After that, a 500-g/mL high concentration BSA solution in PBS was flowed for sufficient time to block nonspecific binding sites on the surface and produce a stable baseline. Control Experiments to Check Nonspecific Binding of Biotin Molecules To quantify the specific binding between biotinylated molecules and streptavidin, it is crucial to check the nonspecific binding between the biotin molecules and the sensing surface. crystal biosensor will provide new capabilities for highly sensitive measurements of biomolecular binding. Keywords: Optical biosensors, high sensitivity, label free, real time, biomolecular binding, small molecule detection, photonic crystal, total internal reflection INTRODUCTION The study of biomolecular binding affinity and kinetics, such as protein-protein binding, the binding of small molecules and drugs to biological targets, or evaluations of DNA hybridization, provides insights into fundamental biological processes and serves as the basis for diagnostic and drug discovery applications.1C3 For measurements of very small molecules (with molecular weights of 500 Da or below) or those at very low concentrations (within the femtomolar to nanomolar range), biomolecular detection usually requires labeling (such as fluorescent tags4); however, the conjugation of these tags may alter or inhibit the functionality of the target molecules. In contrast, label-free-based detection provides more accurate quantitative and kinetic measurements by monitoring the binding of analytes in their natural forms. A number of sensing technologies,5,6 including interferometry,7,8 plasmon sensing,9,10 nanowires,11 waveguides,12,13 microcavity resonators,14,15 and photonic crystals,16C18 have been developed, but the direct detection of small molecule binding still remains challenging. The most widely used commercial system for label-free binding analysis is the surface Plasmon resonance (SPR)-based biosensor.6,9 It detects the binding of analytes to ligands immobilized on a continuous metal surface in a total-internal-reflection geometry. The excited surface plasmon modes are very sensitive to the influence of bound molecules around the refractive index of the dielectric medium c-Kit-IN-2 adjacent to the metal film, with a sensitivity exceeding 103 nm per refractive index unit (RIU). However, because of large absorption in the metal film, the SPR resonance mode is broad (a few tens of nm), which restricts the conventional SPR-based sensors detection sensitivity and precludes its use for applications that require detection of small molecules (<1000 Da) or c-Kit-IN-2 low surface coverage (smaller than 1 pg/mm2) of bound molecules.9 Although sophisticated c-Kit-IN-2 engineering has improved the performance of commercial SPR-based systems for small molecule detection,19 inherently improving the sensors sensitivity is still the key to further advances. An example is the recently reported sensor system based on plasmonic nanorod metamaterials;10 it benefits from both an increase in the bulk refractive-index change and the larger sensing area of nanorod metamaterials matrix and achieves an order of magnitude higher sensitivity than conventional SPR sensors. This results in an improved detection limit size of approximately 250 Da (as measured with D-biotin binding), however, this is still not low enough for many biological Rabbit polyclonal to ACTBL2 applications. One key to obtaining higher sensitivity is to narrow the optical resonance below that exhibited by classical surface plasmon resonances. For example, microtoroid resonators using whispering-gallery-mode (WGM) exhibit ultrahigh Q values (>108) and extremely narrow resonances (<1 pm), enabling very high resolution.14, 15 However, a problem with ultrahigh-Q resonators is that the light is tightly confined by the resonator and only a very small portion of the optical mode interacts with the analyte answer; this leads to a relatively low sensitivity (a few nm/RIU) and thus restricts the detection limit.20 In addition, ultrahigh-Q resonators generally suffer thermal instability21 and difficulties with consistency between experiments arising from coupling light into and out of the resonators. Given the problems associated with both ultrahigh-Q and ultralow-Q resonators in sensing applications, moderate-Q photonic crystal-based biosensors have been widely studied.5, 16C18. Here, we focus our attention on a simple one-dimensional photonic crystal (1-D PC) structure (i.e., alternating pairs of dielectric layers). In this structure, the Q.