Carboxyl-Terminated Dendrimer-Coated Bioactive Interface for Protein Microarray:  High-Sensitivity Detection of Antigen in Complex Biological Samples

Abstract

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.

A comparison of mono and multivalent linkers and their effect on the colloidal stability of nanoparticle and immunoassays performance

“…We realized that the availability and the lifetime of the NHS esters on the dendritic linker would determine the reaction efficiency and yields in the conjugation with the biomolecule that needs to be labeled. The activation reaction with EDC and NHS is most efficient at slightly acidic pH and usually performed in MES buffer at pH 4.7-6.0 [41,42,27]. With that in mind, we decided to explore on the effect of some other variables present in the EDC/NHS activation step such as the molar excess of NHS per COOH group, the ratio of the EDC/NHS co-reactants and the reaction time needed for the COOH activation.…”

“…Multivalent dendrimers are attracting a great deal of interest for applications in biomedical diagnostics [27][28][29][30], pharmacology / drug delivery [31,32] [33], cancer research [34], and nanomedicine [35]. Their use in research is still unabated now, 30 years after their first reported synthesis by Vogtle [36].…”

TIRF microscopy as a screening method for non-specific binding on surfaces

“…Polyethylene(glycol) derivatives are also known in surface chemistry as effective suppressors of non-specific binding of biomolecules [15][16][17][18]. Their function is associated less with electrostatic forces but rather with vast hydration and effects of change of configurational entropy when large molecules adsorb upon them.…”

“…In this process, TEOS was grafted onto a Zeonor substrate and allowed to cross-link with molecules of acrylic acid. The polymerization reaction was initiated by plasma and resulted in the formation of exceptionally stable, hydrophilic film with high density of Ð COOH groups.Polyethylene(glycol) derivatives are also known in surface chemistry as effective suppressors of non-specific binding of biomolecules [15][16][17][18]. Their function is associated less with electrostatic forces but rather with vast hydration and effects of change of configurational entropy when large molecules adsorb upon them.…”

Dendrimer porphyrin-based self-assembled nano-devices for biomedical applications

“…DP-coated surfaces can load more proteins and more protein activity than planar surfaces. [64][65][66][67] Furthermore, we determined the relative amounts of dendrimer immobilized on the surface and monitored the enzymecatalyzed reactions using fluorescence microscopy by means of the fluorescent properties of the DP. 66 To prepare a DP-coated surface, the surface of a silicon wafer was covered with positively charged amine groups using 3-aminopropyltriethoxysilane deposited DP via electrostatic interaction in a silanization reaction (Figure 12).…”