Isolation of microorganisms from biological specimens by dielectrophoresis

textEvery environment of the biosphere supports a particular mix of microorganisms called a microbiome. These diverse microbial communities play critical roles in the health of ecosystems and in higher organisms, including humans. Disruption or translocation of microbiomes may cause lethal infections, contaminate food and drug supplies, and adversely impact industrial activities. Microbiome detection and molecular characterization have emerged as priorities in many fields. Available methods cannot quickly and efficiently extract rare microorganisms in real specimens. Therefore, microbial detection and analysis require long incubation periods or the use of technically challenging molecular biotechnologies. These strategies are impractical in situations requiring immediate intervention. The intrinsic electric and dielectric properties of microbes permit their isolation by the phenomenon of dielectrophoresis in microfluidic devices. These microsystems have the potential to enhance microbial analysis but are plagued by low processing rates and the inability to interface with biological specimens containing high levels of interfering cells and debris. In this study, a method was created to discriminate between target microbes and undesired cells on the basis of their differential susceptibility to permeabilizing agents that altered cell dielectrophoretic responses. Fabrication techniques were developed to manufacture high-aspect ratio microfluidic channels that allowed the physical forces of gravity, diffusion and dielectrophoresis to be exploited to control cell positions over microscale distances normal to a Poiseuille flow gradient. Because the positioning effects were exploited in only one dimension, the other two dimensions of the channels could be scaled up to create large channel cross-sectional areas that supported rapid specimen processing rates while maintaining high separation efficiencies expected for the microscale effects. These strategies were applied in various ways to isolate microbes from whole blood, platelets, stool, saliva, and skin specimens. The dielectrophoretic extraction of microbes enabled by this approach was used to enable electrical impedance detection of ~100 bacteria in less than five hours. As a result, important technological barriers that have limited the applicability of dielectrophoresis in clinical and industrial settings were overcome by increasing throughput and addressing sample preparation requirements. These proof-of-concept data demonstrate the potential for accelerating microbial isolation and detection in diagnostics, screening, and microbiome research.Biomedical Engineerin