Sensors and Actuators B 207 (2015) 740–747 Contents lists available at ScienceDirect Sensors and Actuators B: Chemical jo u r nal homep age: www.elsevier.com/locate/snb Self-referencing SPR-sensor based on integral measurements of light intensity reflected by arbitrarily distributed sensing and referencing spots  Shavkat Nizamov, Vitali Scherbahn, Vladimir M. Mirsky ∗ Department of Biotechnology, Brandenburg University of Technology Cottbus – Senftenberg, 01968 Senftenberg, Germany a r t i c l e i n f o Article history: Received 11 July 2014 Received in revised form 3 October 2014 Accepted 6 October 2014 Available online 12 October 2014 Keywords: Surface plasmon resonance SPR Self-referencing Patterned surface Biosensor Affinity sensor a b s t r a c t A new approach for self-referencing in SPR biosensors is reported. The method is technologically simple and applicable for a wide range of existing SPR instrumentation with Kretschmann configuration. It is based on the micropatterning of the sensor area with sensing and referencing areas whose shape and distribution can be chosen arbitrarily and their characteristic sizes are larger than the plasmons propagation length. Provided that roughly a half of the area is used as the sensing area while its optical thickness is different from that of the referencing area, an integral measurement of the intensity of the reflected light over such a patterned surface near the summary resonance conditions exhibits self- referencing properties. An over ten-fold suppression of the effect caused by the variation of the bulk refractive index was observed. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Since the introduction of biosensors based on the surface plas- mon resonance [1], they quickly become one of the most popular transducing approaches in affinity sensors [2]. SPR transducers are sensitive to the refractive index of surrounding media. Because an increase in the refractive index near sensing surface is a direct con- sequence of adsorption, this approach can be applied for different analytes: biopolymers (proteins, DNA, etc.), toxins, drugs, ions, etc. [3]. However, such versatility has also a drawback: the SPR sen- sors are sensitive to many other effects which also influence the refractive index of media. In the typical case of implementation of SPR biosensing tech- nologies, a parallel p-polarized light beam is reflected from thin metallic layer deposited on the surface of glass (see Fig. 1a). The reflectivity (ratio of reflected and incident light intensities) in such case depends on the coupling condition of incident light to  The topic was presented at the 7th European conference on Optical Sensors and Biosensors, Athens, Greece, 13–16 April 2014 only as a generally written abstract without disclosure of the principle. ∗ Corresponding author. Tel.: +49 3573 85917; fax: +49 3573 85 809. E-mail address: mirsky@b-tu.de (V.M. Mirsky). surface plasmons, which is described by well-known relation for the wavenumber k x : k x = 2   ε 1 ε 2 ε 1 + ε 2 = 2  √ ε 0 sin , where  is the free-space wavelength of incident light,  its inci- dence angle, ε 0 , ε 1 and ε 2 are dielectric permittivities of the glass, metal film and aqueous solution correspondingly. A plot of the reflectivity of SPR biosensor versus incidence angle (and/or wave- length) shows a strongly pronounced dip, whose exact position and shape depend also on ε 2 thus providing information on the envi- ronment near exposed metal surface (see Fig. 1a). On Fig. 1 such curves are shown for SPR biosensor consisting of 50 nm gold layer with 2 nm Cr adhesion layer deposited on the coupling prism with high refractive index (F2, n = 1.6). The gold layer is exposed to water. A deposition of a thin organic layer on the gold surface shifts the curve to the right (see Fig. 1b). This shift characterizes the thickness and the refractive index of the layer. In many cases both incidence angle and wavelength of the incident light are fixed. Such case is shown in Fig. 1c: the light with the wavelength 650 nm is incident at an angle of 64 ◦ . In response to increasing thickness of the organic layer a shift of the SPR curve to the right is observed, this leads to an increase in the intensity of the reflected light. Usually the point with the highest slope on the descending part of SPR curve is taken (like shown at 64 ◦ ). This is not a mandatory requirement, moreover, http://dx.doi.org/10.1016/j.snb.2014.10.022 0925-4005/© 2014 Elsevier B.V. All rights reserved.