Review Optimization of fluorescence response in the design of molecular biosensors Alexander P. Demchenko * TUBITAK Research Institute for Genetic Engineering Biotechnology, 41470 Gebze-Kocaeli, Turkey Faculte ´ de Pharmacie, UMR 7034, Universite ´ Louis Pasteur, 67401 Illkirch, France A.V. Palladin Institute of Biochemistry, 9 Leontovicha str., 01030 Kiev, Ukraine Available online 29 December 2004 Fluorescence is the most sensitive and easily available method to study intermolecular interactions [1,2]. It has many applications in the fields of molecular sensing and biosensing [3]. The most interesting developments in this field involve the design of direct reagent-independent molecular biosensors. Similar to other types of biosen- sors, such as electrochemical and piezoelectric sensing and surface plasmon resonance [4,5], fluorescence sen- sors are based on biospecific interaction between macro- molecules (molecular recognition); however, they also possess characteristic features that offer significant advantages in practical use. First, they are molecular sensors; that is, their operation (both sensing and response) takes place at the molecular level. They can be easily integrated into supramolecular and macro- scopic devices, although their basic operation does not require this. Therefore, they can be used for homoge- neous assays in solution. Second, they are direct, mean- ing that they do not require any intermediate signal transduction factors or steps in recording the sensing event. In addition, they are reagent-independent (some- times called reagentless) sensors, so that their operation does not require the supply and consumption of addi- tional reagents. Incorporation into sensor molecules of a fluorescent dye that changes its emission properties during the recognition event is the most promising approach for detecting sensor–analyte binding. The integration of molecular sensors into more complex units and devices usually does not change the basic sensor performance at the molecular level. Therefore, for every application in integrated schemes, design of the sensor must begin for basic sensor elements at the molecular level. A very attractive additional feature of direct sensors is the possibility of using nearly all possible biopolymer and mimetic systems in which molecular recognition can be realized or simulated. This has led to the development of a wide range of molecular biosensor technologies that include the use of antibodies and their fragments, ligand-binding proteins (natural and raised by selection using several scaffolds), enzymes and their chemically modified products, synthetic peptides, and natural and synthetic polynucleotides. A wide variety of targets have also been analyzed, from small ions to supramolecular structures and living cells. Although indirect biosensors and immunosensors are highly sensitive in analyte assays due to amplification effects that can be obtained in multistep-coupled reactions [6–8], the concept of direct 0003-2697/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.11.041 * Fax: +90 262 646 3929. E-mail address: dem@rigeb.gov.tr. ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 343 (2005) 1–22 www.elsevier.com/locate/yabio