordinates to Eu3TC and enhances its luminescence intensity as a result of the displacement of water from the inner coordination sphere of the central metal. The assay is performed in a time-resolved (gated) mode, which is shown to yield larger signal changes than steady-state measurement of fluorescence. The limit of detection for ALP is 4 µmol L—1. Based on this scheme, a model assay for theophylline as inhibitor for ALP was developed with a linear range from 14 to 68 µmol L— 1 of theophylline. (Journal of Biomolecular Screening 2008:9-16)

Luminescence recognition of different organophosphorus pesticides by the luminescent Eu(III)–pyridine-2,6-dicarboxylic acid probe

Luminescence quenching of a novel long lived Eu(III)-pyridine-2,6-dicarboxylic acid probe of 1:2 stoichiometric ratio has been studied in 0.10 volume fraction ethanol-water mixture at pH 7.5 (HEPES buffer) in the presence of the organophosphorus pesticides chlorfenvinphos (P1), malathion (P2), azinphos (P3), and paraxon ethyl (P4). The luminescence intensity of Eu(III)-(PDCA)(2) probe decreases as the concentration of the pesticide increases. It was observed that the quenching due to P3 and P4 proceeds via both diffusional and static quenching processes. Direct methods for the determination of the pesticides under investigation have been developed using the luminescence quenching of Eu(III)-pyridine-2,6-dicarboxylic acid probe in solution. The linear range for determination of the selected pesticides is 1.0-35.0 μM. The detection limits were 0.24-0.55 μM for P3, P4, and P1 and 2.5 μM for P2, respectively. The binding constants (K), and thermodynamic parameters of the OPs with Eu(III)-(PDCA)(2) were evaluated. Positive and negative values of entropy (ΔS) and enthalpy (ΔH) changes for Eu(III)-(PDCA)(2)-P1 ternary complex were calculated. As the waters in this study do not contain the above mentioned OPs over the limit detectable by the method, a recovery study was carried out after the addition of the adequate amounts of the organophosphorus pesticides under investigation.

Intrinsically Referenced Fluorimetric Sensing and Detection Schemes: Methods, Advantages and Applications

The precision of analytical methods using fluorescent probes or biomolecular labels often is compromised of a variety of conceivable interferences that may originate from the instrumental system, the sample, or the underlying sensor chemistry. Instrumental drifts of the optoelectronic system, photobleaching of luminophores, or high intrinsic color and background fluorescence of the sample cannot be eliminated even by extensive calibration procedures. As a result, intrinsically referenced methods are preferred to improve optical chemo- or biosensor technology, and fluorescent bioimaging. Intrinsic referencing is often accomplished by ratiometric techniques. These include (1) dual wavelength probes, (2) dual luminophore sensors, (3) lifetime-based assays, (4) dual lifetime referencing (in the time and frequency-domain), and (5) fluorescence anisotropy. Applications and advantages of the various approaches are outlined in this review, with a focus on widely used sensing methods for oxygen, pH, carbon dioxide, calcium, glucose, or temperature, and on biomolecular screening. In addition to ratiometric methods, fluorescence correlation spectroscopy represents another attractive tool to determine analyte concentrations via fluorescent probes. Many of these ratiometric approaches have the potential to pave the way for the development of calibration-free sensor and imaging schemes.

High-throughput sensing microtiter plate for determination of biogenic amines in seafood using fluorescence or eye-vision

A new optical sensing microplate was developed for rapid screening for the presence of biogenic amines (BAs) in seafood samples with high sensitivity. The deposition of a sensing spot (containing a chameleon dye (Py-1) in a polymeric cocktail) on the bottom of the wells of a standard microplate renders the plate a new sensing tool for a rapid and parallel detection of up to 96 (real) samples. This sensing microplate enables (1) a semi-quantitative readout of analyte concentration by eye-vision, (2) a rapid fluorescence readout of 96 samples with standard instrumentation in less than two minutes (unlike chromatographic and electrophoretic methods), (3) a statistically robust data evaluation (with 8-12 replicates) and (4) a rapid parallel sample preparation with standard 8 or 12-channel micropipettes. On reaction with biogenic amines, the dye shows a significant visible color change from blue over green to red color. The appearance of red color favorably coincides with the concentration of BAs that can induce symptoms of poisoning. The linear ranges of fluorescence calibration data for six biogenic amines cover the clinical toxicological relevant range of BAs that is too low to be detected by the human nose. The LODs range from 0.16 to 0.56 μg mL(-1), with correlation coefficients (r(2)) between 0.985 and 0.999. Finally, the evolution of spoilage of four fish samples (monitored by determination of their BA status) and the increase of their total amine content were found to agree well with previous data on time-dependent evolution of BAs in fish.

A New Fluorescent PET Probe for Hydrogen Peroxide and its Use in Enzymatic Assays for L-Lactate and D-Glucose

We present a new probe for the determination of hydrogen peroxide (HP). It is based on the yellow fluorophore 4‐amino‐1,8‐napththalimide, coupled to p‐anisidine (as a redox‐active group) to form a probe that is based on photoinduced electron transfer (PET). The preparation of the probe (which we refer to as “HP Green”) was accomplished in four steps with good yield. Its fluorescence is independent of pH in the physiological range and quenched by a PET process that occurs between the p‐anisidine redox moiety and the naphthalimide luminophore. If the p‐anisidine group is oxidized by HP, PET is suppressed and fluorescence intensity is strongly increased. Addition of horseradish peroxidase (HRP) enhances the oxidation of HP Green and further improves the detection limit of HP. The use of HRP and HP Green enables the determination of HP concentration in a range of 0.1 to 5 μM, with a limit of detection (LOD) as low as 64 nM (16 pmol per well in microtiter plates). HP Green and HRP also enable sensitive enzymatic assays of oxidase substrates in a kinetic format, as shown for L‐lactate and D‐glucose. L‐Lactate concentration can be rapidly determined between 0.5 and 10 μM after 6 minutes of incubation at 30 °C, with an LOD of 164 nm (41 pmol per well). This LOD is more than sixfold lower than that of the best commercial assays for lactate. The detection range for D‐glucose is 2 to 30 μm, and the LOD is 644 nM (161 pmol per well). These are among the lowest concentrations detectable for oxidase‐based assays. The hexanoic acid moiety in HP Green may be further used to immobilize the probe in order to obtain sensor layers for continuous assays.

A Fluorescent Probe for Diacetyl Detection

A water-soluble fluorescent probe, rhodamine B hydrazide (RBH), was prepared and its properties for recognition of diacetyl were studied. The method employs the reaction of diacetyl with RBH, a colorless and non-fluorescent rhodamine B spiro form derivative to give a pink-colored fluorescent substance. In weakly acidic media, RBH reacts more selectively with diacetyl than with other carbonyls, causing a large increase in fluorescence intensity and thereby providing an easy assay for the determination of diacetyl.

A novel luminescent terbium-3-carboxycoumarin probe for time-resolved fluorescence sensing of pesticides methomyl, aldicarb and prometryne

The luminescence arising from lanthanide cations offers several advantages over organic fluorescent molecules: sharp, distinctive emission bands allow for easy resolution between multiple lanthanide signals; long emission lifetimes (μs-ms) make them excellent candidates for time-resolved measurements; and high resistance to photo bleaching allow for long or repeated experiments. A time-resolved (gated) luminescence-based method for determination of pesticides methomyl, aldicarb and prometryne in microtiterplate format using the long-lived terbium-3-carboxycoumarin in 1:3 metal:ligand ratio has been developed. The limit of detection is 1.20×10(6), 5.19×10(5) and 2.74×10(6)ng L(-1) for methomyl, prometryne and aldicarb, respectively. The quantum yield (QY=0.08) of Tb(III)-3-carboxycoumarin was determined using 3-(2-benzothiazolyl)-7-diethylamino-coumarin (coumarin 6). Stern-volmer studies at different temperatures indicate that collisional quenching dominates for methomyl, aldicarb and prometryne. Binding constants were determined at 303, 308 and 313 K by using Lineweaver-Burk equation. A thermodynamic analysis showed that the reaction is spontaneous with negative ΔG. Effect of some relevant interferents on the detection of pesticides has been investigated.

Enzyme-Based Test Strips for Visual or Photographic Detection and Quantitation of Gaseous Sulfur Mustard

Sulfur mustard is a chemical agent of high military and terroristic significance. No effective antidote exists, and sulfur mustard can be fairly easily produced in large quantity. Rapid field testing of sulfur mustard is highly desirable. Existing analytical devices for its detection are available but can suffer from low selectivity, laborious sample preparation, and/or the need for complex instrumentation. We describe a new kind of test strip for rapid detection of gaseous sulfur mustard that is based on its degradation by the enzyme haloalkane dehalogenase that is accompanied by a change of local pH. This change can be detected using pH indicators contained in the strips whose color changes from blue-green to yellow within 10 min. In addition to visual read-out, we also demonstrate quantitative reflectometric readout by using a conventional digital camera based on red-green-blue data acquisition. Organic haloalkanes, such as 1,2-dichloroethane, have a negligible interfering effect. The visual limit of detection is 20 μg/L, and the one for red-green-blue read-out is as low as 3 μg/L. The assays have good reproducibility ±6% and ±2% for interday assays and intraday assays, respectively. The strips can be stored for at least 6 months without loss of function. They are disposable and can be produced fairly rapidly and at low costs. Hence, they represent a promising tool for in-field detection of sulfur mustard.