Nabil A. Amro

Positioning protein molecules on surfaces: a nanoengineering approach to supramolecular chemistry.

We discuss a nanoengineering approach for supramolecular chemistry and self assembly. The collective properties and biofunctionalities of molecular ensembles depend not only on individual molecular building blocks but also on organization at the molecular or nanoscopic level. Complementary to “bottom-up” approaches, which construct supramolecular ensembles by the design and synthesis of functionalized small molecular units or large molecular motifs, nanofabrication explores whether individual units, such as small molecular ligands, or large molecules, such as proteins, can be positioned with nanometer precision. The separation and local environment can be engineered to control subsequent intermolecular interactions. Feature sizes as small as 2 × 4 nm2 (32 alkanethiol molecules) are produced. Proteins may be aligned along a 10-nm-wide line or within two-dimensional islands of desired geometry. These high-resolution engineering and imaging studies provide new and molecular-level insight into supramolecular chemistry and self-assembly processes in bioscience that are otherwise unobtainable, e.g., the influence of size, separation, orientation, and local environment of reaction sites. This nanofabrication methodology also offers a new strategy in construction of two- and three-dimensional supramolecular structures for cell, virus, and bacterial adhesion, as well as biomaterial and biodevice engineering.

Fabrication of Nanometer-Sized Protein Patterns Using Atomic Force Microscopy and Selective Immobilization

A new methodology is introduced to produce nanometer-sized protein patterns. The approach includes two main steps, nanopatterning of self-assembled monolayers using atomic force microscopy (AFM)-based nanolithography and subsequent selective immobilization of proteins on the patterned monolayers. The resulting templates and protein patterns are characterized in situ using AFM. Compared with conventional protein fabrication methods, this approach is able to produce smaller patterns with higher spatial precision. In addition, fabrication and characterization are completed in near physiological conditions. The adsorption configuration and bioreactivity of the proteins within the nanopatterns are also studied in situ.

Characterization of AFM tips using nanografting

Abstract We introduce a new method, nanografting, to characterize atomic force microscopy tips. Our technique includes three main steps. First, a self-assembled monolayer (SAM) of thiol is imaged using AFM with a low imaging force. Second, under a high load, a line of new thiols is fabricated within the matrix SAM in a single scan. Finally, the resulting line is imaged by the same AFM tip under a reduced force. From the topographic image of the line, one can extract information regarding the top portion of the AFM tip and the tip–surface contact area during fabrication. The advantages of this approach include its simplicity, high speed, and the ability to characterize the very top portion of the tip. In addition, tips with multiple asperities, which are difficult to investigate using other approaches, can be easily identified and characterized via nanografting.

Nanomolar scale nitric oxide generation from self-assembled monolayer modified gold electrodes

A SAM-modified gold electrode has been developed for the first time for quantitative NO generation of a nanomolar amount that is proportional to the surface area of the electrode.

Scanning probe lithography of self-assembled monolayers

Systematic studies on scanning probe lithography (SPL) methodologies have been performed using self-assembled monolayers (SAMs) on Au as examples. The key to achieving high spatial precision is to keep the tip-surface interactions strong and local. Approaches include three atomic force microscopy (AFM) based methods, nanoshaving, nanografting, and nanopen reader and writer (NPRW), which rely on the local force, and two scanning tunneling microscopy (STM) based techniques, field-induced desorption and electron-induced desorption, which use electric field and tunneling electrons, respectively, for nanofabrication. The principle of these procedures, the critical steps in controlling local tip-surface interactions, and nanofabrication media will be discussed. The advantages of SPL will be illustrated through various examples of production and modification of SAM nanopatterns.

Contact resonance imaging - A simple approach to improve the resolution of AFM for biological and polymeric materials

Abstract It is frequently observed that high resolution is difficult to achieve when using atomic force microscopy (AFM) to image “soft-and-sticky” surfaces, such as polymers and biomaterials. A new and simple method, contact resonance imaging (CRI), is introduced to address these issues. In CRI, the sample is modulated at a resonance frequency of the tip-sample contact, while the average position of the tip still remains in contact with the surface, i.e. in the repulsive region of the force–distance curve. The improvement in image resolution is demonstrated using various biological and polymeric specimens under ambient laboratory conditions and in liquid media. The possible mechanism of the resolution improvement is discussed in comparison to other techniques, such as tapping-mode imaging.

Active probes and microfluidic ink delivery for Dip Pen Nanolithography

Dip Pen Nanolithography (DPNTM) is a scanning probe technique for nanoscale lithography: A sharp tip is coated with a functional molecule (the “ink”) and then brought into contact with a surface where it deposits ink via a water meniscus. The DPN process is a direct-write pattern transfer technique with nanometer resolution and is inherently general with respect to usable inks and substrates including biomolecules such as proteins and oligonucleotides. We present functional extensions of the basic DPN process by showing actuated multi-probes as well as microfluidic ink delivery. We present the fabrication process and characterization of such active probes that use the bimorph effect to induce deflection of individual cantilevers as well as the integration of these probes. We also developed the capability to write with multiple inks on the probe array permitting the fabrication of multi-component nanodevices in one writing session. For this purpose, we fabricate passive microfluidic devices and present microfluidic behavior and ink loading performance of these components.

Dip pen nanolithography/spl trade/ and its potential for nanoelectronics

Dip pen nanolithography (DPN/spl trade/) is a patterning technique for nanoscale science and engineering based on scanning probe microscopy. Its main advantages are very high resolution, the unique capability to deposit many different materials directly onto a substrate and low cost of ownership. We present here new research and development efforts that demonstrate the potential of DPN as a tool to produce nanoelectronic devices and circuits. We show the direct deposition of electronic materials as well as the use of external accessories to accelerate the development phase of nanoelectronic components.

Structural basis of the Escherichi coli outer-membrane permeability

We have studied, using AFM, the structural basis of the outer membrane permeability for the bacterium E. col. The surface of the bacteria is visualized with an unprecedented details. Our AFM images clearly reveal that the outer membrane exhibits protrusions, which correspond to patches of LPS containing hundreds to thousands of LPS molecules. The packing of the nearest neighbor patches is tight, and as such the LPS layer provides an effective permeability barrier for the Gram-negative bacteria. We have also studied the mechanism of their permeability increase upon metal depletion. Our AFM images reveal that LPS molecules are released from the boundaries of some patches during the initial EDTA treatment. Further metal depletion produces a very distinct structure at the outer membrane: appearance of irregularly shaped pits. The pits are likely formed as a result of liberation of LPS patches and lipoproteins, exposing areas of peptidoglacan surface. Our study has proven AFM to be a very useful technique in providing structural basis for the functions of organisms.

Nanostructures of organic molecules and proteins on surfaces

Patterning bioreceptors on surfaces is a key step in the fabrication of biosensors and biochips. State-of-the art technology can produce micrometer-sized biostructures, however, further miniaturization at the nanoscale will require new methods and lithographic tools. In this proceeding, we report three approaches: nanopen reader and writer (NPRW), nanografting and latex particle lithography; for creating nanostructures of small molecules, DNA and proteins. Using nanografting and NPRW, nanostructures of thiol molecules or thiolated ssDNA are fabricated within self-assembled monolayers. Proteins attach selectively to nanopatterns of thiol molecules containing bioadhesive groups such as aldehyde or carboxylates. Using latex particle lithography, arrays of protein nanostructures are produced with high throughput on mica and gold substrates. Near-physiological conditions are used in structural characterization, thus the orientation, reactivity and stability of proteins and DNA molecules within nanostructures may be monitored directly via AFM. While AFM-based approaches provide the highest precision, nanoparticle lithography can produce arrays of protein nanostructures with high throughput. The nanostructures of proteins produced by these approaches provide an excellent opportunity for fundamental investigations of biochemical reactions on surfaces, such as antigen-antibody recognition and DNA-protein interactions. These methods provide a foundation for advancing biotechnology towards the nanoscale.