Investigation of human plasma low density lipoprotein by three-dimensional fluorescence spectroscopy.

Human plasma LDL exhibits a diffuse fluorescence (excitation 360 nm) in the 400-600 nm range. Application of three-dimensional fluorescence spectroscopy shows the presence of 7 fluorophores in the lipid and 6 fluorophores in the protein domain. The 430 nm fluorescence in freshly prepared LDL and its apo-B is most likely indicative for remnants of in vivo lipid peroxidation.

A spreader-bar approach to molecular architecture: formation of stable artificial chemoreceptors.

The destructive influence of lateral diffusion on nanostructured monolayers can be prevented by using the spreader-bar technique. This approach allows the formation of stable artificial receptors for barbituric acid by lateral structuring of a dodecanethiol monolayer with molecular spreader-bars from thiobarbituric acid without chemical polymerization (see schematic representation). The new technique may have applications in chemosensors, affinity chromatography, stereoselective catalysis, and molecular electronics.

A fast responding fibre optic glucose biosensor based on an oxygen optrode.

A fast responding glucose biosensor for the continuous determination of glucose is presented. The biosensor is based on an oxygen optrode, which measures the consumption of oxygen via dynamic quenching of the fluorescence of an indicator by molecular oxygen. Glucose oxidase (GOD) is immobilised onto the surface of this oxygen optrode by adsorption to carbon black and by crosslinking with glutardialdehyde. Carbon black is used as an optical isolation to protect the optrode from the interference of ambient light and sample fluorescence. The measurements were performed in a flow through cell with air saturated glucose standard solutions (phosphate buffered saline pH 6.9). The effect of four different qualities of GOD in relation to response times (the time required to reach 90% of the steady-state signal, tau 90, was 8-60 s, the linear analytical range (0.01 to 2 mM glucose) and the long-term stability (tau 1/2 was 1-20 weeks) were investigated. A simple device is presented capable of enlarging the analytical range up to 200 mM glucose concentration.

Composite films of Prussian blue and N-substituted polypyrroles: covalent immobilization of enzymes and application to near infrared optical biosensing.

We demonstrate the feasibility of optical biosensing using a material which, in essence, is a modified inorganic film to which various enzymes were covalently attached. Thin and transparent blue films composed of Prussian blue and incorporated into a network of N-substituted polypyrroles are sensitive to pH in the 5-9 range at 720 nm wavelength and can be modified with enzymes to result in the respective biosensors. Several methods of enzyme immobilization, using bifunctional crosslinking reagents, and various enzymes were tested. The best results were obtained using the one-step carbodiimide method which resulted in highly active, stable and transparent biosensor films for optical determination of urea and acetylcholine. The operational stability exceeded 1 month and even after 2 months of dry storage at room temperature the activity did not drop. The biosensors allow optical determination of the respective substrates in the millimolar concentration range.

Capacitive monitoring of protein immobilization and antigen-antibody reactions on monomolecular alkylthiol films on gold electrodes.

Self-assembled monolayers of omega-mercaptohexadecanoic acid and omega-mercaptohexadecylamine on gold electrodes are stable at neutral pH and display pure capacitive behavior at frequencies around 20 Hz. Different methods of covalent immobilization of proteins on these monolayers are compared. Various reagents including succinimides, thionylchloride, p-nitrophenol and carbodiimides were used to activate the carboxy groups of the adsorbed monolayer of omega-mercaptohexadecanoic acid. Glutaraldehyde, cyanuric chloride and phenylene diisocyanate were used to activate the amino groups of the monolayer of omega-mercaptohexadecylamine. The immobilization of albumin on the activated surface was studied by capacitive measurements. The N-hydroxysuccinimide and carbodiimide methods were identified as most suitable for protein immobilization in that they did not compromise the insulating properties of the alkylthiol layer and led to maximal increase of its dielectric thickness. These approaches were used for a layer-by-layer preparation of a capacitive immunosensor. Specifically, antibodies to human serum albumin were immobilized on the alkylthiol mono-layer. Binding of the antigen led to a decrease of the electrode capacitance. The detection limit of the immunosensor is as low as 15 nM (1 mg/l).

Enzyme biosensor for urea based on a novel pH bulk optode membrane.

A new, absorbance-based enzymatic biosensor membrane for determination of urea is described. A lipophilic, fully LED- and diode laser-compatible pH sensitive dye was incorporated into a plasticized, carboxylated poly(vinyl chloride) membrane and served as the optical transducer of the sensor. Urease was covalently linked to the surface of the pH bulk optode membrane to form a very thin cover. The resulting biosensor membrane allows rapid determination of urea over the 0.3 to 100 mM range. The reproducibility, stability, and effects of pH and buffer concentration on the response of sensor are reported. The preparation of the pH transducer and the immobilization of the enzyme are simple and may easily be adopted to other biosensor types.

An optical biosensor for lysine based on the use of lysine decarboxylase and a cadaverine-sensitive membrane.

We describe an optical biosensor for lysine based on the use of lysine decarboxylase and an optical transducer for detection of cadaverine which is formed as a result of enzymatic action. A plasticized PVC (polyvinyl chloride) membrane containing a lipophilic tartrate as the amine carrier acts as the optical cadaverine sensor. The transport of the cadaverine cation into the membrane is coupled to a transport of a proton (of the indicator dye) out of the membrane. This causes a spectral change of the indicator dye which can be related to the cadaverine concentration, provided the pH is kept constant. The enzymatic reaction is performed in an enzyme reactor which is part of a flow-through system. The dynamic range is from 0.1 to 100 mM for both cadaverine and lysine. While the cadaverine sensor is moderately selective (ethylamines, for example, interfere), the whole sensor system is highly specific for lysine, nicotine being the only major interferent. Unlike other enzyme-based detection schemes where the production of CO2 (in case of decarboxylases) or consumption of oxygen (in case of oxidases) is measured, this scheme is based on the measurement of the organic ammonium ion (cadaverin cation) formed in the enzymatic reaction. The major advantage of this approach is that in many real samples there is a rather low and fairly constant background of organic amines.(ABSTRACT TRUNCATED AT 250 WORDS)

Sol-gel based glucose biosensors employing optical oxygen transducers, and a method for compensating for variable oxygen background.

Various types of thin-film glucose biosensors based on the use of the enzyme glucose oxidase (GOx) have been developed. The luminescent oxygen probe Ru(dpp)--whose emission is quenched by oxygen--is used to measure the consumption of oxygen. Three different combinations of oxygen transducer and sol-gel immobilized GOx were tested. In the first, GOx was sandwiched between a sol-gel layer doped with Ru(dpp) and a second sol-gel layer composed of pure sol-gel (the 'sandwich' configuration). In the second, a sol-gel layer doped with Ru(dpp) was covered with sol-gel entrapped GOx (the 'two-layer configuration'). In the third, both GOx and a sol-gel powder containing GOx were incorporated into a single sol-gel phase (the 'powder configuration'). In all cases, it was found to be essential to add sorbitol which results in a more porous sol-gel in which diffusion is not impaired. The sandwich configuration provides the highest enzyme activity and the largest dynamic range (0.1-15 mM), but suffers from a distinct decrease in sensitivity upon prolonged use. The two-layer configuration has the fastest response time (t90 = 50 s), while the 'powder configuration' provides the best operational lifetime. The storage stability of all configurations exceeds 4 months if stored at 4 degrees C. In an Appendix, equations are derived which describe the response of such sensors, how the effect of varying oxygen supply can be compensated for by making use of two sensors, one sensitive to oxygen only, the other to both oxygen and glucose, and how such sensors can be calibrated using two calibrators only.

Optical triple sensor for measuring pH, oxygen and carbon dioxide.

A triple sensor unit consisting of opto-chemical sensors for measurement of pH, oxygen and carbon dioxide in bioreactors is presented. The pH and the CO2 sensor are based on the color change of a pH-sensitive dye immobilized on a polymeric support. The resulting changes in absorption are monitored through optical fibers. The oxygen sensor is based on the quenching of the fluorescence of a metal-organic dye. All three sensors are fully LED compatible. The sensitive membranes consist of plastic films and can be stored and replaced conveniently. The sensors are sterilizable with hydrogen peroxide and ethanol. In addition, the pH sensor is steam sterilizable. Accuracy, resolution and reproducibility fulfill the requirements for use in biotechnological applications. Calibration procedures for each sensor are presented. The working principle and the performance of all three sensors are described, with particular emphasis given to their application in bioreactors.

The effect of fatty acid chain length on the rate of arylester hydrolysis by various albumins.

A series of synthetic chromogenic and fluorogenic substrates for monitoring the arylesterase-like activity of albumins from various sources has been studied. Except for ovalbumin, all displayed enzyme-like activity. The acetate, butyrate, caprylate, laurate, and palmitate esters of a coumarin dye were found to be efficiently hydrolyzed within the pH range 8.8-9.8, with the non-enzymatic rate being highest for acetate and lowest for laurate. The latter is considered to be the substrate of choice because it is cleaved most quickly by the proteins tested. A considerable increase in hydrolytic activity was observed upon addition of detergents, but not of Ca and Mg ions, while addition of 1 equivalent of lauric acid to BSA resulted in a 30% decrease in its activity. The results are interpreted in terms of the well-known affinity of albumins for long chain fatty acids and provide the basis for a sensitive determination of small amounts of albumins.

A sensitive kinetic assay of serum albumin based on its enzyme-like hydrolytic activity, using a new chromogenic and fluorogenic substrate.

A new albumin assay, based on the unusual enzyme-like activity of the protein that promotes hydrolysis of ester bonds in fatty acid arylesters was designed for clinical routine use. The substrate introduced shows improved analytical wavelengths and is suitable for both photometric and fluorimetric assay. Experiments have been performed with a conventional photometer, a fluorimeter and a Cobas Fara autoanalyzer. Detection limits are as low as 10 micrograms/ml photometrically and 20 ng/ml fluorimetrically. The method provides a sensitive quantitative determination of even minute amounts of albumin in liquid solution, and a simple semiquantitative test may be performed by fixing the dye on a test strip which then is immersed into a sample solution and observing the development of yellow color intensity.

Cell-type specific protoporphyrin IX metabolism in human bladder cancer in vitro.

5-Aminolevulinic acid (ALA)-supported fluorescence endoscopy of the urinary bladder results in a detection rate of bladder cancer superior to that of white light endoscopy. The different accumulation of the metabolite protoporphyrin IX (PPIX) in tumor cells after ALA instillation is poorly understood; however, it is crucial to optimize diagnosis and potential phototherapy. For systematic analysis of cell-type specific PPIX accumulation and metabolism two human bladder carcinoma cell lines (RT4 and J82), a normal urothelial cell line (UROtsa), and a fibroblast cell line (N1) were chosen, and grown in two different growth states to model important tissue components of the urinary bladder, i.e. tumor, normal epithelium and stroma. To quantitate PPIX content, fluorescence intensities measured by flow cytometry were matched with cellular PPIX extraction values, and related to relative ferrochelatase activity, cellular iron content, number of transferrin receptors per cell and porphobilinogen deaminase (PBGD) activity. For in vitro experiments, the initial correlation of relative flow cytometric and spectrometric measurements of PPIX provides a calibration curve for consequent flow cytometric PPIX quantification. Lower fluorescence of normal cells could be explained by significant differences of ferrochelatase activity and iron content in comparison to tumor cells. However, the content of iron was not related to transferrin receptor content. PBGD activity seemed to play a minor role for the differential accumulation of PPIX in urothelial cells. In conclusion, the in vitro culture of urothelial cells and fibroblasts indicates that the most important metabolic step for PPIX accumulation in the urinary bladder is the transition from PPIX to heme. Further investigation of PPIX metabolism does support the validation of photodynamic diagnosis, and might also lead the way to a highly specific tumor related molecule.

Novel diode laser-compatible fluorophores and their application to single molecule detection, protein labeling and fluorescence resonance energy transfer immunoassay.

We describe a series of new long-wave absorbing and fluorescing cyanine dyes and labels (based on a general logic for the design of such dyes), their spectra, covalent and noncovalent linkage to proteins, their use in single molecule detection (SMD) and as donors and acceptors, respectively, in fluorescence resonance energy transfer studies. The new labels represent water-soluble and reactive fluorophores whose quantum yields increase substantially if noncovalently or covalently bound to proteins. Due to their strong absorptions between 550 and 700 nm they are excitable by light-emitting diodes or diode lasers. Their high absorbances (epsilon around 100,000) and adequate fluorescence quantum yields (phi up to 0.68 if bound to proteins) along with their availability as reactive NHS esters make them viable labels for proteins and oligomers, e.g. in context with SMD or fluorescence energy transfer immunoassay which is demonstrated for the system HSA/anti-HSA.

Optical and fibre-optic sensors for vapours of polar solvents.

An optical sensor is described which continuously measures the concentration of vapours of polar organic solvents such as alcohols, ethers, esters and ketones, but does not respond to hydrocarbons and chlorinated hydrocarbons. The detection is based on reversible decolorization of the blue thermal printer paper used in graphic plotters. Two sensing principles are exploited. In the first, a sensor sheet is placed in a flow-through cell. An LED acts as a source of yellow light and a phototransistor measures the light transmitted. In the second, the sensing membrane is placed at the end of a bifurcated fibre-optic and its diffuse reflectance is measured. The sensitivity of the devices towards various kinds of vapours has been studied and the detection limits vary from 10 to 1000 ppm for some of the most common technical solvents.

Novel metal-organic ruthenium(II) diimin complexes for use as longwave excitable luminescent oxygen probes.

New ruthenium(II) diimine complexes are presented which are useful as luminescent oxygen probes. Because their luminescence excitation maxima are between 535 and 570 nm, they can be photo-excited by green LEDs which are much brighter than the blue LEDs used so far for existing Ru diimines. The spectral and photophysical properties as well as the solubility properties of the new probes are investigated and discussed in terms of quenching, photostability, and lifetimes. The probes were incorporated into organic polymers by three different methods, to obtain oxygen-sensitive materials for use in optical oxygen sensing. The membranes were characterized with respect to oxygen sensitivity, luminescence intensity, response times, and stability. Notwithstanding the poor luminescence of the new ruthenium(II) probes, their stability, LED compatibility and efficient quenching by oxygen makes them an interesting alternative to existing luminescent oxygen probes.

Fibre-optic titrations-IV Direct complexometric titration of aluminium(III) with DCTA.

The end-point of the direct complexometric titration of Al(3+) in pH 4.6 solution can be determined by monitoring the fluorescence intensity of the aluminium-morin complex, by use of a bifurcated fibre-optic light guide. The method allows the determination of aluminium in the 1-800 ppm range with good precision. The procedure is applicable even when the solutions are strongly coloured or turbid, but because of the slow complexation kinetics requires a titration time of about 20 min.

The effect of polymeric supports and methods of immobilization on the performance of an optical copper(II)-sensitive membrane based on the colourimetric reagent Zincon.

A comparative study on the effect of different immobilization methods and matrix materials on the performance of copper(II)-sensitive membrane layers is presented. The indicator dye Zincon was immobilized in hydrophilic and hydrophobic polymers by various methods including: (a) physical entrapment of the Zincon-tetraoctylammonium ion pair in plasticized PVC, hydrogel, polystyrene, ethyl cellulose, poly-HEMA, AQ-polymer and in sol-gel glass; (b) electrostatic immobilization on an anion exchanger cellulose; and (c) covalent immobilization on cellulose via a sulfatoethylsulfonyl reactive group. The response to copper(II) ion was evaluated kinetically via the initial slope of the change in absorbance within 1 min. Layers made of hydrogel and PVC provide the highest sensitivity, while covalent immobilization is the most reproducible one, and sol-gel layers display the best mechanical stability.

Non-enzymatic optical sensor for penicillins.

We report on the first penicillin-sensitive fluorosensor not based on the use of an enzyme. Rather, the recognition process relies on the use of an anion carrier (which carries the penicillin anion from the aqueous sample into the membrane), a proton receptor (lipophilic nile blue which accepts the proton, thereby undergoing a change in fluorescence intensity), and a new lipophilic hydroxylic plasticizer material (which facilitates ion transport). All materials are contained in a dyed poly(vinyl chloride) membrane whose fluorescence is monitored. The optical sensor fully reversibly responds to penicillin V over the 0.01-10mM concentration range, and to penicillin G from 0.03 to 10mM. Potential interferences by about 20 other anions have been investigated. Nitrate, salicylate, and ascorbate were found to interfere significantly. These species are, however, usually not present in penicillin bioreactors or drug formulations where penicillin sensing is most important. The sensor does not respond to penicillins containing an aliphatic amino group (such as amoxicillin). The method has been applied for determination of penicillin G in pharmaceutical formulations.

Photometric and fluorometric continuous kinetic assay of acid phosphatases with new substrates possessing longwave absorption and emission maxima.

A direct and continuous kinetic method for the photometric and fluorometric determination of various acid phosphatases is described. It is based on new coumarin-derived phosphates, which after enzymatic hydrolysis undergo dissociation to form intensely colored and strongly fluorescent phenolate anions. The latter have absorption maxima ranging from 385 to 505 nm, and fluorescence maxima between 470 and 595 nm. The new substrates were compared with respect to their rate of enzymatic hydrolysis, optimum pH, and detection limits of acid phosphatase from potato and wheat germ. Detection limits of 0.001 unit/ml were found by photometry, and as low as 0.00006 unit/ml by fluorometry. The principal advantages of the new substrates over existing ones are longwave absorptions and emissions, large Stokes shifts, and the low pKa values of the corresponding phenols, thus allowing a direct and continuous assay of acid phosphatase even in weakly acidic solutions.

A fiberoptic cholesterol biosensor with an oxygen optrode as the transducer.

A biosensor for the continuous optical determination of cholesterol is presented. Cholesterol oxidase is immobilized covalently on a nylon membrane and the consumption of oxygen is measured by following, via fiberoptic bundles, the changes in fluorescence of an oxygen-sensitive dye whose fluorescence is dynamically quenched by molecular oxygen. The dye is dissolved in a very thin silicone membrane placed beneath the enzyme layer. During interaction of the enzyme with cholesterol, oxygen is consumed, which is indicated by the fluorescent dye. At pH 7.25, the analytical range of the sensor is 0.2 to 3 mM and the time to reach a full steady state in a flowing solution ranges from 7 to 12 min.