Expanding the capabilities of surface plasmon resonance microscopy using three-dimensional microfluidic platforms

Surface Plasmon Resonance Microscopy (SPRm) has emerged as a potential high-throughput biosensor platform for studying biomolecule interactions. The parallel processing capability of SPRm is limited by the density of reactions that can be performed at the sensor surface. To address these reaction regions microfluidic networks are necessary to deliver biomolecules within a continuous flow. Existing two-dimensional microfluidic approaches are limited by the density of isolated reaction zones that can be addressed. A three-dimensional microfluidic device called the Continuous Flow Microspotter (CFM) has been developed which enables efficient deposition of 48 separate spots. The CFM can print spots off-line on an SPR sensor substrate or can be directly coupled to a SPRm platform for real-time, in situ performance. A novel approach to on-chip microfluidic fluid manipulation was demonstrated during the development of the CFM. The technique based on the gas permeable properties of PDMS, enables filling of dead-end channels, local manipulation of individual channels, and the ability to inject and remove bubbles in between samples. The multilayer PDMS fabrication techniques commonly used in microfluidic devices were evaluated according to bond strength to improve the manufacturing process for the CFM by reducing the potential of the delamination in the channel architecture. Additional improvements in fluidic control and new fabrication approaches have improved the CFM tip geometry and have reduced deposition variation between spots to less than 5%. Performance metrics based on flow rate, contact time, and concentration were developed for the CFM to guide future use. Computational models were developed to understand protein adsorption spatially across the reaction zone as parameters such as flow rate and concentration vary. An integrated CFM, also referred to as the microfluidic flow cell array (MFCA), coupled directly to SPR has enabled 48 isolated flow cell reactions, simultaneously. Immobilization, capture, and regeneration steps were demonstrated in antibody capture assays to illustrate the potential for drug discovery and clinical applications. A new in-line referencing technique was also demonstrated to help improve sensitivity while capturing from complex samples. The "spot and hop" approach allows local in-spot referencing for each spot in the array, with the ability to maintain referencing for up to four immobilization steps. The MFCA-SPRm platform provides eight times more throughput than the current state of the art SPR biosensor. With the improvement in repeatability and local in-spot referencing, the MFCA-SPRm platform will help expand the use and capabilities of SPR into more clinical and research settings.