Coiled-Coil-Mediated Assembly of an Icosahedral Protein Cage with Extremely High Thermal and Chemical Stability.

The organization of protein molecules into higher-order nanoscale architectures is ubiquitous in Nature and represents an important goal in synthetic biology. Furthermore, the stabilization of enzyme activity has many practical applications in biotechnology and medicine. Here we describe the symmetry-directed design of an extremely stable, enzymatically active, hollow protein cage of Mr ≈ 2.1 MDa with dimensions similar to those of a small icosahedral virus. The cage was constructed based on icosahedral symmetry by genetically fusing a trimeric protein (TriEst) to a small pentameric de novo-designed coiled coil domain, separated by a flexible oligo-glycine linker sequence. Screening a small library of designs in which the linker length varied from 2 to 12 residues identified a construct containing 8 glycine residues (Ico8) that formed well-defined cages. Characterization by dynamic light scattering, negative stain, and cryo-EM and by atomic force and IR-photoinduced force microscopy established that Ico8 assembles into a flexible hollow cage comprising 20 copies of the esterase trimer, 60 protein subunits in total, with overall icosahedral geometry. Notably, the cages formed by Ico8 proved to be extremely stable toward thermal and chemical denaturation: whereas TriEst was unfolded by heating ( Tm ≈ 75 °C) or denatured by 1.5 M guanidine hydrochloride, the Ico8 cages remained folded even at 120 °C or in 8 M guanidine hydrochloride. The increased stability of the cages is a new property that emerges from the higher-order structure of the protein cage, rather than being intrinsic to the components from which it is constructed.

Fabrication and near-field visualization of a wafer-scale dense plasmonic nanostructured array.

Developing a sensor that identifies and quantifies trace amounts of analyte molecules is crucially important for widespread applications, especially in the areas of chemical and biological detection. By non-invasively identifying the vibrational signatures of the target molecules, surface-enhanced Raman scattering (SERS) has been widely employed as a tool for molecular detection. Here, we report on the reproducible fabrication of wafer-scale dense SERS arrays and single-nanogap level near-field imaging of these dense arrays under ambient conditions. Plasmonic nanogaps densely populated the spaces among globular Ag nanoparticles with an areal density of 120 particles per µm2 upon application of a nanolithography-free simple process consisting of the Ar plasma treatment of a polyethylene terephthalate substrate and subsequent Ag sputter deposition. The compact nanogaps produced a high SERS enhancement factor of 3.3 × 107 and homogeneous (coefficient of variation of 8.1%) SERS response. The local near fields at these nanogaps were visualized using photo-induced force microscopy that simultaneously enabled near-field excitation and near-field force detection under ambient conditions. A high spatial resolution of 3.1 nm was achieved. Taken together, the generation of a large-area SERS array with dense plasmonic nanogaps and the subsequent single-nanogap level characterization of the local near field have profound implications in the nanoplasmonic imaging and sensing applications.

Linear and Nonlinear Optical Spectroscopy at the Nanoscale with Photoinduced Force Microscopy.

The enormous advances made in nanotechnology have also intensified the need for tools that can characterize newly synthesized nanoaterials with high sensitivity and with high spatial resolution. Many existing tools with nanoscopic resolution or better, including scanning electron microscopy (SEM), atomic force microscopy (AFM), and scanning tunneling microscopy (STM) methods, can generate highly detailed maps of nanoscopic structures. However, while these approaches provide great views of the morphological properties of nanomaterials, it has proven more challenging to derive chemical information from the corresponding images. To address this issue, attempts have been made to dress existing nanoscopy methods with spectroscopic sensitivity. A powerful approach in this direction is the combination of scan probe techniques with optical illumination, which aims to marry the nanoscopic resolution provided by a sharp tip with the chemical selectivity provided by optical spectroscopy. Examples of this approach include existing techniques such as scattering-type scanning near-field optical microscopy and tip-enhanced Raman spectroscopy. A new and emerging technique in this direction is photoinduced force microscopy (PiFM), which enables spectroscopic probing of materials with a spatial resolution well under 10 nm. In PiFM, the sample is optically excited and the response of the material is probed directly in the near-field by reading out the time-integrated force between the tip and the sample. Because the magnitude of the force is dependent on the photoinduced polarization in the sample, PiFM exhibits spectroscopic sensitivity. The photoinduced forces measured in PiFM are spatially confined on the nanometer scale, which translates into a very high spatial resolution even under ambient conditions. The PiFM approach is compatible with a wide range optical excitation frequencies, from the visible to the mid-infrared, enabling nanoscale imaging contrast based on either electronic or vibrational transitions in the sample. These properties make PiFM an attractive method for the visualization and spectroscopic characterization of a vast variety of nano materials, from semiconducting nanoparticles to polymer thin films to sensitive measurements of single molecules. In this Account, we review the principles of the PiFM technique and discuss the basic components of the photoinduced force microscope. We highlight the imaging properties of the PiFM instrument and demonstrate the inherent spectroscopic sensitivity of the technique. Furthermore, we show that the PiFM approach can be used to probe both the linear and nonlinear optical properties of nano materials. In addition, we provide several examples of PiFM imaging applications.

Field programmable gate array based reconfigurable scanning probe/optical microscope.

The increasing popularity of nanometrology and nanospectroscopy has pushed researchers to develop complex new analytical systems. This paper describes the development of a platform on which to build a microscopy tool that will allow for flexibility of customization to suit research needs. The novelty of the described system lies in its versatility of capabilities. So far, one version of this microscope has allowed for successful near-field and far-field fluorescence imaging with single molecule detection sensitivity. This system is easily adapted for reflection, polarization (Kerr magneto-optical (MO)), Raman, super-resolution techniques, and other novel scanning probe imaging and spectroscopic designs. While collecting a variety of forms of optical images, the system can simultaneously monitor topographic information of a sample with an integrated tuning fork based shear force system. The instrument has the ability to image at room temperature and atmospheric pressure or under liquid. The core of the design is a field programmable gate array (FPGA) data acquisition card and a single, low cost computer to control the microscope with analog control circuitry using off-the-shelf available components. A detailed description of electronics, mechanical requirements, and software algorithms as well as examples of some different forms of the microscope developed so far are discussed.

Apertureless near-field/far-field CW two-photon microscope for biological and material imaging and spectroscopic applications.

We present the development of a versatile spectroscopic imaging tool to allow for imaging with single-molecule sensitivity and high spatial resolution. The microscope allows for near-field and subdiffraction-limited far-field imaging by integrating a shear-force microscope on top of a custom inverted microscope design. The instrument has the ability to image in ambient conditions with optical resolutions on the order of tens of nanometers in the near field. A single low-cost computer controls the microscope with a field programmable gate array data acquisition card. High spatial resolution imaging is achieved with an inexpensive CW multiphoton excitation source, using an apertureless probe and simplified optical pathways. The high-resolution, combined with high collection efficiency and single-molecule sensitive optical capabilities of the microscope, are demonstrated with a low-cost CW laser source as well as a mode-locked laser source.