Rafael A. Vega

A real-time PCR-based method for determining the surface coverage of thiol-capped oligonucleotides bound onto gold nanoparticles.

Here we report a real-time PCR-based method for determining the surface coverage of dithiol-capped oligonucleotides bound onto gold nanoparticles alone and in tandem with antibody. The detection of gold nanoparticle-bound DNA is accomplished by targeting the oligonucleotide with primer and probe binding sites, amplification of the oligonucleotide by PCR, and real-time measurement of the fluorescence emitted during the reaction. This method offers a wide dynamic range and is not dependant on the dissociation of the oligonucleotide strands from the gold nanoparticle surface; the fluorophore is not highly quenched by the gold nanoparticles in solution during fluorescence measurements. We show that this method and a fluorescence-based method give equivalent results for determining the surface coverage of oligonucleotides bound onto 13 or 30 nm gold nanoparticles alone and in tandem with antibody. Quantifying the surface coverage of immobilized oligonucleotides on metallic nanoparticle surfaces is important for optimizing the sensitivity of gold nanoparticle-based detection methods and for better understanding the interactions between thiol-functionalized oligonucleotides and gold nanoparticles.

Functional antibody arrays through metal ion-affinity templates

In the post-genomic era, surface-based proteomics tools in high-throughput formats are becoming crucial for analyzing protein expression, protein–protein interactions, signal-transduction pathways, and the processes underlying cellular functions. Protein microand nanoarrays hold great promise in areas of health-related research, drug discovery, and diagnostics in which well-defined features and their spacing are important for studying surface–cellular interactions and detecting biomacromolecules. Thus far, a variety of techniques have been developed for immobilizing proteins, specifically antibodies, on surfaces. These techniques have relied primarily on antibody-binding proteins (proteins A, G, A/G, and L), 7] geneticengineering technologies to produce unnatural binding tags for directed surface attachment, electrostatically driven adsorption, covalent linking, or a combination thereof. Although these approaches have been widely used, they have drawbacks ranging from cost and complexity to inactivation of the antibody structures due to denaturation, which leads to poor antigen binding. Here, we present a novel method that utilizes metal ions to immobilize unmodified antibodies in a way that preserves their biorecognition properties in the context of microand nanoarrays. This approach is related to ones used for immobilizing antibodies with metal ions (i.e. Zn, Cu, Ni, Co) on three-dimensional chromatographic supports. Researchers have utilized direct-write techniques, such as robotic spotting and dip-pen nanolithography (DPN), in combination with metal ions as linking groups to generate oligonucleotide and virus particle micro-/nanoarrays. Metal ions as surface linking groups have the advantages of being readily accessible, robust, economical, and stable over long periods of time. Moreover, they are not susceptible to denaturation, as are many of the proteins used for antibody immobilization (i.e. proteins A, G, A/G, and L, etc.). In this report, we demonstrate how the Zn ion can be used as a versatile linker to immobilize antibodies on surfaces in an active state. This includes the assembly of a large class of unmodified polyand monoclonal antibodies (goat IgG, rabbit IgG, mouse IgG1, chicken IgY, and mouse IgM) in the context of microand nanoscale features generated by microcontact printing (m-CP) and DPN, respectively. The activity and utility of metal ion-immobilized antibody arrays are demonstrated with protein and viral antigens. Moreover, we show that one can use nanoarrays as a “litmuslike test” for evaluating the activity of surface-immobilized antibodies. The reduced spot area in a nanoarray demands efficient and relatively uniform immobilization of antibody structures in active states to exhibit uniform activity from spot to spot within the array. With larger features, such as those found in microarrays, inefficient immobilization (i.e. smaller percentage of active antibodies) still leads to apparent uniform activity from feature to feature within the array. Finally, we also demonstrate that we can use this approach to make nanoarrays of antibody structures, such as IgY and IgM, that others have claimed cannot be immobilized in active forms using the protein A/G approach.

Electrically Biased Nanolithography with KOH-Coated AFM Tips

This letter provides the first study aimed at characterizing the desorption and nanolithographic processes for SAM-coated, gold-coated silicon substrates oxidatively patterned with an AFM with a tip under potential control. The process either results in recessed patterns where the monolayer has been removed or raised structures where the monolayer has been removed and silicon oxidation has taken place. Eleven different SAMs have been studied, and the type of pattern formed depends markedly upon SAM chain length, end functional group, and applied bias. We show how local pH and choice of monolayer can be used to very effectively control the type of pattern that is ultimately formed. Interestingly, we show that hydroxide anion accessibility to the substrate surface is one of the most significant factors in determining the pattern topography. Moreover, control over the pattern topography can be achieved by controlling the concentration of the KOH in the water meniscus formed at the point of contact between tip and surface in the context of a bias-controlled DPN experiment with a KOH-coated tip. The work provides important insight into the factors that control SAM desorption and also ways of controlling the topography of features made in a potential-controlled scanning probe nanolithographic process.