Multiplexed detection of bacteria and toxins using a microflow cytometer

Area of Science:

• Biotechnology
• Microfluidics
• Analytical Chemistry

Background:

• Multiplexed detection of biothreat agents is crucial for rapid diagnostics.
• Traditional flow cytometry systems can be bulky and expensive.
• Microfabrication offers a path to miniaturized and cost-effective analytical devices.

Purpose of the Study:

• To develop and validate a microfabricated flow cytometer for simultaneous detection of bacteria and toxins.
• To assess the performance of the microfluidic system against a commercial benchtop flow cytometer.
• To demonstrate the utility of fluorescent coded microspheres in a microfluidic immunoassay.

Main Methods:

• Fabrication of a microfluidic chip with passive groove structures for 3D microsphere focusing.
• Utilized antibody-coated microspheres for sandwich immunoassay-based capture of biothreat targets.
• Employed a four-wavelength optical system for coded microsphere identification and phycoerythrin tracer quantification.
• Performed multiplexed assays for detecting specific bacteria (E. coli, Listeria, Salmonella) and toxins (cholera toxin, SEB, ricin).

Main Results:

• The microflow cytometer achieved limits of detection (LODs) of 10^3 cfu/mL for E. coli, 10^5 cfu/mL for Listeria, and 10^4 cfu/mL for Salmonella.
• Toxin LODs were 1.6 ng/mL for cholera toxin, 0.064 ng/mL for SEB, and 1.6 ng/mL for ricin.
• Performance metrics closely approached those of a commercial benchtop flow cytometer, demonstrating system viability.

Conclusions:

• Microfabricated flow cytometry offers a promising platform for sensitive and multiplexed biothreat agent detection.
• The developed system provides a compact and potentially cost-effective alternative to conventional flow cytometers.
• This technology has significant implications for point-of-care diagnostics and field-based surveillance.