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 (lambda(0)/4n-lambda(0)/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.

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.

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.

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.

Metallic nanohole arrays on fluoropolymer substrates as small label-free real-time bioprobes.

We describe a nanoplasmonic probing platform that exploits small-dimension (

High-throughput nanohole array based system to monitor multiple binding events in real time.

We have developed an integrated label-free, real-time sensing system that is able to monitor multiple biomolecular binding events based on the changes in the intensity of extraordinary optical transmission (EOT) through nanohole arrays. The core of the system is a sensing chip containing multiple nanohole arrays embedded within an optically thick gold film, where each array functions as an independent sensor. Each array is a square array containing 10 x 10 nanoholes (150 nm in diameter), occupying a total area of 3.3 mum x 3.3 mum. The integrated system includes a laser light source, a temperature-regulated flow cell encasing the sensing chip, motorized optics, and a charge-coupled detector (CCD) camera. For demonstration purposes, sensing chips containing 25 nanohole arrays were studied for their use in multiplexed detection, although the sensing chip could be easily populated to contain up to 20 164 nanohole arrays within its 0.64 cm2 sensing area. Using this system, we successfully recorded 25 separate binding curves between glutathione S-transferase (GST) and anti-GST simultaneously in real time with good sensitivity. The system responds to binding events in a concentration-dependent manner, showing a sharp linear response to anti-GST at concentrations ranging from 13 to 290 nM. The EOT intensity-based approach also enables the system to monitor multiple bindings simultaneously and continuously, offering a temporal resolution on milliseconds scale that is decided only by the camera speed and exposure time. The small footprint of the sensing arrays combined with the EOT intensity-based approach enables the system to resolve binding events that occurred on nanohole sensing arrays spaced 96 mum apart, with a reasonable prediction of resolving binding events spaced 56 mum apart. This work represents a new direction that implements nanohole arrays and EOT intensity to meet high-throughput, spatial and temporal resolution, and sensitivity requirements in drug discovery and proteomics studies.