Publications

Chip-based in situ hybridization for identification of bacteria from the human microbiome

Meagher, Robert M.; Liu, Peng L.; Light, Yooli K.; Singh, Anup K.

The emerging field of metagenomics seeks to assess the genetic diversity of complex mixed populations of bacteria, such as those found at different sites within the human body. A single person's mouth typically harbors up to 100 bacterial species, while surveys of many people have found more than 700 different species, of which {approx}50% have never been cultivated. In typical metagenomics studies, the cells themselves are destroyed in the process of gathering sequence information, and thus the connection between genotype and phenotype is lost. A great deal of sequence information may be generated, but it is impossible to assign any given sequence to a specific cell. We seek non-destructive, culture-independent means of gathering sequence information from selected individual cells from mixed populations. As a first step, we have developed a microfluidic device for concentrating and specifically labeling bacteria from a mixed population. Bacteria are electrophoretically concentrated against a photopolymerized membrane element, and then incubated with a specific fluorescent label, which can include antibodies as well as specific or non-specific nucleic acid stains. Unbound stain is washed away, and the labeled bacteria are released from the membrane. The stained cells can then be observed via epifluorescence microscopy, or counted via flow cytometry. We have tested our device with three representative bacteria from the human microbiome: E. coli (gut, Gram-negative), Lactobacillus acidophilus (mouth, Gram-positive), and Streptococcus mutans (mouth, Gram-positive), with results comparable to off-chip labeling techniques.