Label-free biosensor technologies allow the detection of molecular interactions and cell-based biological readouts without the need for conventional labels or engineered cell lines with overexpressed targets. A number of label-free detection technologies have been used for monitoring biomolecular interactions, most notably surface plasmon resonance spectroscopy (SPR) and isothermocalorimetry (ITC) [1–4]. With the rapid development of label-free based detection instrumentation in recent years, the power of various label-free technologies for studying biomolecular interactions and endogenous cellular events has been recognized by biomedical investigators, especially scientists involved in high-throughput screening (HTS). This, in turn, has led to a rapidly expanding list of various assay formats for HTS and allowed, for the first time, label-free screening for drug discovery in an HTS format [3, 5]. This chapter reviews basic principles of several major label-free biosensor technologies, with emphasis on microplate-based technologies and their practical applications in small molecule modulator discovery.

Traditional Label-Based Technologies for HTS

Various in vitro biochemical assays and cell-based methods are frequently employed in HTS for small molecule modulator discovery. These assays are often performed in a microplate format (96/384/1536 wells) in order to meet the demand of handling large sample numbers in primary HTS. In vitro biochemical assays rely on the use of an isolated or purified target that is often coupled with a tag (e.g., a fluorophor) for detection. These targets include purified adaptor proteins, enzymes, receptors, or cellular extract containing the target(s) of interest. Common detection methods in biochemical HTS assays include fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), fluorescence intensity (FI), luminescence, and absorbance. FP measures the binding of a macromolecule to a relatively small peptide or ligand labeled with a fluorophore [6]. Binding of a macromolecule to the fluorophore-labeled small ligand/peptide slows down its rotation in solution and generates an FP signal. FRET allows the measurement of interacting molecules within a certain distance (<100 Å) (see Chapter 14 for details). The target and its binding partner are labeled separately, with a donor and a paired acceptor fluorophore containing matching excitation and emission spectra. Binding of the two molecules brings the donor and acceptor into close proximity, allowing energy transfer from the donor to the acceptor and FRET signal production. FRET signals can be derived in order to provide structural proximity information of molecular interactions [7]. FI-based assays measure changes in fluorescence intensity resulting from biochemical reactions. For example, cleavage of an appropriately fluorophore-labeled substrate by an enzyme can lead to a change in fluorescence intensity [8]. Biochemical assays are routinely used for monitoring receptor-ligand interactions, protein-protein interactions, and enzymatic activities (e.g., kinases and proteases).