Surface plasmon resonance imaging (SPRi) is a label-free technique used for the quantitation of binding affinities and concentrations for a wide variety of target molecules. Although SPRi is capable of determining binding constants for multiple ligands in parallel, current commercial instruments are limited to a single analyte stream on multiple ligand spots. Measurement of binding kinetics requires the serial introduction of different analyte concentrations; such repeated experiments are conducted manually and are therefore time-intensive. To address these challenges, we have developed an integrated microfluidic array using soft lithography techniques for high-throughput SPRi-based detection and determination of binding affinities of antibodies against protein targets. The device consists of 264 element-addressable chambers isolated by microvalves. The resulting 700 pL chamber volumes, combined with a serial dilution network for simultaneous interrogation of up to six different analyte concentrations, allow for further speeding detection times. To test for device performance, human alpha-thrombin was immobilized on the sensor surface and anti-human alpha-thrombin IgG was injected across the surface at different concentrations. The equilibrium dissociation constant was determined to be 5.0 +/- 1.9 nM, which agrees well with values reported in the literature. The interrogation of multiple ligands to multiple analytes in a single device was also investigated and samples were recovered with no cross-contamination. Since each chamber can be addressed independently, this array is capable of interrogating binding events from up to 264 different immobilized ligands against multiple analytes in a single experiment. The development of high-throughput protein analytic measurements is a critical technology for systems approaches to biology and medicine.
Current technologies for aptamer discovery typically leverage the systematic evolution of ligands by exponential enrichment (SELEX) concept by recursively panning semi-combinatorial ssDNA or RNA libraries against a molecular target. The expectation is that this iterative selection process will be sufficiently stringent to identify a candidate pool of specific high-affinity aptamers. However, failure of this process to yield promising aptamers is common, due in part to (i) limitations in library designs, (ii) retention of non-specific aptamers during screening rounds, (iii) excessive accumulation of amplification artifacts, and (iv) the use of screening criteria (binding affinity) that does not reflect therapeutic activity. We report a new selection platform, High-Fidelity (Hi-Fi) SELEX, that introduces fixed-region blocking elements to safeguard the functional diversity of the library. The chemistry of the target-display surface and the composition of the equilibration solvent are engineered to strongly inhibit non-specific retention of aptamers. Partition efficiencies approaching 10(6) are thereby realized. Retained members are amplified in Hi-Fi SELEX by digital PCR in a manner that ensures both elimination of amplification artifacts and stoichiometric conversion of amplicons into the single-stranded library required for the next selection round. Improvements to aptamer selections are first demonstrated using human α-thrombin as the target. Three clinical targets (human factors IXa, X, and D) are then subjected to Hi-Fi SELEX. For each, rapid enrichment of ssDNA aptamers offering an order-nM mean equilibrium dissociation constant (Kd) is achieved within three selection rounds, as quantified by a new label-free qPCR assay reported here. Therapeutic candidates against factor D are identified.
Silicon photonic resonators, implemented using silicon-on-insulator substrates, are promising for numerous applications. The most commonly studied resonators are ring/racetrack resonators. We have fabricated these and other resonators including disk resonators, waveguide-grating resonators, ring resonator reflectors, contra-directional grating-coupler ring resonators, and racetrack-based multiplexer/demultiplexers. While numerous resonators have been demonstrated for sensing purposes, it remains unclear as to which structures provide the highest sensitivity and best limit of detection; for example, disc resonators and slot-waveguide-based ring resonators have been conjectured to provide an improved limit of detection. Here, we compare various resonators in terms of sensor metrics for label-free bio-sensing in a micro-fluidic environment. We have integrated resonator arrays with PDMS micro-fluidics for real-time detection of biomolecules in experiments such as antigen-antibody binding reaction experiments using Human Factor IX proteins. Numerous resonators are fabricated on the same wafer and experimentally compared. We identify that, while evanescent-field sensors all operate on the principle that the analyte's refractive index shifts the resonant frequency, there are important differences between implementations that lie in the relationship between the optical field overlap with the analyte and the relative contributions of the various loss mechanisms. The chips were fabricated in the context of the CMC-UBC Silicon Nanophotonics Fabrication course and workshop. This yearlong, design-based, graduate training program is offered to students from across Canada and, over the last four years, has attracted participants from nearly every Canadian university involved in photonics research. The course takes students through a full design cycle of a photonic circuit, including theory, modelling, design, and experimentation.
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