R.P.H. Kooyman

Performance of a highly sensitive optical waveguide Mach-Zehnder interferometer immunosensor

We describe a highly sensitive sensor which uses the evanescent field of a reusable planar optical waveguide as the sensing element. The waveguide used is optimized to obtain a steep dependence of the propagation velocity on the refractive-index profile near the surface. The adsorption of a layer of proteins thus results in a phase change, which is measured interferometrically using a Mach-Zehnder interferometer set-up. The stability of the interferometer is such that phase changes = (1 × 10-2)2pi per hour can be measured. Immunoreactions have been monitored down to concentrations of 5 × 10-11 M of a 40 kDa protein.

Surface plasmon resonance immunosensors: sensitivity considerations

Some aspects of improving surface plasmon resonance response in immunosensing applications are considered. Both from calculations and experiments, it was found that maximum sensitivity is obtained for a silver layer about 55 nm thick in direct contact with the species to be quantified. Application of an intermediate layer with high permittivity can be useful in suppressing background responses. Experimentally, a protein surface-coverage fraction of ca. 0.1 could be measured, corresponding to ca. 10−10 mol1−1 antibody.

A new approach to immunoFET operation

A new method is presented for the detection of an immunological reaction taking place in a membrane, which covers the gate area of an ISFET. By stepwise changing the electrolyte concentration of the sample solution, a transient diffusion of ions through the membrane-protein layer occurs, resulting in a transient membrane potential, which is measured by the ISFET. The diffusion rate is determined by the immobile charge density in the amphoteric protein layer, which changes upon formation of antibody-antigen complexes. No membrane potential is induced at zero fixed charge density as occurs at a protein characteristic pH. Isoelectric points of embedded proteins can be determined by detecting the zero potential response. Up to now, the authors have studied the membrane adsorption of lysozyme, human serum albumin (HSA) and the immune reaction of HSA with the antibody anti-human serum albumin (alpha HSA). The influence of protein parameters on the amplitude of the transient can be described with an empirical equation. Assuming Langmuir behaviour, the protein concentration in the solution can well be correlated with the concentration in the membrane. This new detection method is unique concerning direct measurements of charge densities and isoelectric points of amphoteric macromolecules adsorbed in the membrane. The simple procedure of one incubation stage followed by one detection stage, without separate washing and labelling techniques, gives direct information about specific charge properties of the macromolecules to be studied.

Determination of thickness and dielectric constant of thin transparent dielectric layers using surface plasmon resonance

The determination of the thickness and dielectric constant of thin dielectric layers by means of surface plasmon resonance is discussed. It appears to be impossible to determine these parameters from one surface plasmon response experiment. This is illustrated theoretically. Variation of the refractive index of the solution in which surface plasmon experiments were performed allowed us to determine these parameters separately.

The realization of an integrated Mach-Zehnder waveguide immunosensor in silicon technology

We describe the realization of a symmetric integrated channel waveguide Mach-Zehnder sensor which uses the evanescent field to detect small refractive-index changes (?nmin ? 1 × 10?4) near the guiding-layer surface. This guiding layer consists of ridge structures with a height of 3 nm and a width of 4 ?m made in Si3N4. This layer has a thickness of 100 nm. The sensor device has been tested with glucose solutions of different bulk refractive indices. Results of a slab-model calculation are in good agreement with obtained experimental results. The feasibility of applying this sensor for immunosensing, detecting directly the binding of antigen to an antibody receptor surface, is shown with antibody-antigen binding experiments.

Light interactions with gold nanorods and cells: implications for photothermal nanotherapeutics

Gold nanorods (AuNR) can be tailored to possess an intense and narrow longitudinal plasmon (LP) absorption peak in the far-red to near-infrared wavelength region, where tissue is relatively transparent to light. This makes AuNRs excellent candidates as contrast agents for photoacoustic imaging, and as photothermal therapeutic agents. The favorable optical properties of AuNR which depend on the physical parameters of shape, size and plasmonic coupling effects, are required to be stable during use. We investigate the changes that are likely to occur in these physical parameters in the setting of photothermal therapeutics, and the influence that these changes have on the optical properties and the capacity to achieve target cell death. To this end we study 3 sets of interactions: pulsed light with AuNR, AuNR with cells, and pulsed light with cells incubated with AuNR. In the first situation we ascertain the threshold value of fluence required for photothermal melting or reshaping of AuNR to shorter AuNR or nanospheres, which results in drastic changes in optical properties. In the second situation when cells are exposed to antibody-conjugated AuNR, we observe using transmission electron microscopy (TEM) that the particles are closely packed and clustered inside vesicles in the cells. Using dark-field microscopy we show that plasmonic interactions between AuNRs in this situation causes blue-shifting of the LP absorption peak. As a consequence, no direct lethal damage to cells can be inflicted by laser irradiation at the LP peak. On the other hand, using irradiation at the transverse peak (TP) wavelength in the green, at comparative fluences, extensive cell death can be achieved. We attribute this behavior on the one hand to the photoreshaping of AuNR into spheres and on the other hand to clustering of AuNR inside cells. Both effects create sufficiently high optical absorption at 532 nm, which otherwise would have been present at the LP peak. We discuss implications of these finding on the application of these particles in biomedicine.

Choice of metal and wavelength for surface-plasmon resonance sensors: some considerations.

An optimal metal–waυelength combination depends on the measuring principle of the surface-plasmon resonance (SPR) sensor. Silυer at 800 nm is the best for fixed-angle measurements.

Possibilities and limitations of direct detection of protein charges by means of an immunological field-effect transistor

An outline of the requirements for the construction of an immunological field-effect transistor (ImmunoFET) which should operate on the direct potentiometric sensing of protein charges is given. Selectivity of the ImmunoFET can be obtained by immobilizing antibodies on the gate area of the ISFET, enhancing the surface affinity to the corresponding antigens over other molecules in the solution. A theoretical approach is given based on the Donnan equilibrium description, which provides an insight into the potential and ion distribution in the protein layer on the ImmunoFET. It is shown that the Donnan potential and the internal pH shift, induced by the protein charges, compensate each other to a great extent. If the ISFET shows Nernstian behaviour, it is concluded that a direct detection of protein charge is impossible. In order to construct an ImmunoFET, a reference FET (REFET) or ISFET with low sensitivity would satisfy the detection of the partially compensated Donnan potential in the presence of an adsorbed protein layer. However, the application of such an ImmunoFET is limited to samples with low ionic strength.

Determination of dielectric permittivity and thickness of a metal layer from a surface plasmon resonance experiment

In this paper we present a fast method for the determination of dielectric permittivity epsilon= -epsilon(r) + iepsilon(i) and thickness d of metal layers from surface plasmon resonance reflection curves. The method is an iteration process using starting parameters derived directly from a reflection curve. The method is tested with simulations and is applied to experimental results. Accuracies reached for silver layers between 25-100 nm and gold layers between 40-75 nm are better than: epsilon(r) +/-1%; epsilon(i) +/-13% and d +/-8%.

New detection method for atrazine pesticides with the optical waveguide Mach-Zehnder immunosensor

Concentrations of analytes can be determined within a few minutes using on-line analysis of the immunobinding kinetics in a solid phase immunoassay. This approach has been applied to the detection of atrazine. Atrazine is detected, at concentrations around the European Community limit (0.1 ?g/l) by a competitive assay. To this end, the two channels of a Mach-Zehnder waveguide sensor are used simultaneously in a difference measurement. The advantage of this way of measuring is discussed with the atrazine measurements.