An embedded microfluidic valve for dynamic control of cellular communication.

The communication between different cell populations is an important aspect of many natural phenomena that can be studied with microfluidics. Using microfluidic valves, these complex interactions can be studied with a higher level of control by placing a valve between physically separated populations. However, most current valve designs do not display the properties necessary for this type of system, such as providing variable flow rate when embedded inside a microfluidic device. While some valves have been shown to have such tunable behavior, they have not been used for dynamic, real-time outputs. We present an electric solenoid valve that can be fabricated completely outside of a cleanroom and placed into any microfluidic device to offer control of dynamic fluid flow rates and profiles. After characterizing the behavior of this valve under controlled test conditions, we developed a regression model to determine the required input electrical signal to provide the solenoid the ability to create a desired flow profile. With this model, we demonstrated that the valve could be controlled to replicate a desired, time-varying pattern for the interface position of a co-laminar fluid stream. Our approach can be performed by other investigators with their microfluidic devices to produce predictable, dynamic fluidic behavior. In addition to modulating fluid flows, this work will be impactful for controlling cellular communication between distinct populations or even chemical reactions occurring in microfluidic channels.

Smart Magnetic Microrobots Learn to Swim with Deep Reinforcement Learning.

Swimming microrobots are increasingly developed with complex materials and dynamic shapes and are expected to operate in complex environments in which the system dynamics are difficult to model and positional control of the microrobot is not straightforward to achieve. Deep reinforcement learning is a promising method of autonomously developing robust controllers for creating smart microrobots, which can adapt their behavior to operate in uncharacterized environments without the need to model the system dynamics. This article reports the development of a smart helical magnetic hydrogel microrobot that uses the soft actor critic reinforcement learning algorithm to autonomously derive a control policy which allows the microrobot to swim through an uncharacterized biomimetic fluidic environment under control of a time varying magnetic field generated from a three-axis array of electromagnets. The reinforcement learning agent learned successful control policies from both state vector input and raw images, and the control policies learned by the agent recapitulated the behavior of rationally designed controllers based on physical models of helical swimming microrobots. Deep reinforcement learning applied to microrobot control is likely to significantly expand the capabilities of the next generation of microrobots.

The Future Application of Organ-on-a-Chip Technologies as Proving Grounds for MicroBioRobots.

An evolving understanding of disease pathogenesis has compelled the development of new drug delivery approaches. Recently, bioinspired microrobots have gained traction as drug delivery systems. By leveraging the microscale phenomena found in physiological systems, these microrobots can be designed with greater maneuverability, which enables more precise, controlled drug release. Their function could be further improved by testing their efficacy in physiologically relevant model systems as part of their development. In parallel with the emergence of microscale robots, organ-on-a-chip technologies have become important in drug discovery and physiological modeling. These systems reproduce organ-level functions in microfluidic devices, and can also incorporate specific biological, chemical, and physical aspects of a disease. This review highlights recent developments in both microrobotics and organ-on-a-chip technologies and envisions their combined use for developing future drug delivery systems.

Engineering control circuits for molecular robots using synthetic biology.

The integration of molecular robots and synthetic biology allows for the creation of sophisticated behaviors at the molecular level. Similar to the synergy between bioelectronics and soft robotics, synthetic biology provides control circuitry for molecular robots. By encoding perception-action modules within synthetic circuits, molecular machines can advance beyond repeating tasks to the incorporation of complex behaviors. In particular, cell-free synthetic biology provides biomolecular circuitry independent of living cells. This research update reviews the current progress in using synthetic biology as perception-action control modules in robots from molecular robots to macroscale robots. Additionally, it highlights recent developments in molecular robotics and cell-free synthetic biology and suggests their combined use as a necessity for future molecular robot development.

Open-source, 3D-printed Peristaltic Pumps for Small Volume Point-of-Care Liquid Handling.

Microfluidic technologies are frequently employed as point-of-care diagnostic tools for improving time-to-diagnosis and improving patient outcomes in clinical settings. These microfluidic devices often are designed to operate with peripheral equipment for liquid handling that increases the cost and complexity of these systems and reduces their potential for widespread adoption in low resource healthcare applications. Here, we present a low-cost (~$120), open-source peristaltic pump constructed with a combination of three dimensional (3D)-printed parts and common hardware, which is amenable to deployment with microfluidic devices for point-of-care diagnostics. This pump accepts commonly available silicone rubber tubing in a range of sizes from 1.5 to 3 mm, and is capable of producing flow rates up to 1.6 mL min-1. This device is programmed with an Arduino microcontroller, allowing for custom flow profiles to fit a wide range of low volume liquid handling applications including precision liquid aliquoting, flow control within microfluidics, and generation of physiologically relevant forces for studying cellular mechanobiology within microfluidic systems.

A 3D-printed portable device for field deployment of living biosensors.

Living biosensors typically use genetically modified organisms (GMOs) to detect infectious biomarkers in clinical samples. GMOs are prohibited to be released into the environment by many laws, which limits the application of living biosensors outside laboratory settings. Here, we reported a robust 3D-printed device that eliminates the risk of exposure of GMOs to potential users and the environment. The device is designed to snugly attach to a common culture tube and consists of two components, a housing and a plunger. The housing contains a stress-focusing cutout and a reagent well to hold the living biosensor. While the plunger is designed to form a two-stage press-fit seal with the housing. The first seal allows to safely transfer the living biosensor from a biosafety lab to the field, and the second seal prevents the leakage of GMOs from the culture tube during the test and before safe disposal. Additionally, a lever-actuated machine was also designed and 3D-printed to assist users operating the device.

Three-dimensional Printing of Thermoplastic Materials to Create Automated Syringe Pumps with Feedback Control for Microfluidic Applications.

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic experimentation is a stable fluid handling system capable of accurately providing an inlet flow rate or inlet pressure. Here, we have developed a syringe pump system capable of controlling and regulating the inlet fluid pressure delivered to a microfluidic device. This system was designed using low-cost materials and additive manufacturing principles, leveraging three-dimensional (3D) printing of thermoplastic materials and off-the-shelf components whenever possible. This system is composed of three main components: a syringe pump, a pressure transducer, and a programmable microcontroller. Within this paper, we detail a set of protocols for fabricating, assembling, and programming this syringe pump system. Furthermore, we have included representative results that demonstrate high-fidelity, feedback control of inlet pressure using this system. We expect this protocol will allow researchers to fabricate low-cost syringe pump systems, lowering the entry barrier for the use of microfluidics in biomedical, chemical, and materials research.

Programming Biomaterial Interactions Using Engineered Living Cells.

We have developed a biomaterials interface that allows the properties of a functionalized surface to be controlled by a population of genetically engineered bacteria. This interface was engineered by linking a genetically modified E. coli strain with a chemically functionalized surface. Critically, the E. coli was engineered to upregulate the production of biotin when induced by a small signaling molecule. This biotin would then interact with the functionalized surface to modulate the surface's binding dynamics. In this chapter, we detail three protocols: one protocol for developing a population of biotin-producing genetically engineered cells, and two protocols for creating different types of functionalized surfaces. These methods will enable scientists to readily explore strategies for controlling surface-based material assembly and modification using a linked culture of engineered cells.

Engineering a living biomaterial via bacterial surface capture of environmental molecules.

Synthetic biology holds significant potential in biomaterials science as synthetically engineered cells can produce new biomaterials, or alternately, can function as living components of new biomaterials. Here, we describe the creation of a new biomaterial that incorporates living bacterial constituents that interact with their environment using engineered surface display. We first developed a gene construct that enabled simultaneous expression of cytosolic mCherry and a surface-displayed, catalytically active enzyme capable of covalently bonding with benzylguanine (BG) groups. We then created a functional living material within a microfluidic channel using these genetically engineered cells. The material forms when engineered cells covalently bond to ambient BG-modified molecules upon induction. Given the wide range of materials amenable to functionalization with BG-groups, our system provides a proof-of-concept for the sequestration and assembly of BG-functionalized molecules on a fluid-swept, living biomaterial surface.

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials.

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

Low-cost feedback-controlled syringe pressure pumps for microfluidics applications.

Microfluidics are widely used in research ranging from bioengineering and biomedical disciplines to chemistry and nanotechnology. As such, there are a large number of options for the devices used to drive and control flow through microfluidic channels. Commercially available syringe pumps are probably the most commonly used instruments for this purpose, but are relatively high-cost and have inherent limitations due to their flow profiles when they are run open-loop. Here, we present a low-cost ($110) syringe pressure pump that uses feedback control to regulate the pressure into microfluidic chips. Using an open-source microcontroller board (Arduino), we demonstrate an easily operated and programmable syringe pump that can be run using either a PID or bang-bang control method. Through feedback control of the pressure at the inlets of two microfluidic geometries, we have shown stability of our device to within ±1% of the set point using a PID control method and within ±5% of the set point using a bang-bang control method with response times of less than 1 second. This device offers a low-cost option to drive and control well-regulated pressure-driven flow through microfluidic chips.

Programming Surface Chemistry with Engineered Cells.

We have developed synthetic gene networks that enable engineered cells to selectively program surface chemistry. E. coli were engineered to upregulate biotin synthase, and therefore biotin synthesis, upon biochemical induction. Additionally, two different functionalized surfaces were developed that utilized binding between biotin and streptavidin to regulate enzyme assembly on programmable surfaces. When combined, the interactions between engineered cells and surfaces demonstrated that synthetic biology can be used to engineer cells that selectively control and modify molecular assembly by exploiting surface chemistry. Our system is highly modular and has the potential to influence fields ranging from tissue engineering to drug development and delivery.

A Model of a Synthetic Biological Communication Interface between Mammalian Cells and Mechatronic Systems.

The creation of communication interfaces between abiotic and biotic systems represents a significant research challenge. In this work, we design and model a system linking the biochemical signaling pathways of mammalian cells to the actions of a mobile robotic prosthesis. We envision this system as a robotic platform carrying an optically monitored bioreactor that harbors mammalian cells. The cellular, optical signal is captured by an onboard fluorescent microscope and converted into an electronic signal. We first present a design for the overall cell-robot system, with a specific focus on the design of the synthetic gene networks needed for the system. We use these synthetic networks to encode motion commands within the cell's endogenous, oscillatory calcium signaling pathways. We then describe a potential system whereby this oscillatory signal could be outputted and monitored as a change in cellular fluorescence. Next, we use the changes resulting from the synthetic biological modifications as new parameters in a simulation of a well-established mathematical model for intracellular calcium signaling. The resulting signal is processed in the frequency domain, with specific frequencies activating cognate robot motion subroutines.

Exploring Host-Microbiome Interactions using an in Silico Model of Biomimetic Robots and Engineered Living Cells.

The microbiome's underlying dynamics play an important role in regulating the behavior and health of its host. In order to explore the details of these interactions, we created an in silico model of a living microbiome, engineered with synthetic biology, that interfaces with a biomimetic, robotic host. By analytically modeling and computationally simulating engineered gene networks in these commensal communities, we reproduced complex behaviors in the host. We observed that robot movements depended upon programmed biochemical network dynamics within the microbiome. These results illustrate the model's potential utility as a tool for exploring inter-kingdom ecological relationships. These systems could impact fields ranging from synthetic biology and ecology to biophysics and medicine.

Sudden motility reversal indicates sensing of magnetic field gradients in Magnetospirillum magneticum AMB-1 strain.

Many motile unicellular organisms have evolved specialized behaviors for detecting and responding to environmental cues such as chemical gradients (chemotaxis) and oxygen gradients (aerotaxis). Magnetotaxis is found in magnetotactic bacteria and it is defined as the passive alignment of these cells to the geomagnetic field along with active swimming. Herein we show that Magnetospirillum magneticum (AMB-1) show a unique set of responses that indicates they sense and respond not only to the direction of magnetic fields by aligning and swimming, but also to changes in the magnetic field or magnetic field gradients. We present data showing that AMB-1 cells exhibit sudden motility reversals when we impose them to local magnetic field gradients. Our system employs permalloy (Ni(80)Fe(20)) islands to curve and diverge the magnetic field lines emanating from our custom-designed Helmholtz coils in the vicinity of the islands (creating a drop in the field across the islands). The three distinct movements we have observed as they approach the permalloy islands are: unidirectional, single reverse and double reverse. Our findings indicate that these reverse movements occur in response to magnetic field gradients. In addition, using a permanent magnet we found further evidence that supports this claim. Motile AMB-1 cells swim away from the north and south poles of a permanent magnet when the magnet is positioned less than ∼30 mm from the droplet of cells. All together, these results indicate previously unknown response capabilities arising from the magnetic sensing systems of AMB-1 cells. These responses could enable them to cope with magnetic disturbances that could in turn potentially inhibit their efficient search for nutrients.

Controlling magnetotactic bacteria through an integrated nanofabricated metallic island and optical microscope approach.

Herein, we demonstrate the control of magnetotactic bacteria through the application of magnetic field gradients with real-time visualization. We accomplish this control by integrating a pair of macroscale Helmholtz coils and lithographically fabricated nanoscale islands composed of permalloy (Ni80Fe20). This system enabled us to guide and steer amphitrichous Magnetospirillum magneticum strain AMB-1 to specific location via magnetic islands. The geometries of the islands allowed us to have control over the specific magnetic field gradients on the bacteria. We estimate that magnetotactic bacteria located less than 1 µm from the edge of a diamond shaped island experience a maximum force of approximately 34 pN, which engages the bacteria without trapping them. Our system could be useful for a variety of applications including magnetic fabrication, self-assembly, and probing the sensing apparatus of magnetotactic bacteria.

Creating biological nanomaterials using synthetic biology.

Synthetic biology is a new discipline that combines science and engineering approaches to precisely control biological networks. These signaling networks are especially important in fields such as biomedicine and biochemical engineering. Additionally, biological networks can also be critical to the production of naturally occurring biological nanomaterials, and as a result, synthetic biology holds tremendous potential in creating new materials. This review introduces the field of synthetic biology, discusses how biological systems naturally produce materials, and then presents examples and strategies for incorporating synthetic biology approaches in the development of new materials. In particular, strategies for using synthetic biology to produce both organic and inorganic nanomaterials are discussed. Ultimately, synthetic biology holds the potential to dramatically impact biological materials science with significant potential applications in medical systems.

Biological colloid engineering: Self-assembly of dipolar ferromagnetic chains in a functionalized biogenic ferrofluid.

We have studied the dynamic behavior of nanoparticles in ferrofluids consisting of single-domain, biogenic magnetite (Fe(3)O(4)) isolated from Magnetospirillum magnetotacticum (MS-1). Although dipolar chains form in magnetic colloids in zero applied field, when dried upon substrates, the solvent front disorders nanoparticle aggregation. Using avidin-biotin functionalization of the particles and substrate, we generated self-assembled, linear chain motifs that resist solvent front disruption in zero-field. The engineered self-assembly process we describe here provides an approach for the creation of ordered magnetic structures that could impact fields ranging from micro-electro-mechanical systems development to magnetic imaging of biological structures.

Calcium signaling is gated by a mechanical threshold in three-dimensional environments.

Cells interpret their mechanical environment using diverse signaling pathways that affect complex phenotypes. These pathways often interact with ubiquitous 2(nd)-messengers such as calcium. Understanding mechanically-induced calcium signaling is especially important in fibroblasts, cells that exist in three-dimensional fibrous matrices, sense their mechanical environment, and remodel tissue morphology. Here, we examined calcium signaling in fibroblasts using a minimal-profile, three-dimensional (MP3D) mechanical assay system, and compared responses to those elicited by conventional, two-dimensional magnetic tensile cytometry and substratum stretching. Using the MP3D system, we observed robust mechanically-induced calcium responses that could not be recreated using either two-dimensional technique. Furthermore, we used the MP3D system to identify a critical displacement threshold governing an all-or-nothing mechanically-induced calcium response. We believe these findings significantly increase our understanding of the critical role of calcium signaling in cells in three-dimensional environments with broad implications in development and disease.