A high-throughput antibody-based microarray typing platform.

Many rapid methods have been developed for screening foods for the presence of pathogenic microorganisms. Rapid methods that have the additional ability to identify microorganisms via multiplexed immunological recognition have the potential for classification or typing of microbial contaminants thus facilitating epidemiological investigations that aim to identify outbreaks and trace back the contamination to its source. This manuscript introduces a novel, high throughput typing platform that employs microarrayed multiwell plate substrates and laser-induced fluorescence of the nucleic acid intercalating dye/stain SYBR Gold for detection of antibody-captured bacteria. The aim of this study was to use this platform for comparison of different sets of antibodies raised against the same pathogens as well as demonstrate its potential effectiveness for serotyping. To that end, two sets of antibodies raised against each of the "Big Six" non-O157 Shiga toxin-producing E. coli (STEC) as well as E. coli O157:H7 were array-printed into microtiter plates, and serial dilutions of the bacteria were added and subsequently detected. Though antibody specificity was not sufficient for the development of an STEC serotyping method, the STEC antibody sets performed reasonably well exhibiting that specificity increased at lower capture antibody concentrations or, conversely, at lower bacterial target concentrations. The favorable results indicated that with sufficiently selective and ideally concentrated sets of biorecognition elements (e.g., antibodies or aptamers), this high-throughput platform can be used to rapidly type microbial isolates derived from food samples within ca. 80 min of total assay time. It can also potentially be used to detect the pathogens from food enrichments and at least serve as a platform for testing antibodies.

Using aptamers to identify and enter cells.

While nucleic acid binding species (aptamers) have previously been selected against a variety of biomedically important protein targets, the selection of aptamers against complex targets, such as cell surfaces and organisms, is becoming increasingly feasible. A number of aptamers have now been generated that can target specific cell-surface antigens and cell types, including tumor biomarkers and tumor cells. The generation of anti-cell aptamers has important implications for identifying disease-specific biomarkers, generating diagnostics, and developing novel drug delivery strategies.

Aptamer mediated siRNA delivery.

Nucleic acids that bind to cells and are subsequently internalized could prove to be novel delivery reagents. An anti-prostate specific membrane antigen aptamer that has previously been shown to bind to prostate tumor cells was coupled to siRNAs via a modular streptavidin bridge. The resulting conjugates could be simply added onto cells without any further preparation, and were taken up within 30 min. The siRNA-mediated inhibition of gene expression was as efficient as observed with conventional lipid-based reagents, and was dependent upon conjugation to the aptamer. These results suggest new venues for the therapeutic delivery of siRNAs and for the development of reagents that can be used to probe cellular physiology.

Aptamer:toxin conjugates that specifically target prostate tumor cells.

We have used RNA aptamer:gelonin conjugates to target and specifically destroy cells overexpressing the known cancer biomarker prostate-specific membrane antigen (PSMA). Aptamer:toxin conjugates have an IC50 of 27 nmol/L and display an increased potency of at least 600-fold relative to cells that do not express PSMA. The aptamer not only promotes uptake into target cells but also decreases the toxicity of gelonin in non-target cells. These results validate the notion that "escort aptamers" may be useful for the treatment of specific tumors expressing unique antigen targets.

Labeling tumor cells with fluorescent nanocrystal-aptamer bioconjugates.

Aptamers that bind to prostate specific membrane antigen (PSMA) were conjugated to luminescent CdSe and CdTe nanocrystals for cell-labeling studies. The aptamer-nanocrystal conjugates showed specific targeting of both fixed and live cells that overexpressed PSMA. More importantly, aptamers were able to label cells dispersed in a collagen gel matrix simulating tissue. The specific binding abilities and synthetic accessibility of aptamers combined with the photostability and small size of semiconductor nanocrystals offers a powerful and general tool for cellular imaging.