Optical DNA-sensor chip for real-time detection of hybridization events.

An optical sensor system based on evanescent field excitation of fluorophore-labeled DNA-targets specifically binding to immobilized DNA probes has been developed, thus enabling for real-time analysis of hybridization events. Oligonucleotide probes are directly immobilized on the surface of the disposable sensor chip via biotin/neutravidin linkage and hybridize to complementary Cy5-labeled target DNA in the sample; this is recorded as an increase in the fluorescence signal. Under optimized conditions the hybridization rate was constant and directly proportional to the target concentration. When an 18mer oligonucleotide was used as a probe a linear calibration curve was obtained for a 56mer single-stranded DNA target derived from the neomycin phosphotransferase gene, a selection marker in a variety of genetically modified plants, with an estimated lower limit of detection of 0.21 nmol L(-1). No cross-hybridization to a 51mer actin DNA target was observed and even a single-nucleotide mismatch led to a negligible signal. A shutter in the readout device enabled separate detection of targets hybridizing to probes immobilized at the inlet and outlet sides, respectively, of the flow channel. This opens a route toward a real-time DNA array format with analysis times as short as 1-2 min. As a realistic sample a Cy5-labeled 56 bp PCR product was measured after separation of the double-stranded DNA by simple heat denaturation with a detection limit clearly lower than that of traditional gel electrophoresis.

Development of an element-selective monitoring system for adsorbable organic halogens (AOX) with plasma emission spectrometric detection for quasi-continuous waste-water analysis.

An automated quasi-continuously-operating monitor has been developed for element-selective analysis of adsorbable organic halogens (AOX) in water. After extensive optimization the automatic method was applied to the analysis of standard solutions and real waste water samples to prove its analytical applicability. The new instrument is based on the element-selective analysis of halogens by means of a spectroscopic detection system consisting of a microwave-induced helium plasma excitation source (TM010-type; developed in this laboratory) and the plasma emission detector (PED) which operates with oscillating narrow-band interference filters. After enriching the organic components on activated charcoal and pyrolysis in an oxygen stream at 950 degrees C, in accordance with DIN/EN 38409,H14/1485, interfering CO2 and H2O gas generated during combustion is removed from the analytes in the so-called ELSA-system (element-selective AOX-analyzer). For focused injection into the plasma excitation source the analytes (hydrogen halides) are trapped in a deactivated fused silica capillary at -180 degrees C; this is followed by identification and quantification on the basis of element-specific emission of radiation in the VIS and NIR-region (chlorine 837.6 nm, fluorine 685.6 nm). Bromine and iodine could not be detected with satisfactory inter-element selectivity, because of spectral interferences caused by matrix elements, and so results from the respective single-element investigations for determination of AOBr and AOI are not presented. The procedure has been validated and the analytical performance has been examined by calibration with p-chlorophenol and p-fluorophenol. The limit of detection was 1.1 microg (absolute) for chlorine and 6.6 microg (absolute) for fluorine.

A new calcium-sensor based on ion-selective conductometric microsensors--membranes and features.

Based on the concept of ion-selective conductometric microsensors (ISCOM) a new calcium sensor was developed and characterized. ISCOM have a single probe, all-solid-state construction and do not need a reference electrode. These sensors are amenable to miniaturization and integration in the true sense of integrated circuit and microsystem technologies. The detection is accomplished by measurement of the bulk conductance Gm of a thin polymeric membrane containing an ion-complexing agent, where the magnitude of Gm can be related to the content of the primary ion in the analyzed solution. Thin-film platinum electrodes forming an interdigitated electrode are used as the transducer to detect the conductivity of the polymeric membrane. Optimization of the membrane composition was carried out by testing different types of calcium-ionophores, polymers, and plasticizers. The sensor characteristics have been investigated. The limit of detection is about 10(-7) mol L(-1). The dynamic range is 10(-6)-10(-1) mol L(-1) with a response time of less than 5 s. These parameters are comparable to those of corresponding potentiometric calcium selective electrodes (ISE). The Ca(2+)-ISCOM demonstrates good practical relevant selectivities against typical interfering ions for biomedical and environmental applications.

Novel teststrip with increased accuracy.

A new type of a simple and cheap teststrip for liquid samples is described. It is based on microchromatography or microtitration on a porous, capillary-active substrate (e.g., filter paper or a similar absorbent material). In case of microchromatography an analyte-selective indicator (and other auxiliary reagents) is impregnated or immobilized on the capillary-active substrate; in case of micro-titration the capillary-active substrate contains a titrant which reacts stoichiometrically with the analyte. Quantitative analysis is performed by measurement of an area (which had changed its color) rather than by evaluation of the shade or intensity of a color (like in conventional teststrips). In order to show the broad applicability of this new principle teststrips for different analytes like Quaternary Ammonium Compounds (QACs), the cation Ni2+, the anion SO4(2-) and H2O2 are described. The detection limit and working range of the novel teststrip can be adjusted by variation of its size.

Disposable optical sensor chip for medical diagnostics: new ways in bioanalysis.

An optical sensor system is described which is particularly well suited for medical point-of-care diagnostics. The system allows for all kinds of immunochemical assay formats and consists of a disposable sensor chip and an optical readout device. The chip is built up from a ground and cover plate with in- and outlet and, between, of an adhesive film with a capillary aperture of 50 microns. The ground plate serves as a solid phase for the immobilization of biocomponents. In the readout device, an evanescent field is generated at the surface of the ground plate by total internal reflection of a laser beam. This field is used for the excitation of fluorophor markers. The generated fluorescence light is detected by a simple optical setup using a photomultiplier tube. Because of the evanescent field excitation, washing or separation steps can be avoided. With this system the pregnancy hormone chorionic gonadotropin (hCG) could be determined in human serum with a detection limit of 1 ng/mL. Recovery values were 86, 106, and 102% for 5, 50, and 100 ng/mL hCG, respectively. The SD in repeated measurements (n = 10) was 5.6%. Furthermore, the feasibility of the system in competitive-type immunoassays was demonstrated for serum theophylline. A linear calibration curve of signal vs theophylline between 1 and 50 mg/L was obtained. Recovery values varied between 118% (10 mg/L) and 81.0% (20 mg/L).

A disposable biosensor for urea determination in blood based on an ammonium-sensitive transducer.

A potentiometric urea-sensitive biosensor using a NH4(+)-sensitive disposable electrode in double matrix membrane (DMM) technology as transducer is described. The ion-sensitive polymer matrix membrane was formed in the presence of an additional electrochemical inert filter paper matrix to improve the reproducibility in sensor production. The electrodes were prepared from one-side silver-coated filter paper, which is encapsulated for insulation by a heat-sealing film. A defined volume of the NH4(+)-sensitive polymer matrix membrane cocktail was deposited on this filter paper. To obtain the urea-biosensor a layer of urease was cast onto the ion-sensitive membrane. Poly (carbamoylsulfonate) hydrogel, produced from a hydrophilic polyurethane prepolymer blocked with bisulfite, served as immobilisation material. The disposable urea sensitive electrode was combined with a disposable Ag/AgCl reference electrode to obtain the disposable urea biosensor. The sensor responded rapidly and in a stable manner to changes in urea concentrations between 7.2 x 10(-5) and 2.1 x 10(-2)mol/l. The detection limit was 2 x 10(-5) mol/l urea and the slope in the linear range 52 mV/decade. By taking into consideration the influence of the interfering K(+)- and Na(+)-ions the sensor can be used for the determination of urea in human blood and serum samples (diluted or undiluted). A good correlation was found with the data obtained by the spectrophotometric routine method.

Surface investigations on the development of a direct optical immunosensor.

In the present paper surface studies for the development of a direct optical immunosensor for fast diagnosis of a myocardial infarction are presented. A fatty acid binding protein was detected by monoclonal antibodies. The applied measuring system was the grating coupler BIOS-1. Based on commercially available transducer materials protein immobilisation techniques have been developed and characterised by TOF-SIMS, AFM and EM. Three different label-free assay types were investigated. Only one assay leads to a sensitive and regenerable sensor set-up. It was possible to detect concentrations of the fatty acid binding protein down to 330 ng/ml. The general applicability of a direct optical immunosensor in the field of myocardial infarction diagnosis was demonstrated by this.

Development and review of radioimmunoassay of 12-S-hydroxyheptadecatrienoic acid.

For more than 25 years 12-S-hydroxyheptadecatrienoic acid (HHT) has been known to be a product of thromboxanesynthase (TX-Syn) when synthesized with thromboxane A2 (TXA2). Although there are some hints that HHT has anti-aggregatory effects, to date, it has neither been shown to have any specific pathological relevance nor is there much information about its physiological role. This review presents a summary of the physicochemical properties of HHT, its chemical synthesis, the impact of various biological systems on its enzymatic and non-enzymatic production and its physiological function and metabolization, as well as a survey of the most important methods for analyzing this unsaturated hydroxy-fatty acid. Due to the low antibody-raising potency expected in HHT, no immunological system for HHT quantification has been developed so far. In our report we present the development and validation of a sensitive and reliable, competitive radioimmunoassay (RIA) suitable for the quantitative determination of HHT. HHT was produced by an enhanced enzymatic method using platelet-rich plasma (PRP). With an effective and modified liquid-liquid and solid-phase extraction method we were able to produce highly purified HHT (97% purity by GC/MS) in sub-milligram ranges. These fractions were used for the synthesis of BSA-antigen-conjugates and for immunization of rabbits. The tritiated tracer was synthesized using prostaglandin H synthase for the production of prostaglandin H2 (PGH2) followed by an aqueous reaction with Fe(2+)-solution to rear-range PGH2 to HHT. The dynamic range of the assay was from 30-400 pg/tube, with a sensitivity of approximately 40 pg/tube. The evaluation of the assay was performed by a HPLC-RIA method as well as by correlation with a quantitative HPLC method and correlation with TXB2 concentrations in a blood coagulation study. The assay may be useful for the quantification of HHT in several tissues and body fluids under various physiological conditions and may also help to understand the possible physiological role of HHT in biological processes.

Kinetic analysis of immunointeractions with covalently immobilized fatty acid-binding protein using a grating coupler sensor.

Application of a grating coupler sensor (GCS) to the real time investigation of the interaction kinetics of covalently immobilized recombinant bovine heart-type fatty acid-binding protein (H-FABP) and corresponding antibody is described. The immobilization of the antigen is performed by activating the matrix hydroxyl groups with p-toluenesulfonyl chloride (TSC) and afterwards coupling the protein by reaction with its nucleophilic aminogroups. Covalent coupling via TSC permits reproducible measurements of immunointeractions on the same grating coupler sensor chip and complete regeneration after each binding cycle with glycine-hydrochloride. We demonstrate the analysis of binding data obtained on a GCS by linearization as well as direct curve fitting using the integrated rate equation for the determination of apparent rate and affinity constants. With both analysis methods we studied H-FABP/monoclonal anti-H-FABP-antibody interactions and obtained an average apparent association rate constant ka = 4.2 X 10(3) M(-1) s(-1) a dissociation rate constant of kd=1.3 X 10(-4) s(-1) and an equilibrium constant of KD=3 X 10(-8) M.

In situ antigen immobilization for stable organic-phase immunoelectrodes.

A new method based on enzymatic single-step in situ synthesis of hapten-carrier conjugates on electrodes is described yielding stable, reproducible, and reusable organic-phase immunoelectrodes (OPIEs). The electrodes developed were tailored for analyte detection in organic solvents and allow for the analysis of soil extracts without further sample processing and cleanup. Catalyzed by transglutaminase from a variant of Streptoverticillium mobaraense, the reaction proceeds in aqueous solution with and without addition of organic media in only 1.5 hours. In this study, the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was chosen as model compound and chemically amino-functionalized prior to its enzymatic immobilization. The high reproducibility of the immobilization procedure allowed for batch calibration of the immunoelectrodes. Moreover, pure methanol or treatment with diluted sulfuric acid used for regeneration studies did not disturb the hapten layer. The OPIE consists of screen-printed carbon electrodes, monoclonal anti-2,4-D antibodies, and the immunochemical recognition reaction and was optimized with regard to a high stability in organic media. For electrochemical detection, horseradish peroxidase was used as enzyme label together with H2O2 as substrate and hexacyanoferrate (II)/(III) as mediator. The OPIE showed high stability upon storage over 93 days. Response times of 17 s (t95) were found to be advantageous compared to those of other biosensors. Including the immunochemical reactions, the complete assay takes 30 min. A calibration curve for 2,4-D in 30% methanol/buffer obtained with 70 electrodes within 4 weeks revealed a detection limit of 9 mg/L, a sensitivity of 1.3 nA L mg-1 cm-2, and a repeatability of 6.8%. Although we calculated a lowered repeatability for reused electrodes of 13.4% and a slightly decreased sensitivity of 0.9 nA L mg-1 cm-2, multiple-used OPIEs could also be applied for calibration.

Validation of the determination of copper and zinc in blood plasma and urine by ICP MS with cross-flow and direct injection nebulization.

The simultaneous determination of copper and zinc in human plasma and urine by inductively coupled plasma mass spectrometry (ICP MS) is discussed. The performances of a cross-flow nebulizer and a direct-injection nebulizer (DIN) were compared. Flow-injection-DIN-ICP MS analysis of clinical samples using 1-2 mul samples was optimized. Isobaric interferences were discussed and were demonstrated to be eliminated for the (65)Cu and most of the Zn nuclides. The need for standard addition to compensate for signal suppression in the case of some serum samples was indicated. Results obtained by ICP MS using calibration with aqueous standard solutions were found to be in good agreement with those obtained by flame AAS for a batch of real blood plasma and urine samples. The methods developed were validated by analysis of several standard reference materials.

Frequency-modulated simultaneous Atomic Absorption Spectrometry (FremsAAS): Determination of As, Se and Sb.

The evaluation of accuracy and efficiency of the frequency-modulated simultaneous Atomic Absorption Spectrometry (FremsAAS) has been extended to an arrangement with EDL as light sources. Fundamental calibrations have been worked out for As, Se and Sb using a graphite furnace as well as hydride generation in combination with a heated quartz tube as atomization unit. The characteristic data are in good agreement with results obtained by conventional single-channel AAS instruments. Determinations in three standard reference materials with different complex matrices resulted in complete agreement with the certified values.

Near-infrared imaging spectroscopy (NIRIS) and image rank analysis for remote identification of plastics in mixed waste.

An infrared camera with focal plane InSb array detector has been applied to the characterization of macroscopic samples of household waste over distances up to two meters. Per waste sample (singelized), a sequence of images was taken at six optical wavelength ranges in the near infrared region (1100 nm - 2500 nm). The obtained three-dimensional data stack served as individual fingerprint per sample. An abstract factor rotation of this stack of six images into a spectroscopical meaningful intermediate six-element vector by Multivariate Image Rank Analysis (MIRA) finally provided a decision limit for the discrimination of plastics and nonplastics. A correct classification of better than 80% has been reached. The experimental NIRIS set-up has been automated so far to allow an on-line identification of a real world waste sample within a few seconds.

Speciation of mercury, platinum and tin - focus of research and future developments.

A discussion about the elements mercury, platinum and tin and their organometallic compounds, with special attention to important aspects like anthropogenic emissions, use in industry, agriculture and medicine, toxicities, biogeochemical cycles, highlights the necessity of speciation analysis. Methods frequently used to speciate mercury, platinum and tin compounds in different matrices are summarized. Future trends are indicated in order to give an impression about the importance of metal speciation and its increasing impact on some key disciplines.

Investigations for the determination of lead by in situ hydride trapping within a graphite electrothermal atomizer for routine analysis.

A new commercial system consisting of a flow injection analysis system for hydride generation coupled with a transversely heated graphite atomizer-atomic absorption spectrometer for the determination of lead is investigated in detail. The hydride generation is optimized by using an ammonium peroxodisulphate-hydrochloric acid system as oxidant and sodium borohydride as reducing reagent. The addition of sodium cumol sulphonate as surface active substance shows advantages considering efficient plumbane production. The hydride trapping and atomization in a graphite electrothermal atomizer is also optimized. The characteristic concentration was 0.74 mug/l, the detection limit was 0.70 mug/l for 500 mul sample volumes. The relative operation standard deviation of this method was smaller than 2%. Further examinations demonstrate the influence of several heavy metals on the determination of lead. Finally, the measurement of standard reference materials shows the efficiency of the method in combination with decomposition with aqua regia solutions.

Trihalomethane concentrations in swimmers' and bath attendants' blood and urine after swimming or working in indoor swimming pools.

The influence of working or swimming in indoor swimming pools on the concentrations of four trihalomethanes (haloforms) in blood and urine was investigated. Different groups (bath attendants, agonistic swimmers, normal swimmers, sampling person) were compared. The proportions of trihalomethanes in blood and urine correlated roughly with those in water and ambient air. Higher levels of physical activity were correlated with higher concentrations. Within one night after exposure in the pool the blood concentrations usually were reduced to the pre-exposure values. Secretion of trichloromethane in urine was found to be less than 10%.

Glucose sensor in containment technology.

A new transducer concept for miniaturized immobilized enzyme glucose sensors is presented. The enzyme containing membrane is anchored inside the microcontainment of a silicon chip together with the metal electrode. Containment based sensors are ideally suited for integration into microsystems. The fabrication process is planned as a full wafer process allowing transfer to low-cost mass-production with a narrow variability. The functionality of the containment concept is demonstrated by the fabrication of chips with single GOD-based amperometric glucose sensors which have been tested in glucose solution. The advantages of the containment technology are discussed.

Biosensor on the base of polypyrrole modified ultramicroelectrode arrays.

A novel approach for the construction of a micro biosensor on the base of amperometric enzyme microelectrode arrays is described in this paper. The technique contains the electrochemical deposition of different enzymes in a conducting organic polymer, e.g. polypyrrole, on a transducer built up with the help of microfabrication technology. The electrochemical characteristics of these microelectrode arrays can be compared to conventional microelectrodes with the advantage of higher current outputs. For the first time different model enzymes like glucose oxidase, choline oxidase and lactate oxidase have been tested showing the principal possibility to construct a micro biosensor on the base of ultramicroelectrode arrays.

Disposable sodium electrodes.

In this paper we describe a disposable sodium sensor in double matrix membrane technology. This sensor is prepared from filter paper with an evaporated silver conducting line on one side. For insulation the sensor is laminated with a pre-perforated heat-sealing film. A defined volume of an ion-sensitive polymer matrix membrane cocktail is filled into one hole. So an ion-sensitive coated film sensor in double matrix membrane technology is produced. The double matrix membrane is formed by the polymer matrix membrane and by the additional filter paper matrix. The response behaviour of the sodium electrode is comparable with conventional macro ion-selective electrodes. By this technology a mass production of low cost sensors is possible.