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This paper presents several methods for analysis of data from reflectometric interference spectroscopic measurements (RIfS) of water samples. The set-up consists of three sensors with different polymer layers. Mixtures of butanol and ethanol in water were measured from 0 to 12,000 ppm each. The data space was characterized by principal component analysis (PCA). Calibration and prediction were achieved by multivariate methods, e.g. multiple linear regression (MLR), partial least squares (PLS) with additional predictors, and quadratic partial least squares (Q-PLS), and by use of artificial neural networks. Artificial neural networks gave the best results of all the calibration methods used. Calibration and prediction of the concentration of the two analytes by artificial neural nets were robust and the set-up could be reduced to only two sensors without deterioration of the prediction.

pH-responsive polymers enable the dyeless optical detection of acidic or basic pollutants in air. The characterization of the sorption process and the optimization of the response time of the sensitive layers were high-lighted. The swelling of a pH-responsive polysiloxane induced by sorption of gaseous ammonia was investigated by measurement techniques such as spectroscopic ellipsometry (SE) and infrared spectroscopy (IR). Furthermore, the pH-responsive polymer was applied for the detection of gaseous ammonia using the LED-based reflectometric interference spectroscopy set-up (RIfS4lambda). A limit of detection of 0.30 mg/m3 ammonia and a response time (t90%) of 35 s could be verified. The application of pH-responsive polymers can be a powerful alternative to dye-based optical sensing, since photobleaching or leaching of the sensitive functional unit cannot occur applying this approach, and since the properties of the sensitive layer proved to be very promising.

Based on the requirements of biomolecular interaction analysis on direct optical transducers, a streptavidin surface is examined. A general protocol was developed allowing the immobilization of biotinylated compounds using the rife biotin-streptavidin system. This type of surface modification can be applied to all biosensors using glass surfaces as sensor devices. Reflectometric interference spectroscopy (RIfS), a label-free, direct optical method was used to demonstrate the quality of the transducer surfaces. The surface modification is based on an aminofunctionalized polyethylene glycol layer covalently bound to the silica surface of the transducer and shows very little nonspecific binding. Biotin molecules can be easily coupled on such layers. Streptavidin followed by a biotinylated estrone derivative was immobilized by incubation of the biotinylated transducer surface. For the streptavidin layer we obtained interference signals corresponding to a protein monolayer. Finally, using a surface prepared as described above, biomolecular interaction experiments with an antibody against estrone were carried out to show the quality of the transducer surface. With RIfS all of the affinity-based surface modifications can be detected online and time resolved., (Copyright 2000 Academic Press.)

The main interests in the development of new combinatorial assays are the reduction of time for screening and an increase in the number of samples measured in parallel. The variety of detection methods is increasing, but the optimal one has not yet been determined. In the past two years, the first parallel detection methods for non-labelled compounds have been developed.

In the field of environmental analysis there is still great potential for development and application of immunoanalytical techniques (IT). Heterogeneous and homogeneous immunoassays (IA), flow-injection immunoanalysis (FIIA) and immunosensors (IS) with different detection principles have been developed. In this review we focus on fluorescence methods for pesticide monitoring published since 1992. These techniques offer a high degree of selectivity and, in principle, sensitivity. Restrictions on the limits of detection (LOD) due to background signals are minimized by development of solid-phase separation systems, new fluorescent probes, and new instrumentation.

Since the first label-free optical biosensor was commercialized in 1990 a rising number of publications have demonstrated the benefits of direct biomolecular interaction analysis (BIA) for biology and biochemistry. This article first gives an overview of the historical development of different transducer principles used for the detection of BIA. Subsequently, the four major parts of a biosensor system: transducer, sample handling, surface/immobilization chemistry and test formats/data evaluation will be discussed, with a main focus on the test formats and data evaluation. The intention of this review is to present an introduction to the field and to point out the difficulties most frequently encountered.

Label-free heterogeneous phase detection critically depends on the properties of the interfacial layer. We have obtained high-density monomolecular poly(ethylene glycol) (PEG) layers by solvent-free coupling of homo-bifunctional PEGs (2,000 g/mol) at 75 degrees C to silica surfaces silanized with glycidyloxipropyltrimethoxysilane (GOPTS). Characterization by ellipsometry and contact angles revealed that PEG layers up to 3.4 ng/mm2 with low roughness and flexibility were obtained. Specific and non-specific binding at these PEG surfaces was monitored by reflectometric interference spectroscopy (RIfS). No significant non-specific adsorption upon incubation of 1 mg/ml ovalbumin was detectable (< 10 pg/mm2), and 150 pg/mm2 upon incubation of 10% calf serum, less than 10% of the amount adsorbed to the solely silanized surfaces. The terminal functional groups of the PEG layers were utilized to couple ligands and a protein. Specific protein interaction with these immobilized compounds was detected with saturation loadings in the range of protein monolayers (2-4 ng/mm2). The excellent functional properties, the high stability of the layers, the generic and practical coupling procedure and the versatility for immobilizing compounds of very different functionality make these PEG layers very attractive for application in label-free detection with silica or metal-oxide based transducers.

A novel Förster donor-acceptor dye pair for an immunoassay based on resonance energy transfer (RET) is characterized with respect to its photophysical properties. As donor and acceptor, we chose the long-wavelength excitable cyanine dyes Cy5 and Cy5.5, respectively. Due to the perfect spectral overlap, an exceptionally high R(0) value of 68.7 A is obtained in solution. For biochemical applications, antibodies (IgG) are labeled with Cy5, while a tracer for competitive binding is synthesized by labeling bovine serum albumin (BSA) with an analyte derivative and Cy5.5. Binding the dyes to proteins at a low dye/protein ratio increases the fluorescence lifetimes and quantum yields, leading to an enhanced R(0) value of 85.2 A. At higher dye/protein ratios, the formation of nonfluorescent dimeric species causes a decrease in the fluorescence lifetime and quantum yield due to RET from monomeric dyes to dimers within one protein molecule. The Förster distances could be calculated using the dimer absorption spectra to 83.9 and 83.6 A for Cy5 and Cy5.5, respectively. Upon binding of the Cy5-labeled IgG to the tracer, efficient quenching of Cy5 fluorescence is observed. Steady-state and time-resolved measurements reveal that approximately 50% of the quenching results in Förster-type RET, while the residual quenching effect is caused by static quenching processes. The applicability of this dye pair is demonstrated in a homogeneous competitive immunoassay for the pesticide simazine.

Optimization of crushing strength and disintegration time of a high-dose plant extract tablet was reached after extensive experimentation. Effects of the processing parameters, like compression force and tooling, and also of the excipients were found to be significant. Best results for both disintegration time and crushing strength were obtained with a plant extract that was granulated by roller compaction before compression. To gain more information about the different effects, artificial neural networks (ANNs) and a conventional multivariate method (partial least squares [PLS]) were used for data analysis. The topologies of the neural networks of the feed-forward type were optimized manually and by pruning methods. All methods were tested for contemplated parameters, crushing strength, and disintegration time. In general, ANNs were found to be more successful in characterizing the effects that influence crushing strength and disintegration time than the conventional multivariate methods.

Antisense oligonucleotides (ON) are regarded as potential therapeutic agents for controlling gene expression at the mRNA level. The strength of the interaction with the target sequence is one critical factor for the therapeutic efficiency of an ON. Herein, the results of studies on antisense 15mer and 20mer ONs against mdr1b-mRNA are described. The mdr1b is a member of the group that encodes the P-glycoprotein (Pgp), responsible for the phenomenon of multidrug resistance. The effects of backbone modification (DNA, phosphorothioate (PTO)), terminal modifications (hexadecyl, cholesteryl, tocopherol, polyethylenglycol, 2'-O-methyl-modified RNA) and base sequence misalignments (1 to 3 bases) on interaction kinetics and binding strength were investigated. The interaction of an immobilized sense strand with the dissolved antisense ON was monitored with a label-free optical transducer based on thin film interference (RIfS). Association kinetics were detected at a low density of immobilized ON. Thermodynamics were investigated by homogeneous phase titration of sense and antisense ON and subsequent quantification of equilibrium concentrations of unbound ON at a transducer highly loaded with sense ON. Association rate constants varied from 3.1 (+/- 0.2) x 10(4) M-1 s-1 (poly(ethylene glycol)-modified DNA strand) to 4.3 (+/- 0.1) x 10(4) M-1 s-1 (hexadecyl-modified strand). Binding constants varied from 1.9 (+/- 0.1) x 10(8) M-1 (cholesteryl modification) to 5 (+/- 0.4) x 10(7) M-1 (tocopherol modification). Phosphorothioate ON showed a reduction in binding strength of more than 1 order of magnitude. The data presented give valuable information for the efficiency of modified antisense oligonucleotides.

This paper presents the detailed design and characterisation of a regenerable integrated optical surface plasmon resonance immunoprobe as a detector for the triazine herbicide simazine. A sensor design theoretically optimised for use in the aqueous environment is presented and its fabrication described. Experimental results on the sensitivity to changes in bulk refractive index of the analyte and on non-specific binding of ovalbumin are presented. Binding inhibition immunoassays were conducted for simazine and the lower limit of detection determined to be 0.16 microgram/l using anti-simazine IgG antibodies and 0.11 microgram/l using anti-simazine Fab fragments. A sample test cycle of 20 min was established.

IR spectroscopic mapping of resin beads allows destruction-free characterization of polymer-bound combinatorial compound libraries. By choice of different absorptions for IR reconstruction, resin beads with common structural elements can be "fished out" of a library and statistically investigated., (© 1998 WILEY-VCH Verlag GmbH, Weinheim, Fed. Rep. of Germany.)

Protein multilayers were prepared on silica surfaces by up to 20 alternating incubations of a biotin-protein conjugate and polymerized streptavidin. Spectroscopic ellipsometry (350-750 nm) was used to investigate the physical thickness and the dispersion of the protein layer after each incubation step. Both parameters could be determined independently for films formed by 5 to 15 incubations. A single homogeneous protein layer was assumed for evaluation. This determination of layer parameters was limited by high correlation coefficients for less than 5 incubation steps. Deviations from the homogeneous single layer model were found for more than 15 incubation steps. The growth of the layer was reproducible, with a thickness increase of about 18.75 nm per incubation. An almost constant refractive index nD of 1.384 +/- 0.002 was found for the multilayer system. A protein mass deposition of 4.74 ng/mm2 was calculated per incubation step. The protein concentration of the layer was estimated of about 0.27 g/ml. The affinity system investigated is quite simple and may well serve as a prototype system in the characterization of optical and other transducers for affinity reactions. Copyright 1997 Academic Press.

The reflection grating coupler for direct affinity sensing is characterized in detail. The performance of this device and its potential in affinity sensing application are investigated with two affinity-based systems: A self-assembling protein-multilayer system based on avidin-biotin interaction was used to compare the response of the device with theoretical expectations. The analytical performance was characterized by a pesticide immunoassay carried out in an indirect test format with a covalently immobilized triazine derivative. Experimentally determined parameters were in good agreement with model calculations. During the binding of 12 protein monolayers at the surface, the change in effective refractive index Dn(eff) detected for a single layer decreased from approximately 8 x 10(-4) to less than 4 x10(-5) by more than 95%, indicating a filling of the evanescent field. By comparison with bulk refractive-index measurements, a refractive index n(D) approximately 1.38 of the protein multilayer was estimated. Fitting of the model gave a refractive index n(D) = 1.377 of the protein multilayer and an average thickness of 11 nm for a single protein layer. An average noise of Dn(eff) = 8.5 x 10(-7) was detected, corresponding to approximately 1% of the maximum response for a protein monolayer. At a triazine derivative attached to the surface through dextran-based surface chemistry, a maximum antibody loading that corresponds to an Dn(eff) of 1.5 x 10(-3) was observed. In an indirect immunoassay of the herbicide simazine, a detection limit of 0.25 mug/1 of simazine was reached with polyclonal Fab fragments in a concentration of 1 mug/ml.

The discrimination of optical isomers (enantiomers) in the gas phase has been performed using two different analytical tools:  thickness shear mode resonators (TSMRs) and reflectometric interference spectroscopy (RIFS). The selective coatings included both enantiomers ((S)- and (R)-receptor) of a Chirasil-Val derivative (stationary phase material in GC) with octyl side chains. Successful discrimination of the enantiomers of different types of analytes (amino acids and lactates) was achieved. The results of both transduction methods were consistent and in good agreement with GC measurements. In addition, different mixtures of both enantiomers of the respective analyte were measured, and the enantiomeric composition could be quantitatively determined with excellent reliability. Since the sensors allow on-line monitoring (not possible with GC) of enantiomeric purity, an application in industrial synthesis (process control) of such compounds represents an interesting feature, especially with regard to the tested derivatives of lactic acid.

We report on the label- and isotope-free monitoring of DNA interactions with low-molecular-weight ligands. An optical technique based on interference at thin layers was used to monitor in real time binding of ligands at DNA which was immobilized by Coulomb interactions at a positively charged surface. Approximately 2 ng DNA/m2 was irreversibly bound to the surface, which remained stable over several days. This result was confirmed by characterization of the layer using spectroscopic ellipsometry. During incubation of immobilized DNA with a variety of intercalators and other DNA-binding compounds in a flow system, interactions were monitored by reflectometric interference spectroscopy. Binding effects between 10 and 400 pg/ mm2 were detected unambiguously. Nonspecific binding effects were excluded by using a negatively charged reference surface. Variation of intercalator concentration allowed the characterization of interaction with respect to kinetics and thermodynamics by the evaluation of binding rate and equilibrium coverage. The affinity constants were determined in the range between 10(5) and 10(6) M-1, in good agreement to those obtained by homogeneous phase assays. Association rate constants between 10(3) and 10(5) M-1 s-1 and dissociation rate constants between 10(-1) and 10(-2) s-1 were determined by evaluation of the binding curves. Both the fast and simple test format and a universal applicability make the new technique described attractive for detecting and characterizing interaction of low-molecular-weight molecules with DNA.

Odour perception in humans can sometimes discriminate different enantiomers of a chiral compound, such as limonene. Chiral discrimination represents one of the greatest challenges in attempts to devise selective and sensitive gas sensors. The importance of such discrimination for pharmacology is dear, as the physiological effect of enantiomers of drugs and other biologically active molecules may differ significantly. Here we describe two different sensor systems that are capable of recognizing different enantiomers and of qualitatively monitoring the enantiomeric composition of amino-acid derivatives and lactates in the gas phase. One sensor detects changes in mass, owing to binding of the compound being analysed (the 'analyte'), by thickness shear-mode resonance; the other detects changes in the thickness of a surface layer by reflectometric interference spectroscopys. Both devices use the two enantiomers of a chiral polymeric receptor, and offer rapid on-line detection of chiral species with high selectivity.

We present a method for the determination of affinity constants based on equilibrium binding between an analyte and an antibody in liquid phase by a heterogeneous phase detection scheme. Equilibrium concentration of free antibody binding sites was probed kinetically by direct optical detection of specific binding to an immobilised analyte derivative. The additional binding signal due to dissociation of the analyte-antibody complex during detection was minimised by the use of fast flow-through conditions. The concentration of free antibody binding sites was titrated by adding increasing analyte concentrations. The affinity constant was derived from the titration curve by a non-linear least square fit of a model function. The affinity of monoclonal triazine antibodies to several s-triazine pesticides and a relevant metabolite was investigated. Kinetic determination of equilibrium concentration of free binding sites was carried out by reflectometric interference spectroscopy (RIfS) using flow injection analysis. The capabilities of the model were investigated using different analyte-antibody pairs and various antibody concentrations. Both bivalent IgG and monovalent Fab fragments were used to compare different binding models. The applied model corresponds well to the titration curves for affinity constants of 10(7) M(-1) and higher. For lower affinity constants significant deviations due to dissociation of the analyte-antibody complex during detection were observed.

The characterization of low molecular weight ligand interaction with receptor molecules is of importance for the investigation of biological processes and for drug research. We report on the investigation of the binding of low molecular weight ligands to immobilized receptors by label-free detection. Reflectometric interference spectroscopy, an optical transducer which allows the monitoring of a few picograms per square millimetre changes in surface coverage, was used to study two model systems. In both cases detection of the binding event was successful. High affinity binding of biotin to immobilized streptavidin was clearly detectable at receptor surface concentrations as low as 1-2 x 10(10) binding sites/mm2. Linear correlation between the receptor surface concentration and the response to biotin binding was observed. Using immobilized DNA, we investigated the binding of common intercalators with respect to kinetics and thermodynamics by evaluation of the association and the dissociation part of the binding curve. Bi-exponential increase and decrease of intercalator loading was observed, indicating complex interaction kinetics. The four structurally different intercalators showed significant distinction in binding kinetics and equilibrium signals. Improvement of experimental parameters is required to obtain more reliable kinetic data.

The characterisation and optimisation of an optical immunoassay with label free detection based on Reflectometric Interference Spectroscopy (RIfS) is presented. The immunoprobe is operated in a sequential scheme, where Fab-fragments react with analyte molecules in a first step. In a second step the optical transducer is used to quantify the amount of unoccupied Fab- fragments in the reaction mixture binding to the hapten-modified transducer surface. For optimisation of the test, the Fab-fragment concentration was varied between 2x10(-8) mol/l and 2.5x 10(-9) mol/l. Down to a concentration of 5x10(-9) mol/l a reduction in the limit of detection has been observed. At the lowest concentration investigated no further improvement has been found due to a reduced binding of the analyte and a strong decrease of antibody binding at the transducer surface. This finding could be explained by the thermodynamics of the antigen-antibody reaction and the performance of the optical transducer used. The limit of detection obtained is discussed with respect to thermodynamics, transducer characteristics and immunoprobe test format.

The modification of glass-type surfaces by several hydrophilic polymers of different molecular masses and functional properties [chitosan, dextran, poly(oxyethylene), poly(ethyleneimine) and poply(acrylamide)] with respect to the application for direct immunoprobes was investigated. Activation of the surface was carried out by silanisation and the polymers were coupled to the surface via amide bonds. The carboxyl derivative of a hapten was attached to the functional groups of the polymers by carbodiimide-activated coupling. As a reference system, the ligand was directly coupled to the silanised surface. Non-specific protein adsorption, specific binding of antibodies and regeneration were monitored by evaluation of reflectance spectra obtained by white light interference at a thin silica layer (RifS). All polymer modified layers showed improved properties compared to those with direct attachment of the hapten. The non-specific adsorption was reduced to 5-50%. Binding of a specific antibody was significantly increased by the polymer modification: Mass transport limited binding of the specific antibody in low concentrations (30 nM) up to a surface coverage value of 2 ng/mm2 and a maximum surface coverage in the range of a monolayer of IgG (5-6 ng/mm2) was observed for most of the polymers. The surface coverage found for IgG bound specifically to the dextran-modified surface exceeded a protein monolayer.

In this paper we report attempts to detect directly the binding of a low molecular weight substance to a protein binding site. An optical transducer based on reflectometric interference spectroscopy (RIFS) was used to detect the binding of biotin (244 g/mol) to a thin silica film surface coated with streptavidin. RIFS allows measurement of changes in the optical thickness of thin transparent films with high resolution. During immobilization of streptavidin, an increase in layer thickness of about 5 nm was detected. Subsequent incubation with biotin (4 μM) resulted in a thickness increase of about 70 pm. Repeated incubation with biotin gave no further increase in layer thickness. The lowest biotin concentration showing significant effects was 40 nM. Incubation with benzoic acid (40 μM) gave no thickness change. The setup allowed significant detection of thickness increases of 2 pm and above. Therefore, the thickness effects observed in the study could be unambiguously and clearly identified.

A multicomponent detection system using optical biosensors and flow injection analysis is described. The analysis of mixtures containing penicillin and ampicillin was realised by evaluating dynamic measurements of Phenol Red spectra in penicillinase optodes in combination with a diode array spectrometer. A variety of optodes has been produced by changing the composition of the receptor gel and the working pH. A set of characteristic quantities (describing dynamic and static features) could be obtained for each optode. These were used to compare the predictivity of classical multivariate calibration methods as well as of an artificial neural network. In addition, different algorithms were applied for the evaluation of the spectral data in order to select the most appropriate method for feature extraction. In consequence, the information obtained from the multivariate calibration models was used to set up an optimal sensor array consisting of four optodes with different types of penicillinase at different working pH.

Biosensors are by definition a combination of a biological receptor compound and a physical or physicochemical transducer. Therefore, the transducing structure is a critical part of every biosensor. In the development of new and improved biosensing layers the importance of the transducing structure is not restricted to the substrate to which biological structures have to be coupled. A field of even greater importance is the use of transducers as probes providing information on the structure and function of biosensing layers, and their relation to a transducer surface. The aim of this paper is to give an overview on optical transducer principles and optical (surface) analytical techniques relevant as part of biosensing structures as well as probes in the development and optimisation of biosensing layers. Categories discussed are basic optical effects, materials involved, surface chemistry, the principal and technological limits of spatial resolution, and sensitivity. The intimate relation between the spatial resolution of a probe, the resulting size of interaction areas, and the feasibility of array structures is pointed out. Two interferometric methods are presented in principle, and their application to biosensing and some results are discussed in detail. The necessity to characterise receptor layers to get detailed information about the interaction process is pointed out. The close relationship between optimal characterisation of layers by selection of adequate probe technologies and improvement of probe performance, and the development of new biosensing layers is discussed. Finally, an outlook is given for future aspects of improved spatial resolution and multianalyte detection.

Phospholipid bilayers with transport proteins and antigen/antibody interfaces are considered to be suitable biosensor systems. The quality of such membranes or interfaces depends on the properties of the layers. Optical methods have proved to be an appropriate tool for characterizing those layers in situ and in a non-destructive manner. Two systems with potential for biosensor applications are characterized by some of these methods: phospholipid bilayer membranes spread from vesicle solution and protein-antigens both adsorbed on planar solid support. The results of spectral ellipsometry, surface plasmon resonance (SPR) and spectral interferometry are compared with respect to quality of characterization, expenditure of sample preparation and measurement, and time resolution. The phospholipid membranes adsorbed show a relatively low refractive index and a relatively high thickness. Bruggeman effective medium approximation is used to calculate the effective layer thickness. This result is compared to SPR measurements. A correlation between thickness and vesicle concentration may be detected. Further, the test protocol of an immunoassay is examined by spectral interferometry and SPR. Thicknesses determined are compared to results obtained by applying spectral ellipsometry. The data measured by ellipsometry are in agreement with the molecular dimensions of the immunoglobulins. Differences between details can be explained by physical considerations.