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.

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.

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.

As the field of microfluidics continues to grow, there is an increasing demand for public education about this technology. This article presents a quick, simple, safe, and inexpensive method for teaching microfluidics to younger students and the general public. (To listen to a podcast about this article, please go to the Analytical Chemistry multimedia page at pubs.acs.org/page/ancham/audio/index.html .).

We present a novel bonding technique for poly(dimethylsiloxane) (PDMS)-based devices employing chemical surface modifications at room temperature. PDMS surfaces were functionalized to present primary amine groups, and glass or gold substrates were functionalized to present carboxylic acid groups. Irreversible bonding was achieved by bringing the two surfaces in contact and reacting at room temperature to form peptide bonds between the substrates. Shear tests reveal the bond strengths achieved to be comparable to values obtained using conventional bonding methods. We also describe the use of carboxyl-terminated silanes on gold surfaces to bond amine-modified PDMS devices. Water contact angle measurements and X-ray photoelectron spectroscopy (XPS) confirmed the conjugation, a novel result that expands the variety of surface chemistries available for such bonding.

Standard libraries for systematic evolution of ligands by exponential enrichment (SELEX) typically utilize flanking regions that facilitate amplification of aptamers recovered from each selection round. Here, we show that these flanking sequences can bias the selection process, due in part to their ability to interfere with the fold or function of aptamers localized within the random region of the library sequence. We then address this problem by investigating the use of complementary oligonucleotides as a means to block aptamer interference by each flanking region. Isothermal titration calorimetry (ITC) studies are combined with fold predictions to both define the various interference mechanisms and assess the ability of added complementary oligonucleotides to ameliorate them. The proposed blocking strategy is thereby refined and then applied to standard library forms of benchmark aptamers against human α-thrombin, streptavidin, and vascular endothelial growth factor (VEGF). In each case, ITC data show that the new method effectively removes fixed-region mediated interference effects so that the natural binding affinity of the benchmark aptamer is completely restored. We further show that the binding affinities of properly functioning aptamers within a selection library are not affected by the blocking protocol, and that the method can be applied to various common library formats comprised of different flanking region sequences. Finally, we present a rapid and inexpensive qPCR-based method for determining the mean binding affinity of retained aptamer pools and use it to show that introduction of the pre-blocking method into the standard SELEX protocol results in retention of high-affinity aptamers that would otherwise be lost during the first round of selection. Significant enrichment of the available pool of high-affinity aptamers is thereby achieved in the first few rounds of selection. By eliminating single-strand (aptamer-like) structures within or involving the fixed regions, the technique is therefore shown to isolate aptamer sequence and function within the desired random region of the library members, and thereby provide a new selection method that is complementary to other available SELEX protocols.

Photosynthetic reaction centres (RCs) convert light into separated charges with nearly perfect quantum efficiency, and have been used to generate photocurrent. Previous work has shown that electron tunnelling rates between redox centres in proteins depend exponentially on the tunnelling distance. In this work the RC from Rhodobacter sphaeroides was genetically modified with the aim of achieving the shortest tunnelling distances yet demonstrated between the RC's electron-accepting P site and underlying graphite and gold electrodes, and between the electron donor Q site and graphite electrodes. Opposite charges are carried to counter electrodes using mobile mediators, as in dye-sensitised solar cells. Native RCs are bound to graphite surfaces through N-(1-pyrene)iodoacetamide. Although the linker's length is only 4 Å, the electron transfer pathway between the Q electron donor site on the RC and the electrode surface is still too large for current to be significant. A mutant version with the electron acceptor P side close to the graphite surface produced currents of 15 nA cm −2 upon illumination. Direct binding of RCs to a gold surface is shown, resulting in currents of 5 nA cm −2 . In both cases the current was unaffected by mediator concentration but increased with illumination, suggesting that direct electron transfer was achieved. The engineering of an RC to achieve direct electron transfer will help with long term efforts to demonstrate RC-based photovoltaic devices.

Lipid nanoparticles (LNPs) are established in the biopharmaceutical industry for efficient encapsulation and cytosolic delivery of nucleic acids for potential therapeutics, with several formulations in clinical trials. The advantages of LNPs can also be applied in basic research and discovery with a microfluidic method of preparation now commercially available that allows preparations to be scaled down to quantities appropriate for cell culture. These preparations conserve expensive nucleic acids while maintaining the particle characteristics that have made LNPs successful in later stages of genetic medicine development. Additionally, this method and the resulting LNPs are seamlessly scalable to quantities appropriate for in vivo models and development of nucleic acid therapeutics.The present work describes the methodology for preparing LNPs loaded with siRNA, mRNA or plasmids using a commercially available microfluidic instrument and an accompanying transfection kit. Guidelines for application to cultured cells in a well-plate format are also provided.