Microfluidic Acoustophoresis for Flowthrough Separation of Gram-Negative Bacteria using Aptamer Affinity Beads

This protocol describes our microfluidic acoustophoresis technology that can be used for the rapid, efficient separation of Gram-negative bacteria from a medium using aptamer-modified micro beads. Microfluidic acoustophoresis systems enable the Gram-negative bacteria separation with reasonable throughput and non-contact cell isolation. Microfluidic acoustophoresis can be optimized to isolate bacteria from medical and environmental samples.

To begin, design a microfluidic acoustophoresis chip that collects PS beads at the target outlet and discards the rest after injecting a PS sample mixture into the sample inlet. Prepare a chip with the layers above and below the silicon layer bonded to the borosilicate glass. And a third borosilicate glass layer, using anodization, add 1000 volts and 400 degrees Celsius.

Attach a PZT to the borosilicate glass layer along the microfluidic channel using less than 10 microliters of cyanoacrylate adhesive. Inoculate the GN and GP bacteria in Luria-Bertani medium and incubate it at 37 degrees Celsius and 220 revolutions per minute for 16 hours. Centrifuge the cultured bacteria at 9, 056 RCF for one minute at room temperature.

Then wash twice with 1X PBS buffer. Prepare the selected GN and GP bacteria for analysis by resuspending them in the binding buffer. Resuspend the 10 micrometer streptavidin coated micro bead mixture before use.

Vortex the mixture for 20 seconds. Prepare the aptamer by denaturing it at 95 degrees Celsius for three minutes, and then refolding it at zero degrees Celsius for two minutes. Transfer 250 microliters of the resuspended streptavidin coated micro bead mixture to a 1.5 milliliter tube and add 100 microliters of biotinylated DNA aptamer to the tube.

Incubate the mixture at room temperature for 30 minutes while rotating at 25 revolutions per minute. Centrifuge the reaction mixture at 9, 056 RCF for 40 seconds at room temperature. Then wash the tube twice with 200 microliters of Tris-HCl buffer.

Add 10 microliters of 100 milligrams per milliliter BSA to the washed sample tube and incubate for 30 minutes at room temperature while rotating at 25 revolutions per minute. Finally, wash the aptamer modified micro beads twice in Tris-HCl buffer by centrifugation at 9, 056 RCF for 40 seconds at room temperature. Connect PEEK tubes to the two inlets for injecting two samples and buffer, and the two outlets for collecting and discharging waste.

Using a 10 milliliter syringe, fill the microfluidic acoustophoresis channel with bubble free demineralized water. Prepare a precision pressure controller with two or more output channels to control the fluid flow. After preparing the device, inject the sample and buffer by applying a pressure of two kilopascals to the sample inlet and four kilopascals to the buffer inlet using the precision pressure control device.

Using a microscope, focus on a bead and move it to the center of the microfluidic channel using the PZT. Generate a resonance frequency of 3.66 megahertz using a single channel function generator and amplify a typical signal by 16 decibels using a power amplifier. Observe the separation and enrichment processes on the acoustofluidic chip with a fluorescence microscope and a high speed camera operating at 1, 200 frames per second.

The acoustophoretic chip introduced in this study confirmed that, as the voltage of the PZT increased, the central concentration of the 10 micrometer sized beads increased. At five volts of the PZT voltage, most of the 10 micrometer sized beads were concentrated in the center. The ratio of the number of beads collected at the outlet to the total number of injected 10 micrometer sized beads was referred to as the recovery rate.

The ratio of the number of 10 micrometer sized beads to the total number of beads collected was referred to as purity. The results indicated that the device had a high separation efficiency for beads with a size of 10 micrometers. The numbers of GN and GP bacteria bound to each aptamer modified micro bead were measured using a microscope with a high speed camera.

All five GN bacteria were bound to the beads while significantly fewer GP bacteria were bound to the substances tested. Signal strength differed significantly between the GN and GP bacteria. The adhesive used to attach the PZT should be used as little as possible for accuracy of corrugation and the PZT and microchannel should be parallel.

This device can be used to detect live bacteria or bacteria derived biomarkers in the period of early diagnosis of bacterial infectious diseases.