Biosensors based on binding-modulated donor–acceptor distances Chunhai Fan 1 , Kevin W. Plaxco 2 and Alan J. Heeger 3 1 Division of Nanobiology and Nanomedicine, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, P.R. China 2 Department of Chemistry and Biochemistry and Interdepartmental Biomolecular Science and Engineering Program, University of California at Santa Barbara, Santa Barbara, CA 93106, USA 3 Insitute for Polymers and Organics Solids, Department of Physics and Department of Materials, University of California at Santa Barbara, Santa Barbara, CA 93106, USA The promising recognition characteristics exhibited by biomolecules have caused significant interest in bio- molecule-based sensor strategies. Here we review several emerging biosensor designs that use modulated electron or energy transfer to a bio-specific ligand as the signaling mechanism. The efficiencies of both electron transfer and energy transfer are strongly dependent on donor–acceptor distance. When coupled with the large conformational changes sometimes associated with biomolecular recognition, these distance-dependant processes provide a robust means for generating optical and electronic signals. Introduction The detection of biological materials has a crucial role in a wide range of applications, including clinical diagnosis, environmental monitoring, forensic analysis and anti- terrorism. This demand has motivated significant interest in the development of convenient, real-time biosensors. Biosensors are devices that transduce a bio-recognition event, such as an antibody–antigen binding or the formation of a DNA duplex, into measurable electronic or opto-electronic signals. As such, biosensors offer the potential to replace traditional biochemical assays in applications, such as those listed above, in which time and operational convenience are paramount. In many biosensor architectures, signal magnitude is dependent on the efficiency of electron transfer or energy transfer between exogenous (non-biological and therefore readily detected even in the presence of biological contaminants) donors and acceptors (D/A) attached to the biomolecule. Because these transfer efficiencies are a sensitive function of D/A distance – Forster resonance energy transfer (FRET) efficiency falls off with the sixth power of distance and electron transfer (ET) rates fall off exponentially – the modulation of electron- or energy-transfer processes provides a ready means for signal generation. Here, we focus on recent efforts to rationally incorporate recognition- modulated ET and FRET in sensitive, real-time electronic and optical biosensors. Ligand-modulated FRET as a signaling mechanism Competition-based D/A distance change Although FRET has been used as a ‘biomolecular ruler’ since Stryer’s pioneering work in the 1960s [1], a recent flurry of work in the area has shown that FRET is well suited for many optical biosensor applications. Because of their ease of design, competition assays have long been applied to the problem of detecting protein– ligand binding reactions when a suitable binding-induced distance change can be identified [2]. A recent, sensor- relevant example of this was reported by Mauro et al., who proposed a maltose biosensor based on maltose-binding protein (MBP). The donor–acceptor pair was established by employing an emissive semiconductor quantum dot (QD) decorated with MBP and a dye-labeled ligand analogue b-cyclodextrin (b-CD) [3]. Although b-CD binds to the MBP, it is competitively displaced by the natural ligand maltose, leading to the physical separation of the QD–MBP and the b-CD-dye conjugate. This separation effectively eliminates FRET-based quenching of the QD, quantitatively increasing the fluorescence signal in response to maltose in the range of 0.5 to 100 mM (Figure 1a). More recently, they have provided in-depth FRET analysis of this QD–protein bioconjugate nano- assembly, demonstrating the possibility for more efficient and accurate assays via precise control of the system [4,5]. It is noteworthy that the narrow, highly tunable band- widths, extreme resistance to photobleaching and high luminosity of quantum dots [6], combined with the relatively widespread applicability of competition assays [2] suggests that this approach might be useful in the detection of a wide range of protein–ligand interactions. Enzymatic cleavage-based D/A distance change Enzymatic cleavage of a linker can also be harnessed to physically separate a D/A pair, thus providing a sensitive means of detecting many enzymatic activities, an approach that has been used for the sensitive detection of proteases [7] and other enzymes [8]. Historically, however, this approach was limited to the detection of enzymatic activities [9] that could be used to cleave a D/A–linker conjugate. Ghardiri and coworkers have recently extended this sensing strategy to the detection of DNA hybridization via an engineered molecular Corresponding author: Fan, C. (fchh@sinap.ac.cn). Review TRENDS in Biotechnology Vol.23 No.4 April 2005 www.sciencedirect.com 0167-7799/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2005.02.005