Nanoparticle phosphors made of lanthanide oxides are a promising new class of tags in biochemistry because of their large Stokes shift, sharp emission spectra, long luminescence lifetime, and good photostability. We demonstrate the application of these nanoparticles to the visualization of protein micropatterns. Luminescent europium-doped gadolinium oxide (Eu:Gd2O3) nanoparticles are synthesized by spray pyrolysis. The size distribution is from 5 to 200 nm. The particles are characterized by means of laser-induced fluorescent spectroscopy and transmission electron microscopy (TEM). The main emission peak is at 612 nm. The nanoparticles are coated with avidin through physical adsorption. biotinylated bovine serum albumin (BSA-b) is patterned on a silicon wafer using a microcontact printing technique. The wafer is then incubated in a solution of avidin-coated nanoparticles. Fluorescent microscopic images reveal that the nanoparticles are organized onto designated area, as defined by the microcontact printing process. The luminescent nanoparticles do not suffer photobleaching during the observation, which demonstrates their suitability as luminescent labels for fluorescence microscopy studies. More detailed studies are preformed using atomic-force microscopy (AFM) at a single nanoparticle level. The specific and the nonspecific binding densities of the particles are qualitatively evaluated.
Nanoparticles made of lanthanide oxides are promising fluorophores as a new class of tags in biochemistry because of their large Stokes shift, sharp emission spectra, long lifetime and lack of photobleaching. We demonstrate for first time the application of these nanoparticles to the visualization of protein micropatterns. Europium-doped gadolinium oxide (Eu:Gd2O3) nanoparticles were synthesized by spray pyrolysis and were characterized by means of laser-induced fluorescent spectroscopy and TEM. Their main emission peak is at 612 nm. And their size distribution is from 5 nm to 500 nm. The nanoparticles were coated with avidin through physical adsorption. Biotinylated Bovine Serum Albumin (BSA-b) was patterned on a silicon wafer using a micro-contact printing technique. The BSA-b - patterned wafer was incubated in a solution containing the avidin-coated nanoparticles. The specific interaction between biotin and avidin was studied by means of fluorescent microscopy and atomic-force microscopy (AFM). The fluorescent microscopic images revealed that the nanoparticles were organized into designated structures as defined by the microcontact printing process - non-specific binding of the avidin-coated nanoparticles to bare substrate was negligible. The fluorescent pattern did not suffer any photobleaching during the observation process which demonstrates the suitability of Eu:Gd2O3 nanoparticles as fluorescent labels with extended excitation periods - organic dyes, including chelates, suffer bleaching over the same period. More detailed studies were preformed using AFM at a single nanoparticle level. The specific and the non-specific binding densities of the particles were qualitatively evaluated.
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.
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.
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.
Conference Committee Involvement (1)
Physical Chemistry of Interfaces and Nanomaterials II
6 August 2003 | San Diego, California, United States