Gert Ludwig Duveneck

Planar waveguides for ultra-high sensitivity of the analysis of nucleic acids

Abstract In the first part of this paper, the need for analytical techniques capable of highly parallel and sensitive nucleic acid analysis, with the capability of achieving very low limits of detection (LODs) and of resolving small differences in concentration, is described. Whereas the requirement for performing simultaneously multi-analyte detection is solved by the approach of nucleic acid microarrays, requirements on sensitivity can often not be satisfied by classical detection technologies. Inherent limitations of conventional fluorescence excitation and detection schemes are identified, and the implementation of planar waveguides as analytical platforms for nucleic acid microarrays, with fluorescence excitation in the evanescent field associated with the guided excitation light, is proposed. The relevant parameters for an optimization of sensitivity are discussed. In the second part of this paper, the specific formats of our planar waveguide platforms, which are compatible with established industrial standard formats allowing for integration into industrial high throughput environments, are presented, as well as the dedicated optical system for fluorescence excitation and detection that we developed. In a direct comparison with a state-of-the-art scanner, it is demonstrated that the implementation of genomic microarrays on planar waveguide platforms allows for unprecedented, direct detection of low-abundant genes in limited amounts of sample. Otherwise, when using conventional fluorescence excitation and detection configurations, the detection of such low amounts of nucleic acids requires massive sample preparation and signal or target amplification steps.

Novel bioaffinity sensors for trace analysis based on luminescence excitation by planar waveguides

Abstract We have developed a novel generation of optical bioaffinity sensors for ultra trace analysis. These sensors are based on luminescence generation in the evanescent field of high-refractive-index single-mode planar waveguides, With the waveguiding layers and the grating parameters chosen, very sharp discrimination of bulk against surface-confined excitation, in combination with high excitation intensities in the evanescent field, can be achieved, leading to unprecedented sensitivity. Experimental data of the optimization of the transducer parameters will be presented. Incoupling of excitation light is performed using diffractive gratings. Different methods for the detection of both transmitted and luminescence light will be presented. The transmitted excitation light can be detected either at the distal waveguide chip end or using a second outcoupling grating. Both isotropically emitted luminescence, collected by a lens located below the transducer substrate (‘volume detection’), and emission coupled back into the waveguiding layer can be monitored, the latter via a second outcoupling grating. First experimental results obtained in model bioaffinity assays will be presented, demonstrating the feasibility of the different detection methods mentioned above, as well as the superior sensitivity of our novel sensor configuration. In still preliminary experiments, 100 attomoles of fluorescently labelled DNA (16-mer oligonucleotide), applied at 100 femtomolar concentration, can be detected.

Fiber-optic Atrazine immunosensor☆

Abstract Fiber-optic sensors based on the excitation of luminescent chromophores by the evanescent field associated with light guiding in an optical fiber can be used for highly sensitive and selective biochemical affinity assays. Due to the small penetration depth of the evanescent field into the medrium, the generation and detection of luminescence are restricted to the close proximity of the fiber core, i.e., fluorophores in solution beyond the evanescent field will not contribute to the emission signal. Evanescent wave sensors allow the binding of fluorophores to the sensor surface to be monitored in real-time mode. These advantages make this approach especially useful for the determination of substances in complex media, such as blood, river water or soil extracts. An evanescent-wave fiber-optic immunosensor for the detection of the herbicide Atrazine has been developed. In the competitive assay format chosen, fluorescein-labeled and nonlabeled Atrazine in solution compete for the binding sites of anti-Atrazine antibodies immobilized on the surface of the optical fiber. A signal reproducibility of better than 5% within the working range of the sensor (0.5–200 nM Atrazine concentration) is achieved. The sensor performance in complex media has been investigated using samples of surface water and soil extracts.

Fiber optic sensor for oxygen determination in liquids

Abstract A fiber optic sensor for the determination of oxygen in liquids is presented. Potential applications range from environmental analytics to medical diagnostics and process control. The sensing principle is based on dynamic luminescence quenching by oxygen. A ruthenium complex used as oxygen-sensitive dye is immobilized on an optical fiber by adsorption and subsequent coating with a membrane. Excitation of the Ru complex is performed both by the evanescent field associated with light guided in the optical fiber and by direct irradiation, dependent on the refractive index profile of the fiber/membrane/medium system. The emitted light is collected by the same fiber. The temperature-controlled sensor is exposed to a liquid stream of defined oxygen concentration. Oxygen is detectable in the range 0–800 Torr, with a resolution of 2 Torr in the 0–100 Torr range and of 2% for oxygen partial pressures above 100 Torr. Response times are of the order of 30 s. Generally, sensor signals are affected by sample medium. Optimized sensors exhibit a good performance, with no effects of protein adsorption or background fluorescence, even in serum.

Sensing and reference pads for integrated optical immunosensors

For integrated optical immunosensors, a much more reliable interpretation of the sensor output signal can be achieved by introducing reference pads near the sensing pads, in order to separate specific from nonspecific effects. The results of theoretical and experimental investigations are reported for immunosensors based on measuring changes of the effective refractive index due to the binding of analyte molecules. The emphasis is on the correction for variations of the light wavelength, the angle of incidence, and the temperature for integrated optical grating coupler chips. Using the binding of rabbit immunoglobulin to immobilized protein A as a regenerable model bioaffinity system, the influence of consecutive assay cycles on the two pads has been investigated. Experiments have been performed using sensor chips consisting of TiO2 waveguiding films on fused silica and on replicated polycarbonate substrates.

A model system for the development of an optical biosensor based on lipid membranes and membrane-bound receptors

Abstract The reproducible and easy immobilization of receptors on sensor surfaces is a prerequisite for the development of receptor-based fibre optic biosensors. Using a fused silica fiber as the transducer, binding processes of luminescently labeled ligands can be monitored by evanscent wave sensor (EWS) technology. The vesicle fusion technique was chosen for the immobilization of membrane-bound receptors in order to preserve their binding specificity and activity, by embedding them in an environment similar to a lipid bilayer. The results of initial studies of repetitive cycles of lipid layer deposition and removal, indicating good reproducibility of lipid layer formation on the fiber, are presented. Using the binding of fluorescently labeled streptavidin to a biotinylated lipid layer as a model system for receptor-ligand interaction, good sensitivity, combined with low non-specific binding were observed.

Automated optical sensing system for biochemical assays

In this paper, we present a new system called FOBIA that was developed and optimized with respect to automated operation of repetitive assay cycles with regenerable bioaffinity sensors. The reliability and precision of the new system is demonstrated by an application in a competitive assay for the detection of the triazine herbicide Atrazine. Using one sensor in more than 300 repetitive cycles, a signal precision better than 5% was achieved.

Novel generation of luminescence-based biosensors: single-mode planar waveguide sensors

First results with a new generation of bioaffinity sensors based on luminescence excitation using single-mode planar waveguides are presented. Planar waveguides show superior physical characteristics, concerning sensitivity and required sample volumes, and can principally be fabricated in low-costs mass production processes. Additionally, they allow for different detection geometries, e.g., different configurations of fluorescence detection and simultaneous determination of the transmitted excitation light. The transducer characteristics, possible detection geometries and our present experimental configuration are explained. Results with different bioaffinity systems applied on the novel transducer generation are presented, demonstrating the capability of detecting attomole amounts of analytes within a few minutes.

Planar waveguides as efficient transducers for bioaffinity sensors

Specific detection of extremely low amounts of antigens or disease markers allowing the diagnosis of diseases and infections at a very early stage has become a major driving force for the development of new generations of biochemical sensors. To match this goal, we propose the substitution of the widely used fiber-shaped evanescent field sensors by planar single-mode metal oxide waveguides. In combination with bioaffinity assays, this transducer geometry offers benefits such as enhanced sensitivity, ease of sensor handling and preparation, sample volume reduction, versatility, and low cost per test. Recently, planar waveguides have been used in sensor schemes exploiting the changes of the so-called effective refractive index (caused by changes in mass of surface-bound biomolecules): grating couplers, surface plasmon resonance, and interferometers. However, compared to luminescence-based sensor schemes, the sensitivities of these label-free methods are inferior. In this paper we report on a new generation of luminescence- based bioaffinity sensors for human diagnostics including optimization of the planar evanescent field transducers, the design of a compact sensor system, and a first study of the binding of fluorophore-labeled IgG to protein A immobilized on the transducer.

Reference and sensing pads for integrated optical immunosensors

For integrated optical immunosensors, a much more reliable interpretation of the sensor output signal can be achieved by introducing reference pads located near the sensing pads, in order to separate specific from nonspecific effects. Results of theoretical and experimental investigations are reported for immunosensors based on measuring changes of the effective refractive index due to the binding of analyte molecules. The emphasis is on the correction for temperature variations which is an important example of compensation for nonspecific effects. Using the binding of rabbit immunoglobulin to immobilized protein A as a regenerable model bioaffinity system, the influence of consecutive assay cycles on the two pads has been investigated. Experiments have been performed using sensor chips consisting of TiO2 waveguiding films on fused silica and replicated polycarbonate substrates.