Here, we demonstrated the development of microfluidic acoustophorsis device that enables the efficient and rapid separation of gram-negative bacteria from cultured media. Bacterial infections cause many detrimental risks due to their virulent factors. The gold standard for finding the causing pathogen in a blood sample is blood culture, which may take hours to days. Shortening the time to diagnosis would significantly reduce mortality.
Our design enhances the separation efficiency by incorporating a lengthier, square shaped microchannel. Both inlets and outlets have a trifurcated junction, which makes simultaneous separation of the targets from a mixed sample possible by focusing the target particles at the center of the microchannel and the unwanted near the microchannel walls. From this architecture, the simultaneous excitation of two orthogonal resonances can generate 2-D acoustic standing wave by single piezoelectric actuation, focusing particles tightly on nodal points. Microbeads modified with a RNA aptamer,GN6, which has specificity to gram-negative bacteria were incubated with bacteria culture sample and then were injected into the acoustophoretic channel for simultaneous separation and washing. For the proof of principal, we tested separation performance of microfluidic acoustophoresis device using 10 different types of Gram-negative bacteria and 5 different types of Gram-positive bacteria. After separation of target bound bead using the device, we investigated and counted number of bacteria bound in each microbead in single bead observation manner. This protocol showed high specificity towards all Gram-negative bacterial as high sensitivity of as 103cells/ml of the bacterial and no (or limited) specificity of the Gram-positive bacteria.