Reflectometric interference spectroscopy (RIfS) is a physical method based on the interference of white light at thin films, which is used to investigate molecular interaction.

Principle

The underlying measuring principle corresponds to that of the

Michelson interferometer The Michelson interferometer is a common configuration for optical interferometry and was invented by the 19/20th-century American physicist Albert Abraham Michelson. Using a beam splitter, a light source is split into two arms. Each of those li ...

Realization

White light is directed vertically onto a multiple-layer system of a SiO₂, a high-refractive Ta₂O₅ and an additional SiO₂ layer (this additional layer can be chemically modified). The partial beams of the white light are reflected at each phase boundary and then refracted (transmitted). These reflected partial beams superimpose which results in an interference spectrum that is detected using a diode array spectrometer.
Through chemical modification the upper SiO₂ layer is changed in a way to allow interaction with target molecules. This interaction causes a change in the thickness of the physical layer d and the

refractive index In optics, the refractive index (or refraction index) of an optical medium is a dimensionless number that gives the indication of the light bending ability of that medium. The refractive index determines how much the path of light is bent, or ...

n within this layer. The product of both defines the optical thickness of the layer: n • d.
A change in the optical thickness results in a modulation of the interference spectrum. Monitoring this change over time allows to observe the binding behaviour of the target molecules.

Application

RIfS is used especially as a detection method in chemo- and

biosensor A biosensor is an analytical device, used for the detection of a chemical substance, that combines a biological component with a physicochemical detector. The ''sensitive biological element'', e.g. tissue, microorganisms, organelles, cell recep ...

s. Chemosensors are particularly suitable for measurements under difficult conditions and in the gaseous phase. As sensitive layers, mostly non-selective measuring polymers are used which sort the analytes according to size (the so-called molecular sieve effect when using microporous polymers) or according to polarity (e.g. functionalized

polydimethylsiloxane Polydimethylsiloxane (PDMS), also known as dimethylpolysiloxane or dimethicone, belongs to a group of polymeric organosilicon compounds that are commonly referred to as silicones. PDMS is the most widely used silicon-based organic polymer, as its ...

s). When performing non-selective measurements, a sum signal from several analytes is measured which means that multivariate data analyses such as

neural network A neural network is a network or circuit of biological neurons, or, in a modern sense, an artificial neural network, composed of artificial neurons or nodes. Thus, a neural network is either a biological neural network, made up of biological ...

s have to be used for quantification. However, it is also possible to use selectively measuring polymers, so-called

molecular imprinted polymer A molecularly imprinted polymer (MIP) is a polymer that has been processed using the molecular imprinting technique which leaves cavities in the polymer matrix with an affinity for a chosen "template" molecule. The process usually involves initiatin ...

s (MIPs) which provide artificial recognition elements. When using

polymer A polymer (; Greek '' poly-'', "many" + ''-mer'', "part") is a substance or material consisting of very large molecules called macromolecules, composed of many repeating subunits. Due to their broad spectrum of properties, both synthetic a ...

s such as

polyethylene glycol Polyethylene glycol (PEG; ) is a polyether compound derived from petroleum with many applications, from industrial manufacturing to medicine. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular we ...

s or

dextran Dextran is a complex branched glucan (polysaccharide derived from the condensation of glucose), originally derived from wine. IUPAC defines dextrans as "Branched poly-α-d-glucosides of microbial origin having glycosidic bonds predominantly C-1 ...

s are applied onto the layer system, and on these recognition elements for biomolecules are immobilized. Basically, any molecule can be used as recognition element (proteins such as

antibodies An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...

, DNA/RNA such as

aptamer Aptamers are short sequences of artificial DNA, RNA, XNA, or peptide that bind a specific target molecule, or family of target molecules. They exhibit a range of affinities ( KD in the pM to μM range), with little or no off-target binding ...

s, small organic molecules such as

estrone Estrone (E1), also spelled oestrone, is a steroid, a weak estrogen, and a minor female sex hormone. It is one of three major endogenous estrogens, the others being estradiol and estriol. Estrone, as well as the other estrogens, are synthesized ...

, but also

lipids Lipids are a broad group of naturally-occurring molecules which includes fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, and others. The functions of lipids include ...

such as

phospholipid Phospholipids, are a class of lipids whose molecule has a hydrophilic "head" containing a phosphate group and two hydrophobic "tails" derived from fatty acids, joined by an alcohol residue (usually a glycerol molecule). Marine phospholipids typ ...

membranes). RIfS, like SPR is a label-free technique, which allows the time-resolved observation of interaction among the binding partners without the use of fluorescence or radioactive labels.

Literature

* G. Gauglitz, A. Brecht, G. Kraus and W. Nahm. Sensor. Actuat. B-Chem. 11, 1993 * A. Jung. Anal. Bioanal. Chem. 372 1, 2002 * F. Gesellchen, B. Zimmermann, F. W. Herberg. Methods in Molecular Biology, 2005 * T. Nagel, E. Ehrentreich-Forster, M. Singh, et al. Sensors and Actuators B-Chemical 129 2, 2008 * P. Fechner, F. Pröll, M. Carlquist and G. Proll. Anal. Bioanal. Chem. Nov 1, 2008

External links

Barolo.ipc.uni-tuebingen.de
Spectroscopy