A rapid, automated, multi-analyte Microflow Cytometer is being developed as a portable, field-deployable sensor for onsite
diagnosis of biothreat agent exposure and environmental monitoring. The technology relies on a unique method for
ensheathing a sample stream in continuous flow past an interrogation region where optical fibers provide excitation and
collect emission. This approach efficiently focuses particles in the interrogation region of the fluidic channel, avoids
clogging and provides for subsequent separation of the core and sheath fluids in order to capture the target for
confirmatory assays and recycling of the sheath fluid. Fluorescently coded microspheres provide the capability for highly
multiplexed assays. Optical analysis at four different wavelengths identified six sets of the coded microspheres
recognizing Escherichia coli, Listeria, and Salmonella as well as cholera toxin, staphylococcal enterotoxin B (SEB), and
ricin, and assay results were compared with those of a commercial Luminex analysis system.
A multi-analyte diagnostic system based on a novel microflow cytometer is under development as a portable, fielddeployable
sensor for environmental monitoring and for rapid point-of-care and on-site diagnosis of exposure to
biothreat agents. The technology relies on a unique method for ensheathing a sample stream in continuous flow past an
illuminated interrogation region. This sheathing approach efficiently focuses particles in the interrogation region of the
fluidic channel and minimizes clogging by complex samples. Fluorescently coded microspheres provide the capability
for highly multiplexed assays. In this report, separation of six microsphere sets was demonstrated with determination of
immunoassays on three of the six sets; comparison to the commercial platform was made.
The increasing demand for portable devices to detect and identify pathogens represents an
interdisciplinary effort between engineering, materials science, and molecular biology. Automation
of both sample preparation and analysis is critical for performing multiplexed analyses on real world
samples. This paper selects two possible components for such automated portable analyzers:
modified silicon structures for use in the isolation of nucleic acids and a sheath flow system suitable
for automated microflow cytometry.
Any detection platform that relies on the genetic content (RNA and DNA) present in complex
matrices requires careful extraction and isolation of the nucleic acids in order to ensure their
integrity throughout the process. This sample pre-treatment step is commonly performed using
commercially available solid phases along with various molecular biology techniques that require
multiple manual steps and dedicated laboratory space. Regardless of the detection scheme, a major
challenge in the integration of total analysis systems is the development of platforms compatible
with current isolation techniques that will ensure the same quality of nucleic acids. Silicon is an
ideal candidate for solid phase separations since it can be tailored structurally and chemically to
mimic the conditions used in the laboratory.
For analytical purposes, we have developed passive structures that can be used to fully ensheath one
flow stream with another. As opposed to traditional flow focusing methods, our sheath flow profile
is truly two dimensional, making it an ideal candidate for integration into a microfluidic flow
cytometer. Such a microflow cytometer could be used to measure targets captured on either
antibody- or DNA-coated beads.