The recent Ebola crisis in West Africa highlights challenges associated with pathogen diagnostics in the developing world, particularly logistical challenges with sample transport, availability of resources, and skilled labor. We present innovations in assay chemistry, microfluidic consumables, and smart phone-based instrumentation to enable a new generation of portable diagnostic devices.
The threats of disease outbreaks and bioterrorism demand field-deployable technology capable of rapid, sensitive, and accurate diagnosis. In order to address such public health concerns, we present a portable centrifugal microfluidic platform and demonstrate sensitive detection of E. coli down to single digit starting copies using isothermal amplification via loop-mediated isothermal amplification (LAMP). The platform, which is composed of a compact optical system for laser induced fluorescence (LIF) detection, a quiet brushless motor, and an efficient non-contact heater, offers an easy-to-use system capable of performing sensitive pathogen screening in a lab-free environment.
Uncultivable microorganisms likely play significant roles in the ecology within the human body, with subtle but important implications for human health. Focusing on the oral microbiome, we are developing a processor for targeted isolation of individual microbial cells, facilitating whole-genome analysis without the need for isolation of pure cultures. The processor consists of three microfluidic modules: identification based on 16S rRNA fluorescence in situ hybridization (FISH), fluorescence-based sorting, and encapsulation of individual selected cells into small droplets for whole-genome amplification. We present here a technique for performing microscale FISH and flow cytometry, as a prelude to single cell sorting.
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.
Uncultivable microorganisms likely play significant roles in the ecology within the human body, with subtle but important implications for human health. Focusing on the oral microbiome, we are developing a processor for targeted isolation of individual microbial cells, facilitating whole-genome analysis without the need for isolation of pure cultures. The processor consists of three microfluidic modules: identification based on 16S rRNA fluorescence in situ hybridization (FISH), fluorescence-based sorting, and encapsulation of individual selected cells into small droplets for whole genome amplification. We present here a technique for performing microscale FISH and flow cytometry, as a prelude to single cell sorting.