Dale N. Larson

Emerging tools for real-time label-free detection of interactions on functional protein microarrays.

The availability of extensive genomic information and content has spawned an era of high‐throughput screening that is generating large sets of functional genomic data. In particular, the need to understand the biochemical wiring within a cell has introduced novel approaches to map the intricate networks of biological interactions arising from the interactions of proteins. The current technologies for assaying protein interactions – yeast two‐hybrid and immunoprecipitation with mass spectrometric detection – have met with considerable success. However, the parallel use of these approaches has identified only a small fraction of physiologically relevant interactions among proteins, neglecting all nonprotein interactions, such as with metabolites, lipids, DNA and small molecules. This highlights the need for further development of proteome scale technologies that enable the study of protein function. Here we discuss recent advances in high‐throughput technologies for displaying proteins on functional protein microarrays and the real‐time label‐free detection of interactions using probes of the local index of refraction, carbon nanotubes and nanowires, or microelectromechanical systems cantilevers. The combination of these technologies will facilitate the large‐scale study of protein interactions with proteins as well as with other biomolecules.

Metallic Nanohole Arrays on Fluoropolymer Substrates as Small Label-Free Real-Time Bioprobes

We describe a nanoplasmonic probing platform that exploits small-dimension (<or=20 microm (2)) ordered arrays of subwavelength holes for multiplexed, high spatial resolution, and real-time analysis on biorecognition events. Nanohole arrays are perforated on a super smooth gold surface (roughness rms < 2.7 A) attached on a fluoropolymer (FEP) substrate fabricated by a replica technique. The smooth surface of gold provides a superb environment for fabricating nanometer features and uniform immobilization of biomolecules. The refractive index matching between FEP and biological solutions contributes to approximately 20% improvement on the sensing performance. Spectral studies on a series of small-dimension nanohole arrays from 1 microm (2) to 20 microm (2) indicate that the plasmonic sensing sensitivity improves as the gold-solution contact area increases. Our results also demonstrate that nanohole arrays with a dimension as small as 1 microm (2) can be used to effectively detect biomolecular binding events and analyze the binding kinetics. The future scientific opportunities opened by this nanohole platform include highly multiplexed analysis of ligand interactions with membrane proteins on high quality supported lipid bilayers.

Multiplexed plasmonic sensing based on small-dimension nanohole arrays and intensity interrogation

We performed multiplexed sensing on nanohole array devices to simultaneously obtain information on molecular absorption, scattering, and refractive-index change, which were distinguished by using different array structures with distinct optical behavior. Up to 25 arrays were fabricated within a 65 microm x 50 microm area to provide real-time information of the local surface environment. The performance of multiplexed sensing was examined by flowing NaCl, Coomassie blue, bovine serum albumin, and liposome solutions that exhibit different visible light absorption/scattering properties and different refractive indices. Experimental artifacts from light source fluctuation, sample injections, and light scattering induced by aggregates in solutions were detected by monitoring superwavelength holes or nanohole arrays with different periodicity and hole diameters.

Breaking the diffraction barrier outside of the optical near-field with bright, collimated light from nanometric apertures

The optical diffraction limit has been the dominant barrier to achieving higher optical resolution in the fields of microscopy, photolithography, and optical data storage. We present here an approach toward imaging below the diffraction barrier. Through the exposure of photosensitive films placed a finite and known distance away from nanoscale, zero-mode apertures in thin metallic films, we show convincing, physical evidence that the propagating component of light emerging from these apertures shows a very strong degree of collimation well past the maximum extent of the near-field (λ0/4n–λ0/2n). Up to at least 2.5 wavelengths away from the apertures, the transmitted light exhibits subdiffraction limit irradiance patterns. These unexpected results are not explained by standard diffraction theory or nanohole-based “beaming” rationalizations. This method overcomes the diffraction barrier and makes super-resolution fluorescence imaging practical.

Nanohole arrays of mixed designs and microwriting for simultaneous and multiple protein binding studies

We demonstrate using nanohole arrays of mixed designs and a microwriting process based on dip-pen nanolithography to monitor multiple, different protein binding events simultaneously in real-time based on the intensity of Extraordinary Optical Transmission of nanohole arrays. The microwriting process and small footprint of the individual nanohole arrays enabled us to observe different binding events located only 16 microm apart, achieving high spatial resolution. We also present a novel concept that incorporates nanohole arrays of different designs to improve confidence and accuracy of binding studies. For proof of concept, two types of nanohole arrays, designed to exhibit opposite responses to protein bindings, were fabricated on one transducer. Initial studies indicate that the mixed designs could help to screen out artifacts such as protein intrinsic signals, providing improved accuracy of binding interpretation.

Thermal Management Design of a Nanoscale Biocalorimeter

A nanoscale calorimeter design based on temperature induced changes in a surface plasmon based photonics effect has the potential to decrease the mass of experimental compounds consumed and to increase the throughput of experiments investigating drug development. This calorimeter is based on a demonstrated surface plasmon biosensor in which index of refraction changes as small as 10−5 % caused by biochemical reactions on the sensor surface are detected. To achieve this sensitivity require that the device’s temperature be held constant to within ± 0.001 K. In the biosensor the temperature was held constant to measure the concentration changes. For the calorimeter the concentration is held constant and temperature changes are monitored. In the calorimeter design the nanohole array sensor will be used as a sensitive thermometer that will be used to determine the enthalpy of binding, equilibrium binding constant and entropy changes of biochemical reactions. The numerical analysis described in this work demonstrates that nanoscale calorimetry is possible. The simulations demonstrate that two designs can produce temperature rises of 5.5 and 40 C, respectively well above the (10−3 ) C resolution of the sensors. These results were obtained using less than three orders of magnitude less reactants than is currently being used in calorimetry studies which is a significant advance of this technology.Copyright

Using Signal Processing Techniques to Enhance the TemperatureMeasurements Using Nanohole Array Sensors

A signal processing approach to reducing noise in the observed transmission through nanohole array sensors used in a microscale calorimeter is presented. The results demonstrate that using rectification and moving average processes, a well-defined Extraordinary Optical Transmission (EOT)-temperature calibration curve can be developed in the presence of a moving light fringe pattern.