Introduction

When faced with the challenge of developing a sensor, a difficult question to answer is how to transduce a biological event into a quantifiable signal. The reflection of electromagnetic radiation from a surface, also known as reflectometry, is a basic sensing principle which has found various applications over the years, from time-domain-reflectrometry to check the quality of electrical lines and aircraft wiring, to x-ray reflectometry to determine the structure of any type of thin film.

Reflectometric interference spectroscopy (RIfS) allows to determine the thickness of a thin transparent film on a reflective surface by measuring the spectrum of white light reflected from the sample. When a substrate is irradiated, part of the light is immediately reflected from its surface, but part of it enters into the transparent film, and is only cast back after hitting the second interface. Now, if the reflected light is collected in the right angle, an interference pattern arises in its spectrum, the modulation of which depends on the pathlength traveled by the refracted beam. In other words, the thickness of the transparent layer. This occurrence is shown in figure 3.4. RIfS is a well established method to monitor the coatings of lenses or semiconductors. Despite its simplicity, there were not many advocators for its application in the field of biosensing in the past.

Prof. Günter Gauglitz of the Analytical Chemistry Department of the University of Tübingen was the first to use RIfS to directly monitor antigen-antibody interactions in 1991 (rabbit IgG with anti-rabbit IgG). However, the method was first mentioned in 1989, when used to sense inorganic reactions [63, 64]. Figure 3.5 A shows the sensor system developed in the group of Prof. Gauglitz. A BK7 glass slide coated with a functionalised polymer film (green dots) is irradiated from the bottom. The light is reflected at the glass-polymer (grey arrow) and polymer-water interface (green arrow). When an analyte

Figure 3.5.:A: RIfS set-up invented by Prof. Günter Gauglitz [2]. A polymer coated BK7 glass is irradiated from the bottom. The binding of an analyte at the polymer water interface is detected B: RIfS set-up invented by Prof. Michael J. Sailor [62].

Porous silicon is irradiated from the top. Analyte binding to the pore walls causes a change in the refractive index of the porous layer, resulting in a spectral shift of the interferogram. The drawings were made in accordance to the schematics shown in the cited publications.

is introduced in the aquaous phase above the polymer film (red molecules), it may bind to the receptors present on the polymer, and causes the film thickness to rise, which results in a change of the RIfS signal (red arrow). Over the years this set-up underwent only minor changes.

From the extensive body of work Prof. Gauglitz and his group accomplished with reflectometry sensing in the past twenty years, only some landmarks will be briefly men-tioned. After optimising their transducer element, the main focus of investigation lay on the development of immunosensors for low molecular weight analytes [65]. In 1994, they reported on a competitive immunoassay to sense dinitrophenol/anti-dinitrophenol interaction, and on the direct detection of biotin binding to immobilised streptavidin in 1996 [66, 67]. In the following year, they published an assay which allows to label-free monitor DNA-ligand interaction [68]. Apart from their work on different specific antigen-antibody interactions, the latest published paper on anti-β2-glycoprotein-I an-tibodies came out in 2012, they focused on optimising the instrument itself [69]. The first report on an instrument designed for high throughput pharmaceutical screening was released in 1997, which was later developed into a parallel set-up allowing for the use of 96 well-plates in 2002 [70–72]. In this parallelised instrument, the sensing does not rely on the measurement of the entire white light spectrum, but in a simplified way on the detection of just four selected wavelengths. In 1994, they published a study, using biomembranes spread from vesicle solution as sensing platform, but apparently dismissed this option later on, due to difficulties with the reproducible formation of a lipid mem-brane on the sensor surface [73].

3.2 RIfS: Introduction

In 2004, Prof. Jacob Piehler of the Biophysics Department of the University of Os-nabrück published a study in which he showed the ligand induced assembly of the type I interferon receptor on supported lipid bilayers with RIfS [74]. Furthermore, he reported on a set-up which combined the technique with total internal reflection fluorescence spec-troscopy [75].

Prof. Michael J. Sailor of the Chemistry and Biochemistry Department of the Uni-versity of California in San Diego showed in 1992 that micron-dimension porous silicon structures exhibit luminescence in the visible spectral region [76]. Five years later, he introduced a biosensor based on RIfS which allowed for the detection of interactions in-side a porous silicon film. The set-up can be seen in figure 3.5 B. A porous silicon chip is irradiated from the top. Light is partially reflected from the surface and again at the interface between porous and bulk silicon, causing an interference pattern to arise in the light gathered from the sample, the modulation of which is dependent on the thickness and refractive index of the porous layer. By adsorbing material to the pore walls, the refractive index of the porous layer is altered, causing a shift in the interference pat-tern. At the time, they had applied the sensor for the detection of biotin, the steroid digoxigenin, short DNA oligonucleotides, streptavidin and several antibodies [62].

In years to come, the research efforts of the group of Prof. Sailor focused on the improvement of the porous transducer chip of their biosensor. Their investigations com-prise surface modifications, optimising pore sizes, double layer etching of porous silicon and testing different pore materials such as anodic aluminum oxide and titania nan-otube arrays [77–81]. His latest publication from 2011 dealt with the combination of RIfS with electroadsorption based on conductive carbonised porous silicon films. By ap-plying a voltage to a conductive optical film, they were able to sense the accumulation of positively charged proteins on the pore walls with RIfS, thus demonstrating that this combination may be used to identify molecules based on their size, charge and diffusional characteristics [34].

Several other research groups made use of the two sensing approaches poineered by Gauglitz and Sailor without significantly changing the instrumental assemblies [82–84].

Recently a commercial version of Gauglitz set-up was brought to the market by Analytik Jena (Konrad-Zuse-Str. 1, 07745, Jena) called BIAffinity, which led to a considerable increase in the number of publicated studies performed with reflectometric interference spectroscopy [85,86].