Dynamic Control of Cell-Free Synthetic Biology-Based Biohybrid Microrobots by An Embedded Micro-Peristaltic Pump

Introduction: Traditionally, gene circuits rely on intact, living cells to facilitate gene expression. Alternatively, cell-free transcription-translation (TXTL) systems harness the necessary gene expression machinery of living cells. This characteristic eliminates the need for intact living cells for in vitro protein synthesis, providing researchers across different fields a unique opportunity to explore novel experimental strategies. For example, other researchers fabricated in silicon DNA compartments as artificial cells for gene expression in TXTL.
Synthetic biology has equipped cells with complex behaviors that can complete specific tasks based on programmed molecular algorithms. We hypothesized that the TXTL system affords great possibility to develop microrobots with capabilities similar to those of cells. Our objective is to develop cell-free synthetic biology-based autonomous biohybrid microrobots to improve therapeutic and diagnostic tools. Previously, we developed a microparticle-based microrobot implemented with DNA-based molecular algorithms, empowering the microrobot to perform perception-action and communication behavior in the TXTL system. By exploiting the surface chemistry of biotin-streptavidin binding on microrobots, we could selectively control and modify the molecular assembly of DNA circuits on these microrobots. Our next goal is to further expand the sensing capabilities of these biohybrid microrobots by controlling their movement to a target area in a microfluidic chip simulating the physiological environment in vivo. One aspect of controlling the microrobots' movement in nanoliter-scale fluids is to utilize the fluid dynamics in which microrobots are engaged. Here, we introduced a low-cost micro-peristaltic pump formed by three embedded microfluidic valves to selectively manipulate the fluids' dynamic to drive the microrobots’ movement.

Materials and

Methods: Fabricating the PDMS nuts
The solenoid valves were manufactured by following the protocol outlined by DeAngelis et al. The thread pattern of an electric solenoid was replicated by curing PDMS around bolts with the same thread. Then, the PDMS nuts were cut, and plasma bonded to a thin layer of PDMS to form individual valves.
Embedding the PDMS nuts into a microfluidic chip
Subsequentially, the wafer with microfluidic patterns was spin-coated to form an approximately 80 μm thick PDMS layer, which was partially cured for 30 min in a 65 °C oven. The PDMS nuts were then placed on top of the partially cured PDMS layer where the channel of interest was located. Finally, more PDMS was gently poured around the nuts and placed in a 65 °C oven for 90 min to fully cure every part, followed by plasma bonding to a cover slide to assemble the chip. Finally, the electric solenoids were screwed into the PDMS nuts to create functional valves.
Dynamic control
A syringe pump-driven flow was introduced into the chip. Two motor driver shields connected to an Arduino microcontroller controlled the electric solenoids. A custom Python script was used to send commands to and collect data from the microcontroller. Real-time videos were obtained using an inverted microscope with a 10X objective lens and a camera in the eyepiece.

Results, Conclusions, and Discussions: Our previous work developed a regression model for this microfluidic valve correlating input electrical signals with flow profiles, demonstrating the valve’s capability to generate desired time-varying fluid patterns for a co-laminar fluid stream.
Building on this discovery, here, we employed three microfluidic valves in a row. By controlling the plungers inside the solenoids up and down with different voltage input profiles, we successfully formed this portable microfluidic peristaltic pump, which didn’t need any compressed air source and was compatible with any microfluidic chip. Here we placed the peristaltic pump on a simplified y-channel microfluidic chip to demonstrate fluid control. By varying pumping forces, patterns, and frequencies, we can identify a broader set of fluid actuation profiles and observe more complex fluid behaviors, providing a new opportunity to understand the human body's fluid dynamics better. Additionally, we can assess the performance of the micro-peristaltic pump not only under uniform distributed pressure conditions but also under decreasing/increasing pressure gradient conditions to simulate the physiological microenvironment in the body. Subsequently, we can implement microparticles in the microfluidic chip to study different microrobot actuation conditions, simulating cell movement in body fluids. We envision this micro-peristaltic pump playing a pivotal role in regulating microrobot behavior across various scientific studies, such as biosensing, drug delivery, and more.

In summary, this work provides a novel mechanism of control over microrobot actuation and represents a new tool for governing cell-free synthetic biology gene expression. The resulting cell-free synthetic biology-enabled microrobots, with this and other control mechanism, will gain the ability to perceive, analyze, and respond to biological or chemical inputs of interest in complex environments, enhancing the possibility of understanding body fluid dynamics and developing miniaturized diagnostic and treatment devices.

Acknowledgements (Optional):