Acoustofluidic medium exchange for preparation of electrocompetent bacteria using channel wall trapping

Abstract

Comprehensive integration of process steps into a miniaturised version of synthetic biology workflows remains a crucial task in automating the design of biosystems. However, each of these process steps has specific demands with respect to the environmental conditions, including in particular the composition of the surrounding fluid, which makes integration cumbersome. As a case in point, transformation, i.e. reprogramming of bacteria by delivering exogenous genetic material (such as DNA) into the cytoplasm, is a key process in molecular engineering and modern biotechnology in general. Transformation is often performed by electroporation, i.e. creating pores in the membrane using electric shocks in a low conductivity environment. However, cell preparation for electroporation can be cumbersome as it requires the exchange of growth medium (high-conductivity) for low-conductivity medium, typically performed via multiple time-intensive centrifugation steps. To simplify and miniaturise this step, we developed an acoustofluidic device capable of trapping the bacterium Escherichia coli non-invasively for subsequent exchange of medium, which is challenging in acoustofluidic devices due to detrimental acoustic streaming effects. With an improved etching process, we were able to produce a thin wall between two microfluidic channels, which, upon excitation, can generate streaming fields that complement the acoustic radiation force and therefore can be utilised for trapping of bacteria. Our novel design robustly traps Escherichia coli at a flow rate of 10 μL min^-1 and has a cell recovery performance of 47 ± 3% after washing the trapped cells. To verify that the performance of the medium exchange device is sufficient, we tested the electrocompetence of the recovered cells in a standard transformation procedure and found a transformation efficiency of 8 × 10⁵ CFU per μg of plasmid DNA. Our device is a low-volume alternative to centrifugation-based methods and opens the door for miniaturisation of a plethora of microbiological and molecular engineering protocols.

Fig. 1. Design and working principle of cell trapping in the acoustofluidic device. (a) Sketch of the design. (b) Micrograph series of bacteria trapping (E. coli in white, ESI Video S1), showing the channel before (upper panel) and during acoustic trapping (lower panel). The piezo was excited at 1.7 MHz with 46 V_PP, the bacteria concentration was ∼4 × 10⁸ cells per mL and the scale bar corresponds to 100 μm. (c) Photograph of the acoustofluidic device with an 8 mm scale bar.

Fig. 2. Numerical modelling of the acoustofluidic device. (a) Sketch of the device. (b) Optical microscopy image of the device cross-section. (I) Channel depth = 192.6 ± 0.4 μm, (II) channel width = 198.0 ± 0.3 μm, (III) wall top thickness = 13.0 ± 0.15 μm, (IV) wall bottom thickness = 6.15 ± 0.15 μm. The scale bar corresponds to 100 μm. (c) Simulated geometry and (d) typical mesh near a corner (see blue rectangle in (c)). Please refer to ESI Fig. S1 for the mesh study. At 2 MHz the model attained 631′961 degrees of freedom and had a computation time of 25 s and 22 s for the acoustic and streaming simulations, respectively, on a PC with 32 GB RAM and Intel Xeon E 2186G processor. (e) Frequency spectrum of acoustic energy density and wall displacement. A strong resonance is clearly visible at 1.8 MHz. (f) Displacement of the wall (disp), (g) Gor'kov potential (U) with acoustic radiation force (white arrows) and (h) streaming velocity (v_str) at f_res = 1.8 MHz.

Fig. 3. Overview of automated semi-continuous medium exchange. (a) Sketch of the acoustofluidic device: cell suspension is fed through the top inlet and ultrapure water through the bottom inlet. In step 1 (capture), acoustic forces trap the bacteria at the thin vibrating wall. In step 2 (wash) a flow of ultrapure water flushes the channel, leading to the desired medium exchange for the cells that remain trapped by the acoustic forces. Finally, in step 3 (release), the acoustics is turned off, bacteria are suspended into the new medium and leave the device with the flow. (b) Fluorescence microscopy pictures of the channel section in which the capturing of the E. coli (white) is performed. I) We flush the channel with cell solution before the start of the experiment while the acoustics are off. II) We turn on the acoustics leading to cell accumulation at the vibrating wall. III) End of capturing step. IV) End of washing step: cells are retained at the vibrating wall and channel is flushed with ultrapure water. V) Release step: we turn off the acoustics; thus, cells detach from the vibrating wall. VI) End of release step: cells left the device. The scale bars correspond to 100 μm.

Fig. 4. Graph of fraction of initial cells recovered at the outlet during each step of the medium exchange protocol with and without acoustics at a capturing flow rate of 10 μL min⁻¹. We calculated the cell concentrations from CFUs on non-selective plates according to eqn (9). Green: Experiments without acoustics as a control. Blue: Experiments with an excitation frequency of 1.89 MHz at 40 V_PP. Capture: Cells that are not trapped and leave the device during the capture step. Wash: Cells that leave the device during the wash step. Release: Cells that leave the device during the release step suspended in the new medium. All experiments were performed in triplicates to verify the reproducibility of our method.