Dynamic mass redistribution: a novel physiological signal of cells for cell systems biology and pharmacology Ye Fang*, Ann M. Ferrie, Guangshan Li, Florence Verrier, Elizabeth Tran, Jun Xi, Meenal Soni, Norman Fontaine, Ronald Verkleeren * Corresponding author. E-mail: fangy2@corning.com Biochemical Department, Science and Technology Division, Corning Incorporated, NY 14831 RECEIVED DATE: January 15, 2007 Abstract: This article presents a brief overview of label-free optical biosensor-based cell assay technologies. Theoretical analysis suggests that a resonant waveguide grating (RWG) biosensor detects ligand-induced dynamic mass redistribution (DMR) within the bottom portion of an adherent cell layer. Pharmacological studies suggest that the DMR signal can serve as a novel physiological readout for monitoring receptor activation, and examining ligand pharmacology. Chemical biology studies support the hypothesis that the DMR signal is an integrated response that consists of contributions from many cellular events induced by the ligand, thus providing alternative means to study cell systems biology. Orthogonal confirmations using conventional cell biology approaches have led to identifying specific cellular event(s) that dominate the DMR signals observed for three classes of receptors: epidermal growth factor receptor, Gq-coupled receptors and Gs-coupled receptors. Keywords: optical biosensor; cell-based assay; cell systems biology; cell systems pharmacology; dynamic mass redistribution Introduction Surface plasmon resonance (SPR) and resonant waveguide grating (RWG) are the two most popular types of optical biosensors. Since their birth almost three decades ago [1,2], both technologies have gained widespread uses for biomolecular interaction analysis due to the superior sensitivity to mass changes upon an analyte’s binding to receptors immobilized on the sensor surface. However, the ability of optical biosensors to examine living cells has largely been unrealized. To improve the R&D productivity of pharmaceutical and biotech industries, drug discovery paradigms have started shifting from target-oriented to systems biology-focused approaches, which greatly emphasize the use of whole cell systems for drug screens and testing [3]. This creates both a need in drug discovery and a potential solution in optical biosensors. Recently, we have demonstrated that RWG biosensors are capable of examining the health and activities of living cells, particularly cell signaling [4- 12]. This article reviews the principle and applications of biosensor-based cell assays, with special focus on fundamental understanding of optical signatures obtained for several classes of receptors. Theoretical analysis suggests that the optical signal is related to dynamic mass redistribution [4] The theories of operation for optical biosensor-based affinity profiling have been extensively discussed [1-2]. However, theoretical analysis for whole cell sensing has lagged behind. We have developed a theory, based on well-known optical and cellular biophysics, to explain the nature of optical signals manifested by RWG biosensors. First, the sensor configuration is considered as a non- conventional three-layer system: a substrate, a waveguide film in which a grating structure is embedded, and a cell layer (Fig.1). Figure 1 A three-layer configuration for detecting ligand-induced DMR in living cells using RWG biosensor. Cells are anchored to the sensor surface through interaction with the extracellular matrix (ECM). This is because a living cell has large dimensions (typically tens of microns), and cells are cultured directly onto the surface of a RWG biosensor until high confluency is reached. The interaction of cells with the surface is primarily mediated through three types of contacts: focal, close and extracellular matrix (ECM) contacts, such that the cell membrane is separated from the substrate by several nanometers to 100nm or more. Second, the biosensor exploits an evanescent wave to detect ligand-induced alterations of the cell layer at or near the sensor surface. The evanescent wave is an electromagnetic field, created by the total internal reflection of guided light at a solution-surface interface, with a well-characterized short penetration depth or sensing volume. Based on the sensor configuration and the physical properties of cells, the penetration depth of the TM 0 (transverse magnetic or p-polarized) mode is found to be around 150nm, meaning that the biosensor only senses the bottom portion of the cell layer. Third, a ligand-induced change in effective refractive index (i.e., the detected signal) is, to first order, directly proportional to the change in refractive index of the bottom portion of cell layer: c n C S N ) ( (1) where S(C) is the sensitivity to the cell layer, and n c is the ligand-induced change in local refractive index of the cell layer sensed by the biosensor. Fourth, the n c value is directly proportional to change in local concentrations of cellular targets or molecular assemblies within Broadband light Reflected light Detectable DMR Waveguide glass cell ECM