Protein microarrays are promising tools that can potentially enable high throughput proteomic screening in areas such as disease diagnosis and drug discovery. A critical aspect in the development of protein microarrays is the optimization of the array's surface chemistry to achieve the high sensitivity required for detection of proteins in cell lysate and other complex biological mixtures. In the present study, a high-density antibody array with minimal nonspecific cellular protein adsorption was prepared using a glass surface coated with a poly(propyleneimine) dendrimer terminated with carboxyl group (PAMAM-COOH). The carboxyl-terminated dendrimer-modified surface has almost similar nonspecific cellular protein adsorption when compared to an inert PEG-modified surface. In addition, the multiple functional sites available for reaction on the dendrimer surface facilitated high-density immobilization of antibodies and efficient capture of bioanalytes. Various molecules were tested for their ability to block or deactivate the reactive carboxyl surface after antibody immobilization to further reduce the nonspecific binding. A short oligoethylene glycol (NH2-d4-PEG-COOH), was found to significantly improve the signal-to-noise ratio of the assay, resulting in higher sensitivity. The properties and functional qualities of the various surfaces were characterized by contact angle and AFM measurements. Nonspecific protein adsorption and protein immobilization as a function of dendrimer generations and sensitivity of antigen capturing from a buffer (1 pM) as well as from the complex cell lysate (10 pM) system were examined. Our detailed experimental studies demonstrated a facile method of preparing surfaces with high protein loading and low nonspecific protein binding for the development of high sensitivity protein microarrays.

Protein microarray offers a means for high-throughput profiling of cellular proteins to provide insights into the mechanisms of biological processes. This study describes the design and fabrication of a robust platform, spatially addressable protein array (SAPA), by exploring the specificity of ssDNA hybridization for self-assembly of semi-synthetic ssDNA-antibody conjugates which capture antigens from complex biological samples. This approach does not involve the direct immobilization of antibodies nor antigen, but instead captures the target antigens in the solution phase followed by self-directed assembly of the complex onto the surface. In an effort to optimize the platform, the effects of surface chemistry, nonspecific protein adsorption, facile preparation, and purification of ssDNA-conjugated antibody and capture of the antigen from a complex biological sample such as cell lysate were examined. This platform allowed antigen detection in cell lysate with high sensitivity (1 pM). The method described herein can be extended to the high-throughput detection of other interacting molecules in solution phase and their subsequent assembly onto any substrate.

The double emulsion process has commonly been applied to encapsulate water-soluble bioactive agents into polymeric microspheres. However, the integrity of many of these agents may be destroyed by the highly energetic procedures such as sonication that are routinely used to produce stable water-in-oil (w/o) emulsion. The aim of this research was to pursue the possibility of replacing the sonication by a mild emulsification procedure such as vortex mixing, with the use of certain materials to help to obtain stable w/o emulsion. The following materials were examined: poly(lactide-co-ethylene glycol) (PELA) as the polymer, ethyl acetate and acetone as the solvents, poly(vinyl alcohol) (PVA) and d-alpha tocopheryl polyethylene glycol 1000 succinate (Vitamin E TPGS) as the emulsifiers in w/o emulsion. The experimental results, with human serum albumin (HSA) as the encapsulated agent, showed that, when vortex mixing was used, these materials could significantly improve w/o emulsion stability and help to obtain satisfactory encapsulation effects, i.e. high encapsulation efficiency (EE) and low initial release burst. A delicate structure, i.e. liposomes, which is very sensitive to sonication, was then incorporated into microspheres by the 'modified double emulsion process'. It was found that the liposomes were intact and the encapsulation effects were good. Therefore, it can be concluded that the modified double emulsion process could be advantageous for the encapsulation of delicate substances.