Native protein nanolithography that can write, read and erase

The development of systematic approaches to explore protein-protein interactions and dynamic protein networks is at the forefront of biological sciences. Nanopatterned protein arrays offer significant advantages for sensing applications, including short diffusion times, parallel detection of multiple targets and the requirement for only tiny amounts of sample. Atomic force microscopy (AFM) based techniques have successfully demonstrated patterning of molecules, including stable proteins, with submicrometre resolution. Here, we introduce native protein nanolithography for the nanostructured assembly of even fragile proteins or multiprotein complexes under native conditions. Immobilized proteins are detached by a novel vibrational AFM mode (contact oscillation mode) and replaced by other proteins, which are selectively self-assembled from the bulk. This nanolithography permits rapid writing, reading and erasing of protein arrays in a versatile manner. Functional protein complexes may be assembled with uniform orientation at dimensions down to 50 nm. Such fabrication of two-dimensionally arranged nano-objects with biological activity will prove powerful for proteome-wide interaction screens and single molecule/virus/cell analyses.     

Design of a differential distributed amplifier and oscillator using close-packed interleaved transmission lines

The design of a differential distributed amplifier using close-packed transmission lines is discussed along with the implementation of a differential distributed oscillator. Efficient layout techniques for the implementation of these circuits are demonstrated. A differential distributed amplifier with 25 GHz of bandwidth and 7.5 dB of gain is presented as a design example. The amplifier can also be configured as a 9.2 GHz distributed oscillator with -6 dBm single-ended output power, phase noise of -103 dBc/Hz at a 1 MHz offset, and a 17% tuning range.     

On-chip crosstalk mitigation for densely packed differential striplines using via fence enclosures

The measured crosstalk characteristics for close-packed via fence enclosed differential stripline structures in a standard digital CMOS process are reported. The transmission lines achieve a packing pitch of 16 /spl mu/m of interconnect width per differential pair. The nearest neighbour far-end differential crosstalk is measured to be better than -43 dB and the near-end differential crosstalk is better than -37 dB below the drive signal at frequencies up to 20 GHz for 600 /spl mu/m lines. This is sufficient for use in high-density, high-speed analogue and digital integrated circuits.     

Atomic force microscopy of a hydrated bacterial surface protein

The protein surface layer of the bacterium Deinococcus radiodurans (HPI layer) was examined with an atomic force microscope (AFM). The measurements on the air-dried, but still hydrated layer were performed in the attractive imaging mode in which the forces between tip and sample are much smaller than in AFM in the repulsive mode or in scanning tunnelling microscopy (STM). The results are compared with STM and transmission electron microscopy (TEM) data.     

Rapid prototyping of arrayed microfluidic systems in polystyrene for cell-based assays

Microfluidic cell-based systems have enabled the study of cellular phenomena with improved spatiotemporal control of the microenvironment and at increased throughput. While poly(dimethylsiloxane) (PDMS) has emerged as the most popular material in microfluidics research, it has specific limitations that prevent microfluidic platforms from achieving their full potential. We present here a complete process, ranging from mold design to embossing and bonding, that describes the fabrication of polystyrene (PS) microfluidic devices with similar cost and time expenditures as PDMS-based devices. Emphasis was placed on creating methods that can compete with PDMS fabrication methods in terms of robustness, complexity, and time requirements. To achieve this goal, several improvements were made to remove critical bottlenecks in existing PS embossing methods. First, traditional lithographic techniques were adapted to fabricate bulk epoxy molds capable of resisting high temperatures and pressures. Second, a method was developed to emboss through-holes in a PS layer, enabling creation of large arrays of independent microfluidic systems on a single device without need to manually create access ports. Third, thermal bonding of PS layers was optimized in order to achieve quality bonding over large arrays of microsystems. The choice of materials and methods was validated for biological function in two different cell-based applications to demonstrate the versatility of our streamlined fabrication process.     

Scanning tunnelling microscopy of biomacromolecules

When imaging biomacromolecules with a STM, coating of specimens with a conductive layer is a convenient preparation method which provides a good rate of success. Utilizing evaporated platinum/carbon as a coating film we have investigated two biomacromolecules of very different appearance. The first of these is the HPI-layer, a natural two-dimensional protein crystal with a period of 18 nm, which is found on the surface of the bacterium Deinococcus radiodurans. The second specimen is type IV collagen which forms long triple-helical strands approx. 1·5nm in diameter. The resulting STM pictures compare very well with electron microscopical images.     

AFM imaging in solution of protein-DNA complexes formed on DNA anchored to a gold surface

A chemical procedure for anchoring DNA molecules to gold surfaces was used to facilitate the imaging of DNA and DNA-protein complexes in buffer solution by tapping mode atomic force microscopy (TMAFM). For preparing flat gold surfaces, a novel approach was employed by evaporating small amounts of gold onto freshly cleaved mica to give flat films that were stable under aqueous buffer conditions. The thickness of the investigated films ranged from 1 to 10 nm. For typical films of 4-6 nm, which were stable under aqueous buffer conditions, the root mean square (RMS) roughness ranged between 0.25 and 0.5 nm, as measured by atomic force microscopy (AFM). This roughness is comparable to that of obtained by the template stripped gold (TSG) technique, which is widely used in scanning probe microscopy but involves more preparation steps. In order to visualize DNA and DNA-protein complexes by TMAFM, the DNA was chemisorbed to the gold surface through a linker carrying a terminal thiol group at the 5'-end of each of the DNA strands. The modified DNA fragments were bound to the gold films and imaged in buffer solution, while unmodified DNA could not be visualized. Since the DNA was not dried during the process, it can be assumed that its native conformation was retained. This mode of anchoring did not prevent interaction with proteins, as confirmed by the observation that the topology of a complex formed by adding the protein to a surface-anchored DNA was the same as that obtained by anchoring a pre-formed complex to the gold surface. We attribute this observation to the fact that the DNA is anchored to the gold surfaces only through its ends, therefore the DNA-support interaction is minimized but imaging is still possible.     

A scanning tunneling microscope (STM) for biological applications: design and performance

The design of a scanning tunneling microscope (STM) for biological applications, operating at ambient pressure, is described. The STM is combined with an "auxiliary" light microscope to facilitate finding and identifying specimen areas of interest. The performance of the STM has been tested with evaporated gold films and with graphite. We have evaluated evaporated carbon and platinum/carbon films deposited on glass or mica to be used as specimen supports. First applications to biological material coated with a conducting film of platinum/carbon are described.