Integrating microfluidics and synthetic biology: advancements and diverse applications across organisms.

Synthetic biology is the design and modification of biological systems for specific functions, integrating several disciplines like engineering, genetics, and computer science. The field of synthetic biology is to understand biological processes within host organisms through the manipulation and regulation of their genetic pathways and the addition of biocontrol circuits to enhance their production capabilities. This pursuit serves to address global challenges spanning diverse domains that are difficult to tackle through conventional routes of production. Despite its impact, achieving precise, dynamic, and high-throughput manipulation of biological processes is still challenging. Microfluidics offers a solution to those challenges, enabling controlled fluid handling at the microscale, offering lower reagent consumption, faster analysis of biochemical reactions, automation, and high throughput screening. In this review, we diverge from conventional focus on automating the synthetic biology design-build-test-learn cycle, and instead, focus on microfluidic platforms and their role in advancing synthetic biology through its integration with host organisms - bacterial cells, yeast, fungi, animal cells - and cell-free systems. The review illustrates how microfluidic devices have been instrumental in understanding biological systems by showcasing microfluidics as an essential tool to create synthetic genetic circuits, pathways, and organisms within controlled environments. In conclusion, we show how microfluidics expedite synthetic biology applications across diverse domains including but not limited to personalized medicine, bioenergy, and agriculture.

An Automated Single-Cell Droplet-Digital Microfluidic Platform for Monoclonal Antibody Discovery.

Monoclonal antibody (mAb) discovery plays a prominent role in diagnostic and therapeutic applications. Droplet microfluidics has become a standard technology for high-throughput screening of antibody-producing cells due to high droplet single-cell confinement frequency and rapid analysis and sorting of the cells of interest with their secreted mAbs. In this work, a new method is described for on-demand co-encapsulation of cells that eliminates the difficulties associated with washing in between consecutive steps inside the droplets and enables the washing and addition of fresh media. The new platform identifies hybridoma cells that are expressing antibodies of interest using antibody-characterization assays to find the best-performing or rare-cell antibody candidates.

An electrochemical aptasensor for Δ9-tetrahydrocannabinol detection in saliva on a microfluidic platform.

We present a novel "on-off", cost-effective, rapid electrochemical aptasensor combined with a microfluidics cartridge system for the detection of Δ9-THC (Δ9-tetrahydrocannabinol) in human saliva via differential pulse voltammetry. The assay relied on the competitive binding between the Δ9-THC and a soluble redox indicator methylene blue, using an aptamer selected via FRELEX. We found that the aptasensor can detected 1 nM of Δ9-THC in PBS in a three-electrode cell system, while the sensitivity and both the dissociation constant (Kd) and association constant (Kb) were dependent on the aptamer density. The aptamer also showed great affinity towards Δ9-THC when tested against cannabinol and cannabidiol. The same limit of detection of 1 nM in PBS was achieved in small volume samples (∼60 µL) using the aptamer-modified gold screen-printed electrodes combined with the microfluidic cartridge setup, however, the presence of 10% raw human saliva had a negative effect which manifested in a 10-fold increase in the LOD due to interfering elements. Filtering the saliva, improved the tested volume to 50% and the LOD to 5 nM of Δ9-THC which is lower than the concentrations associated with impairment (6.5-32 nM). The aptasensor showed a good storage capability up to 3 days, however, the reusability significantly dropped from 10 cycles (freshly prepared) to 5 cycles. The results clearly demonstrate the feasibility of the aptasensor platform with the microfluidics chamber towards a point-of-care testing application for the detection of Δ9-THC in saliva.

Integrating machine learning and digital microfluidics for screening experimental conditions.

Digital microfluidics (DMF) has the signatures of an ideal liquid handling platform - as shown through almost two decades of automated biological and chemical assays. However, in the current state of DMF, we are still limited by the number of parallel biological or chemical assays that can be performed on DMF. Here, we report a new approach that leverages design-of-experiment and numerical methodologies to accelerate experimental optimization on DMF. The integration of the one-factor-at-a-time (OFAT) experimental technique with machine learning algorithms provides a set of recommended optimal conditions without the need to perform a large set of experiments. We applied our approach towards optimizing the radiochemistry synthesis yield given the large number of variables that affect the yield. We believe that this work is the first to combine such techniques which can be readily applied to any other assays that contain many parameters and levels on DMF.

Droplet digital microfluidic system for screening filamentous fungi based on enzymatic activity.

Fungal cell-wall-degrading enzymes have great utility in the agricultural and food industries. These cell-wall-degrading enzymes are known to have functions that can help defend against pathogenic organisms. The existing methods used to discover these enzymes are not well adapted to fungi culture and morphology, which prevents the proper evaluation of these enzymes. We report the first droplet-based microfluidic method capable of long-term incubation and low-voltage conditions to sort filamentous fungi inside nanoliter-sized droplets. The new method was characterized and validated in solid-phase media based on colloidal chitin such that the incubation of single spores in droplets was possible over multiple days (2-4 days) and could be sorted without droplet breakage. With long-term culture, we examined the activity of cell-wall-degrading enzymes produced by fungi during solid-state droplet fermentation using three highly sensitive fluorescein-based substrates. We also used the low-voltage droplet sorter to select clones with highly active cell-wall-degrading enzymes, such as chitinases, ß-glucanases, and ß-N-acetylgalactosaminidases, from a filamentous fungi droplet library that had been incubated for >4 days. The new system is portable, affordable for any laboratory, and user-friendly compared to classical droplet-based microfluidic systems. We propose that this system will be useful for the growing number of scientists interested in fungal microbiology who are seeking high-throughput methods to incubate and sort a large library of fungal cells.

A Synthetic Biosensor for Detecting Putrescine in Beef Samples.

Biogenic amines (BAs) are toxicological risks present in many food products. Putrescine is the most common foodborne BA and is frequently used as a quality control marker. Currently, there is a lack of regulation concerning safe putrescine limits in food as well as outdated food handling practices leading to unnecessary putrescine intake. Conventional methods used to evaluate BAs in food are generally time-consuming and resource-heavy with few options for on-site analysis. In response to this challenge, we have developed a transcription factor-based biosensor for the quantification of putrescine in beef samples. In this work, we use a naturally occurring putrescine responsive repressor-operator pair (PuuR-puuO) native to Escherichia coli. Moreover, we demonstrate the use of the cell-free putrescine biosensor on a paper-based device that enables rapid low-cost detection of putrescine in beef samples stored at different temperatures. The results presented demonstrate the potential role of using paper-based biosensors for on-site testing, particularly as an index for determining meat product stability and quality.

Viral Generation, Packaging, and Transduction on a Digital Microfluidic Platform.

Viral-based systems are a popular delivery method for introducing exogenous genetic material into mammalian cells. Unfortunately, the preparation of lentiviruses containing the machinery to edit the cells is labor-intensive, with steps requiring optimization and sensitive handling. To mitigate these challenges, we introduce the first microfluidic method that integrates lentiviral generation, packaging, and transduction. The new method allows the production of viral titers between 106 and 107 (similar to macroscale production) and high transduction efficiency for hard-to-transfect cell lines. We extend the technique for gene editing applications and show how this technique can be used to knock out and knock down estrogen receptor gene─a gene prominently responsible for 70% of breast cancer cases. This new technique is automated with multiplexing capabilities, which have the potential to standardize the methods for viral-based genome engineering.

Digital Microfluidics Chips for the Execution and Real-Time Monitoring of Multiple Ribozymatic Cleavage Reactions.

In this paper, we describe the design and performance of two digital microfluidics (DMF) chips capable of executing multiple ribozymatic reactions, with proper controls, in response to short single-stranded DNA inducers. Since the fluorescence output of a reaction is measurable directly from the chip, without the need for gel electrophoresis, a complete experiment involving up to eight reactions (per chip) can be carried out reliably, relatively quickly, and efficiently. The ribozymes can also be used as biosensors of the concentration of oligonucleotide inputs, with high sensitivity, low limits of quantification and of detection, and excellent signal-to-noise ratio. The presented chips are readily usable devices that can be used to automate, speed up, and reduce the costs of ribozymatic reaction experiments.

Expanding the limits towards 'one-pot' DNA assembly and transformation on a rapid-prototype microfluidic device.

DNA assembly and transformation are crucial to the building process in synthetic biology. These steps are significant roadblocks when engineering increasingly complex biological systems. To address this, recent development of widespread 'biofoundry' facilities has employed automation equipment to expedite the synthetic biology workflow. Despite significant progress, there is a clear demand for lower-cost and smaller-footprint automation equipment. The field of microfluidics have emerged to provide automation capabilities to meet this demand. However, we still lack devices capable of building large multi-gene systems in a consolidated process. In response to this challenge, we have developed a digital microfluidic platform that performs "one-pot" Golden Gate DNA assembly of large plasmids and transformation of E coli. The system features a novel electrode geometry and modular design, which make these devices simple to fabricate and use, thus improving the accessibility of microfluidics. This device incorporates an impedance-based adaptive closed loop water replenishment system to compensate for droplet evaporation and maintain constant assembly reaction concentrations, which we found to be crucial to the DNA assembly efficiency. We also showcase a closed-loop temperature control system that generates precise thermodynamic profiles to optimize heat shock transformation. Moreover, we validated the system by assembling and transforming large and complex plasmids conferring a biosynthetic pathway, resulting in performance comparable to those of standard techniques. We propose that the methods described here will contribute to a new generation of accessible automation platforms aimed at speeding up the 'building' process, lowering reagent consumption and removing manual work from synthetic biology.

Real-Time Optogenetics System for Controlling Gene Expression Using a Model-Based Design.

Optimization of engineered biological systems requires precise control over the rates and timing of gene expression. Optogenetics is used to dynamically control gene expression as an alternative to conventional chemical-based methods since it provides a more convenient interface between digital control software and microbial culture. Here, we describe the construction of a real-time optogenetics platform, which performs closed-loop control over the CcaR-CcaS two-plasmid system in Escherichia coli. We showed the first model-based design approach by constructing a nonlinear representation of the CcaR-CcaS system, tuned the model through open-loop experimentation to capture the experimental behavior, and applied the model in silico to inform the necessary changes to build a closed-loop optogenetic control system. Our system periodically induces and represses the CcaR-CcaS system while recording optical density and fluorescence using image processing techniques. We highlight the facile nature of constructing our system and how our model-based design approach will potentially be used to model other systems requiring closed-loop optogenetic control.

Is microfluidics the "assembly line" for CRISPR-Cas9 gene-editing?

Acclaimed as one of the biggest scientific breakthroughs, the technology of CRISPR has brought significant improvement in the biotechnological spectrum-from editing genetic defects in diseases for gene therapy to modifying organisms for the production of biofuels. Since its inception, the CRISPR-Cas9 system has become easier and more versatile to use. Many variants have been found, giving the CRISPR toolkit a great range that includes the activation and repression of genes aside from the previously known knockout and knockin of genes. Here, in this Perspective, we describe efforts on automating the gene-editing workflow, with particular emphasis given on the use of microfluidic technology. We discuss how automation can address the limitations of gene-editing and how the marriage between microfluidics and gene-editing will expand the application space of CRISPR.

One Cell, One Drop, One Click: Hybrid Microfluidics for Mammalian Single Cell Isolation.

Generating a stable knockout cell line is a complex process that can take several months to complete. In this work, a microfluidic method that is capable of isolating single cells in droplets, selecting successful edited clones, and expansion of these isoclones is introduced. Using a hybrid microfluidics method, droplets in channels can be individually addressed using a co-planar electrode system. In the hybrid microfluidics device, it is shown that single cells can be trapped and subsequently encapsulate them on demand into pL-sized droplets. Furthermore, droplets containing single cells are either released, kept in the traps, or merged with other droplets by the application of an electric potential to the electrodes that is actuated through an in-house user interface. This high precision control is used to successfully sort and recover single isoclones to establish monoclonal cell lines, which is demonstrated with a heterozygous NCI-H1299 lung squamous cell population resulting from loss-of-function eGFP and RAF1 gene knockout transfections.

A fucosyltransferase inhibition assay using image-analysis and digital microfluidics.

Sialyl-LewisX and LewisX are cell-surface glycans that influence cell-cell adhesion behaviors. These glycans are assembled by α(1,3)-fucosyltransferase enzymes. Their increased expression plays a role in inflammatory disease, viral and microbial infections, and cancer. Efficient screens for specific glycan modifications such as those catalyzed by fucosyltransferases are tended toward costly materials and large instrumentation. We demonstrate for the first time a fucosylation inhibition assay on a digital microfluidic system with the integration of image-based techniques. Specifically, we report a novel lab-on-a-chip approach to perform a fluorescence-based inhibition assay for the fucosylation of a labeled synthetic disaccharide, 4-methylumbelliferyl ß-N-acetyllactosaminide. As a proof-of-concept, guanosine 5'-diphosphate has been used to inhibit Helicobacter pylori α(1,3)-fucosyltransferase. An electrode shape (termed "skewed wave") is designed to minimize electrode density and improve droplet movement compared to conventional square-based electrodes. The device is used to generate a 10 000-fold serial dilution of the inhibitor and to perform fucosylation reactions in aqueous droplets surrounded by an oil shell. Using an image-based method of calculating dilutions, referred to as "pixel count," inhibition curves along with IC50 values are obtained on-device. We propose the combination of integrating image analysis and digital microfluidics is suitable for automating a wide range of enzymatic assays.

Integration of World-to-Chip Interfaces with Digital Microfluidics for Bacterial Transformation and Enzymatic Assays.

Digital microfluidics (DMF) represents an alternative to the conventional microfluidic paradigm of transporting fluids in enclosed channels. One of the major benefits of DMF is that fluid motion and control is achieved without external pumps. The automation component of DMF have pushed the barriers of this "lab-on-chip" technology. However, integration with external components (i.e., "world-to-chip") interfaces have been a challenge. Two common "world-to-chip" challenges are (1) delivering biological samples to DMF devices and (2) accurately controlling temperatures on device. To address these challenges, this work describes two "world-to-chip" interface features that have been integrated on a DMF platform: a reagent delivery system and a thermal control apparatus. This platform enables a variety of biological or chemical experiments to be conducted on-chip while reducing manual intervention. Specifically, our platform increases reagent volumes available to device reservoirs volume by at least 50-fold eliminating the need to manually refill reservoirs while improving droplet dispensing reproducibility. In addition, we have integrated a closed-loop temperature control system that offers precise temperature control on-chip. To validate our "world-to-chip" interface, we have automated bacterial transformation and enzymatic assay protocols, showing that such a system enhances DMF performance. Overall, we propose that this system will improve biological experimentation which requires fluidic and temperature control integrated on DMF platforms.

An integrated droplet-digital microfluidic system for on-demand droplet creation, mixing, incubation, and sorting.

Droplet microfluidics is a technique that has the ability to compartmentalize reactions in sub nano- (or pico-) liter volumes that can potentially enable millions of distinct biological assays to be performed on individual cells. In a typical droplet microfluidic system, droplets are manipulated by pressure-based flows. This has limited the fluidic operations that can be performed in these devices. Digital microfluidics is an alternative microfluidic paradigm with precise control and manipulation over individual droplets. Here, we implement an integrated droplet-digital microfluidic (which we call 'ID2M') system in which common fluidic operations (i.e. droplet generation, cell encapsulation, droplet merging and mixing, droplet trapping and incubation, and droplet sorting) can be performed. With the addition of electrodes, we have been able to create droplets on-demand, tune their volumes on-demand, and merge and mix several droplets to produce a dilution series. Moreover, this device can trap and incubate droplets for 24 h that can consequently be sorted and analyzed in multiple n-ary channels (as opposed to typical binary channels). The ID2M platform has been validated as a robust on-demand screening system by sorting fluorescein droplets of different concentration with an efficiency of ∼96%. The utility of the new system is further demonstrated by culturing and sorting tolerant yeast mutants and wild-type yeast cells in ionic liquid based on their growth profiles. This new platform for both droplet and digital microfluidics has the potential to be used for screening different conditions on-chip and for applications like directed evolution.

An automated microfluidic gene-editing platform for deciphering cancer genes.

Gene-editing techniques such as RNA-guided endonuclease systems are becoming increasingly popular for phenotypic screening. Such screens are normally conducted in arrayed or pooled formats. There has been considerable interest in recent years to find new technological methods for conducting these gene-editing assays. We report here the first digital microfluidic method that can automate arrayed gene-editing in mammalian cells. Specifically, this method was useful in culturing lung cancer cells for up to six days, as well as implementing automated gene transfection and knockout procedures. In addition, a standardized imaging pipeline to analyse fluorescently labelled cells was also designed and implemented during these procedures. A gene editing assay for interrogating the MAPK/ERK pathway was performed to show the utility of our platform and to determine the effects of knocking out the RAF1 gene in lung cancer cells. In addition to gene knockout, we also treated the cells with an inhibitor, Sorafenib Tosylate, to determine the effects of enzymatic inhibition. The combination of enzymatic inhibition and guide targeting on device resulted in lower drug concentrations for achieving half-inhibitory effects (IC50) compared to cells treated only with the inhibitor, confirming that lung cancer cells are being successfully edited on the device. We propose that this system will be useful for other types of gene-editing assays and applications related to personalized medicine.

An Automated Induction Microfluidics System for Synthetic Biology.

The expression of a recombinant gene in a host organism through induction can be an extensively manual and labor-intensive procedure. Several methods have been developed to simplify the protocol, but none has fully replaced the traditional IPTG-based induction. To simplify this process, we describe the development of an autoinduction platform based on digital microfluidics. This system consists of a 600 nm LED and a light sensor to enable the real-time monitoring of  the optical density (OD) samples coordinated with the semicontinuous mixing of a bacterial culture. A hand-held device was designed as a microbioreactor to culture cells and to measure the OD of the bacterial culture. In addition, it serves as a platform for the analysis of regulated protein expression in E. coli without the requirement of standardized well-plates or pipetting-based platforms. Here, we report for the first time, a system that offers great convenience without the user to physically monitor the culture or to manually add inducer at specific times. We characterized our system by looking at several parameters (electrode designs, gap height, and growth rates) required for an autoinducible system. As a first step, we carried out an automated induction optimization assay using a RFP reporter gene to identify conditions suitable for our system. Next, we used our system to identify active thermophilic ß-glucosidase enzymes that may be suitable candidates for biomass hydrolysis. Overall, we believe that this platform may be useful for synthetic biology applications that require regulating and analyzing expression of heterologous genes for strain optimization.

Image-based feedback and analysis system for digital microfluidics.

Digital microfluidics (DMF) is a technology that provides a means of manipulating nL-µL volumes of liquids on an array of electrodes. By applying an electric potential to an electrode, these discrete droplets can be controlled in parallel which can be transported, mixed, reacted, and analyzed. Typically, an automation system is interfaced with a DMF device that uses a standard set of basic instructions written by the user to execute droplet operations. Here, we present the first feedback method for DMF that relies on imaging techniques that will allow online detection of droplets without the need to reactivate all destination electrodes. Our system consists of integrating open-source electronics with a CMOS camera and a zoom lens for acquisition of the images that will be used to detect droplets on the device. We also created an algorithm that uses a Hough transform to detect a variety of droplet sizes and to detect singular droplet dispensing and movement failures on the device. As a first test, we applied this feedback system to test droplet movement for a variety of liquids used in cell-based assays and to optimize different feedback actuation schemes to improve droplet movement fidelity. We also applied our system to a colorimetric enzymatic assay to show that our system is capable of biological analysis. Overall, we believe that using our approach of integrating imaging and feedback for DMF can provide a platform for automating biological assays with analysis.

On-chip integration of droplet microfluidics and nanostructure-initiator mass spectrometry for enzyme screening.

Biological assays often require expensive reagents and tedious manipulations. These shortcomings can be overcome using digitally operated microfluidic devices that require reduced sample volumes to automate assays. One particular challenge is integrating bioassays with mass spectrometry based analysis. Towards this goal we have developed µNIMS, a highly sensitive and high throughput technique that integrates droplet microfluidics with nanostructure-initiator mass spectrometry (NIMS). Enzyme reactions are carried out in droplets that can be arrayed on discrete NIMS elements at defined time intervals for subsequent mass spectrometry analysis, enabling time resolved enzyme activity assay. We apply the µNIMS platform for kinetic characterization of a glycoside hydrolase enzyme (CelE-CMB3A), a chimeric enzyme capable of deconstructing plant hemicellulose into monosaccharides for subsequent conversion to biofuel. This study reveals NIMS nanostructures can be fabricated into arrays for microfluidic droplet deposition, NIMS is compatible with droplet and digital microfluidics, and can be used on-chip to assay glycoside hydrolase enzyme in vitro.

Next generation tools to accelerate the synthetic biology process.

Synthetic biology follows the traditional engineering paradigm of designing, building, testing and learning to create new biological systems. While such approaches have enormous potential, major challenges still exist in this field including increasing the speed at which this workflow can be performed. Here, we present recently developed microfluidic tools that can be used to automate the synthetic biology workflow with the goal of advancing the likelihood of producing desired functionalities. With the potential for programmability, automation, and robustness, the integration of microfluidics and synthetic biology has the potential to accelerate advances in areas such as bioenergy, health, and biomaterials.