We describe an exciting new application domain for deep reinforcement learning (RL): droplet routing on digital microfluidic biochips (DMFBs). A DMFB consists of a two-dimensional electrode array, and it manipulates droplets of liquid to automatically execute biochemical protocols for clinical chemistry. However, a major problem with DMFBs is that electrodes can degrade over time. The transportation of droplet transportation over these degraded electrodes can fail, thereby adversely impacting the integrity of the bioassay outcome. We demonstrated that the formulation of droplet transportation as an RL problem enables the training of deep neural network policies that can adapt to the underlying health conditions of electrodes and ensure reliable fluidic operations. We describe an RL-based droplet routing solution that can be used for various sizes of DMFBs. We highlight the reliable execution of an epigenetic bioassay with the RL droplet router on a fabricated DMFB. We show that the use of the RL approach on a simple micro-computer (Raspberry Pi 4) leads to acceptable performance for time-critical bioassays. We present a simulation environment based on the OpenAI Gym Interface for RL-guided droplet routing problems on DMFBs. We present results on our study of electrode degradation using fabricated DMFBs. The study supports the degradation model used in the simulator.

Epigenetics, the study of inheritable mechanisms that regulate gene expression, has clinical ramifications from cancer to autoimmune disorders to psychiatric pathologies. The main tool to study epigenetics is chromatin immunoprecipitation (ChIP), which probes the relationship between DNA and its structural nucleosome-forming histone proteins. Standard benchtop ChIP has three major drawbacks: (1) it requires a large input volume of cells, (2) it is very time consuming and work intensive, and (3) it is low throughput. Digital microfluidic biochips (DMFB) have proven to be successful at utilizing small volumes of reagents and samples to perform high throughput bioanalyses and assays of macromolecules. Their ease of configurability, automation, and high sensitivity make them an ideal platform for ChIP adaptation. Previously, we demonstrated the first step towards ChIP implementation on a DMFB by detecting specifically modified nucleosomes, the building blocks of chromatin, in a semi-quantitative nucleosome immunoprecipitation (NuIP) assay. Herein, we modify the prior 'sweep-through' DMFB design to enhance the capture and detection of analyte-bound magnetic beads. With this modification, we can increase both the level of detection and granularity when probing a sample. Furthermore, this new 'pull-through' design presents a novel method for bead collection using an on-chip magnetic source.

A prototype aerosol detection system is presented that is designed to accurately and quickly measure the concentration of selected inorganic ions in the atmosphere. The aerosol detection system combines digital microfluidics technology, aerosol impaction and chemical detection integrated on the same chip. Target compounds are the major inorganic aerosol constituents: sulfate, nitrate and ammonium. The digital microfluidic system consists of top and bottom plates that sandwich a fluid layer. Nozzles for an inertial impactor are built into the top plate according to known, scaling principles. The deposited air particles are densely concentrated in well-defined deposits on the bottom plate containing droplet actuation electrodes of the chip in fixed areas. The aerosol collection efficiency for particles larger than 100 nm in diameter was higher than 95%. After a collection phase, deposits are dissolved into a scanning droplet. Due to a sub-microliter droplet size, the obtained extract is highly concentrated. Droplets then pass through an air/oil interface on chip for colorimetric analysis by spectrophotometry using optical fibers placed between the two plates of the chip. To create a standard curve for each analyte, six different concentrations of liquid standards were chosen for each assay and dispensed from on-chip reservoirs. The droplet mixing was completed in a few seconds and the final droplet was transported to the detection position as soon as the mixing was finished. Limits of detection (LOD) in the final droplet were determined to be 11 ppm for sulfate and 0.26 ppm for ammonium. For nitrate, it was impossible to get stable measurements. The LOD of the on-chip measurements for sulfate was close to that obtained by an off-chip method using a Tecan spectrometer. LOD of the on-chip method for ammonium was about five times larger than what was obtained with the off-chip method. For the current impactor collection air flow (1 L/min) and 1 h collection time, the converted LODs in air were: 0.275 μg/m3 for sulfate, 6.5 ng/m3 for ammonium, sufficient for most ambient air monitoring applications.

Hsu BN and Fair RB* Author Affiliations Department of Electrical and Computer Engineering, Durham, North Carolina, USA Received: May 31, 2019 | Published: June 21, 2019 Corresponding author: Fair RB, Department of Electrical and Computer Engineering, Durham, North Carolina, USA DOI: 10.26717/BJSTR.2019.19.003247 Also View In:

Two methods were studied for selectively measuring the on-chip absorbance of trace sulfate analytes in droplets on a digital microfluidics (DMF) platform. In one method, the direction of measurement was perpendicular to the flat upper and lower surfaces of the DMF platform (vertical), and in the second method, the measurement direction was parallel to the DMF platform surfaces (horizontal). The channel height or the vertical light path length was 0.24 mm, and the droplet diameter was 1 mm. The DMF system employed a silicone oil transport medium whereby a thin, non-uniform oil layer formed between the droplet and the upper/lower plates which was unstable, resulting in randomly formed local oil lenses. The mobile oil lenses caused vertical absorbance measurement errors and uncertainties. The effects of the oil lenses were verified by simulation. Horizontal absorbance measurements were taken with embedded optical fibers (0.2 mm in diameter) aligned over the bottom chip surface in contact with the sides of the droplet, resulting in a horizontal light path length approximately three times that of the vertical light path. Because no oil lenses could form on the droplet’s sides, the stability of repeated horizontal measurements outperformed repeated vertical measurements made on the same droplet and on multiple droplets actuated into the measurement positions. Comparisons were based on measurement standard deviations and limits of detection (LOD). The following LODs and measurement standard deviations were achieved for horizontal measurements of multiple sulfate concentrations in 1.5 µl droplets: 7 ppm for sulfate (0.3–2.7%) and an R 2 value of 0.957 from a least square data fit. Measurements on a commercial plate reader gave comparable results (200 µl liquid in each well, LOD equals 11 ppm, CV equals to 0.2–4%), even though the absorbance path was larger (0.7 mm). This LOD value means that the chip could detect 10.5 ng of sulfate. LOD values on vertical measurements were also similar, but large measurement errors from numerous outlier points yielded an R 2 value of 0.735 and large average measurement standard deviations (36%).

This work demonstrates sparse cell detection in mL samples, using magnetic bead manipulation on an ElectrowettingOn-Dielectric (EWD) chip. Sparse sample detection was performed in two steps: cell capture off chip from the starting solution with a microelectromagnet and on-chip fluorescent signal detection on an EWD chip. In the first step, immunological reactions enable the binding between target cells and antibody-coated magnetic beads, which enabled sample capture with high cell survival rates. In the second step, fluorescent detection is achieved on an EWD chip via fluorescent signal measurement and two-dimensional magnetic bead concentration. Magnetic bead concentration is controlled with an integrated microelectromagnet, a planar set of half-circle-shaped current-carrying wires embedded in an actuation electrode of an EWD device. This two-dimensional wire structure serves as a microelectromagnet capable of segregating magnetic beads into an area on the order of 10 μm2 with a resulting improvement in Signal-To-Noise Ratio (SNR) of 30 times. Simple device integration ensures that the magnetic bead manipulation and the EWD function can be operated simultaneously without introducing additional steps in the EWD chip fabrication process. Immunological reaction kits were selected in order to ensure the compatibility of target cells, magnetic beads and EWD functions. The magnetic bead choice ensures the binding efficiency and survivability of target cells. The magnetic bead selection and binding mechanism used in this work can be applied to a wide variety of samples with a simple switch of the type of antibody. Sparse cell fluorescent measurements with good SNR are made possible by using fluorescent stains and a method of concentrating cells attached to beads into a small detection area. Theoretical limitation of the entire sparse sample detection system is as low as 1 colony forming unit/mL (CFU/mL). DOI: 10.29011/ 2577-2260. 100045

This paper reports on the use of a digital microfluidic platform to perform multiplex automated genetic engineering (MAGE) cycles on droplets containing Escherichia coli cells. Bioactivated magnetic beads were employed for cell binding, washing, and media exchange in the preparation of electrocompetent cells in the electrowetting-on-dieletric (EWoD) platform. On-cartridge electroporation was used to deliver oligonucleotides into the cells. In addition to the optimization of a magnetic bead-based benchtop protocol for generating and transforming electrocompetent E. coli cells, we report on the implementation of this protocol in a fully automated digital microfluidic platform. Bead-based media exchange and electroporation pulse conditions were optimized on benchtop for transformation frequency to provide initial parameters for microfluidic device trials. Benchtop experiments comparing electrotransformation of free and bead-bound cells are presented. Our results suggest that dielectric shielding intrinsic to bead-bound cells significantly reduces electroporation field exposure efficiency. However, high transformation frequency can be maintained in the presence of magnetic beads through the application of more intense electroporation pulses. As a proof of concept, MAGE cycles were successfully performed on a commercial EWoD cartridge using variations of the optimal magnetic bead-based preparation procedure and pulse conditions determined by the benchtop results. Transformation frequencies up to 22% were achieved on benchtop; this frequency was matched within 1% (21%) by MAGE cycles on the microfluidic device. However, typical frequencies on the device remain lower, averaging 9% with a standard deviation of 9%. The presented results demonstrate the potential of digital microfluidics to perform complex and automated genetic engineering protocols.

Featured Article: Srinivasan V, Pamula V, Fair R. An integrated digital microfluidic lab-on-a-chip for clinical diagnostics on human physiological fluids, Lab Chip 2004;4:310–15.2 Our 2004 article highlighted here was the first report of a droplet-based microfluidic device that integrated on a monolithic platform all the functions for analyzing human physiological fluids, including sample injection, on-chip reservoirs, droplet formation structures, fluidic transport, mixing of reagent and sample droplets, and optical detection. The device could also be run by a computer. Thus, the device proved the potential for automated analysis of multiple analytes for clinical diagnostics using a programmable lab-on-a-chip. The use of microfluidic lab-on-a-chip technology for clinical applications in the early 2000 timeframe was almost exclusively based on continuous fluid flow in microchannels. Fluid pumping based on electrokinetic phenomena (electrophoretic separation and electroosmotics), external pressure sources, centrifugal effects, and passive capillary flow were being investigated. However, continuous-flow based devices offered very little flexibility in terms of scalability, reconfigurability, and suitability for use with a variety of liquids. For instance, physiological liquids with high ionic strength, such as blood and urine, could not be pumped using electrokinetic effects due to excessive Joule heating. In addition, continuous flow systems relied on fixed channels through …

Electrowetting-on-dielectric (EWD) digital microfluidic laboratory-on-a-chip platforms demonstrate excellent performance in automating labor-intensive protocols. When coupled with an on-chip electroporation capability, these systems hold promise for streamlining cumbersome processes such as multiplex automated genome engineering (MAGE). We integrated a single Ti:Au electroporation electrode into an otherwise standard parallel-plate EWD geometry to enable high-efficiency transformation of Escherichia coli with reporter plasmid DNA in a 200 nL droplet. Test devices exhibited robust operation with more than 10 transformation experiments performed per device without cross-contamination or failure. Despite intrinsic electric-field nonuniformity present in the EP/EWD device, the peak on-chip transformation efficiency was measured to be 8.6 ± 1.0 × 108 cfu·μg-1 for an average applied electric field strength of 2.25 ± 0.50 kV·mm-1. Cell survival and transformation fractions at this electroporation pulse strength were found to be 1.5 ± 0.3 and 2.3 ± 0.1%, respectively. Our work expands the EWD toolkit to include on-chip microbial electroporation and opens the possibility of scaling advanced genome engineering methods, like MAGE, into the submicroliter regime.

We report the integration of a lead zirconate titanate, \(\hbox {Pb[Zr}_{x}\hbox {Ti}_{1-x}\hbox {O}_{3}\)] (PZT), piezoelectric transducer disk into the top plate of an otherwise conventional electrowetting-on-dielectric (EWD) digital microfluidics device to demonstrate on-demand induction of circulating fluid flow within single 200 nL droplets. Microparticle image velocimetry was used to measure in-plane velocity distributions for PZT excitation voltages that ranged from 0 to 50 \(\hbox {V}_{\text {RMS}}\). Intra-droplet streaming velocities in excess of 2.0 \(\hbox {mm}\cdot \hbox {s}^{-1}\) were observed without droplet breakup or damage to the EWD device layer. Additionally, we found median intra-droplet streaming velocity to depend quadratically on PZT excitation voltage up to the stress limit of the interfacial boundary. Our approach offers an alternative device architecture for active micromixing strategies in EWD digital microfluidics laboratory-on-chip systems.

Theoretical and experimental approaches verifying the fluidic operation of a partially shielded digital microfluidics device are presented in this paper. This paper is motivated by recent demand from the synthetic biology community for electro-wetting on dielectric (EWD) enabled in-droplet electroporation, but is generalizable to a range of EWD applications that require shielding structures to be patterned on the EWD. An electrode patterned in an additional metal layer on the insulator that supports EWD actuation reduces the effective strength of the EW force due to dielectric shielding at the droplet contact line. A numerical model was developed to predict the impact of the partially shielding electrode on threshold voltage, EW force, fluid velocity, and droplet transport time. Compared with a batch of devices lacking the extra electrode, the presence of the added metal layer resulted in a 29% increase in threshold voltage, an 82% increase in transport time, and a 44% decrease in average transport velocity. Each trend agrees with the simulation results obtained from the fluid transport model. These results support the development of design rules for microfluidic devices that require partially shielding metal layers to integrate with EWD device architectures.

Digital (droplet-based) microfluidics enables the integration of fluid-handling operations and reaction-outcome detection. Despite these benefits, defects and erroneous fluidic operations continue to be major barriers to the adoption and deployment of these devices. We describe the first practical and fully integrated cyberphysical error-recovery system that can be implemented in real time on a field-programmable gate array (FPGA). The hardware-assisted solution is based on an error dictionary containing the error-recovery plans for various anticipated errors. The dictionary is computed and stored in FPGA memory before the start of the biochemical experiment. Errors in droplet operations on the digital microfluidic platform are detected using capacitive sensors, the test outcome is interpreted by control hardware, and corresponding error-recovery plans are triggered in real-time. Experimental results are reported for a fabricated silicon device, and links to videos are provided for the first-ever experimental demonstration of real-time error recovery in cyberphysical digital-microfluidic biochips using a hardware implemented dictionary.

This paper demonstrates an integrated device combining both EWD droplet actuation and intra-droplet magnetic bead manipulation. Magnetic bead manipulation is achieved by using current-carrying wires acting as microelectromagnets. The current wire structure is capable of segregating and separating magnetic beads within a droplet. By adjusting the amount of current in the wire structure, high segregation efficiency within the droplet is shown and the separation of two kinds of beads is realized within a distance of 65 µm. The EWD droplet actuation function and the magnetic bead separation function can be operated independently, which provides flexibility when designing operation protocols. The current wire structure is embedded in an EWD electrode without affecting droplet actuation. The vertical structure of the device and its fabrication process are also the same as a normal EWD device. Magnetic bead segregation efficiency of 96.8 % was achieved for 2.8-µm beads that were collected over a distance of 65 µm during a 20-s current pulse. Droplet splitting is shown to allow complete separation of beads into one of the two daughter droplets. And, intra-droplet separation of 1- and 2.8-µm beads was demonstrated over an average distance of 65 µm. This experiment shows the feasibility of performing separation in droplets with a complex mixture of multiple beads, each having different magnetic contents.

This paper demonstrates a device which is capable of performing both magnetic molecular segregation within a droplet as well as droplet actuation. Segregation of magnetic beads is performed within a droplet while the droplet is dispensed and transported using electrowetting on dielectric (EWD). The reason it is important to investigate such devices is that the capability could be used in current applications, including immunoassays, cell labeling and DNA isolation.

An electrowetting-based digital microfluidic lab-on-a-chip (LoC) platform for automated trace sulfate measurement is presented in this paper. This platform was designed to be integrated with a digital microfluidic impactor for online ambient aerosol sampling and analysis. The LoC uses a discrete droplet format in contrast to the traditional continuous flow micro-fluidic systems. The traditional sulfate measurement method – methylthymol blue (MTB) colorimetric method is modified for its application on the LoC platform. An absorbance measurement system integrated with the chip was designed, consisting of a light emitting diode and a photodiode that detects the color change due to the reaction. The MTB colorimetric assay was modified to be compatible with the micro-fluidic platform and optimized with respect to reagent concentration. The LoC system provides a higher level of automation, consumes less reagent, and produces less waste than the conventional “macro” systems. It has a short analytical cycle of 30 seconds. The system has a limit of detection of 0.54 mg/L and provides a broader linear measurement range (up to 150 mg/L) than the traditional implementation of the methylthymol blue method.

Electrowetting-on-dielectric (EWD) microfluidics is an emerging platform for practical applications such as water quality testing and medical diagnostics. Low power consumption, low sample and reagent volumes, small size, and rapid fluid transport are features of electrowetting microfluidic platforms that will enable the development of cost-effective, rapid time-to-result, and portable point-of-care diagnostic devices. Microresonator sensors are an excellent sensor technology for integration into these microfluidic systems, because they perform high sensitivity detection of proteins, DNA, and other biologically relevant molecules while tolerating a droplet oil encapsulation layer. This paper reports on a SU-8 polymer microresonator embedded in the top plate of the EWD system, which enables addressing of the sensor with a single droplet of in volume and enables droplets to be moved onto and off of the sensor. This system is the first to demonstrate actuation of droplets onto and off of an integrated microresonator sensor. Both photolithographically patterned and electron beam lithographically patterned microresonator sensors were tested, and the effect of a conventional filler medium, silicone oil, on the sensor sensitivity was investigated.

Advances in digital microfluidics and integrated sensing hold promise for a new generation of droplet-based biochips that can perform multiplexed assays to determine the identity of target molecules. Despite these benefits, defects and erroneous fluidic operations remain a major barrier to the adoption and deployment of these devices. We describe the first integrated demonstration of cyberphysical coupling in digital microfluidics, whereby errors in droplet transportation on the digital microfluidic platform are detected using capacitive sensors, the test outcome is interpreted by control hardware, and software-based error recovery is accomplished using dynamic reconfiguration. The hardware/software interface is realized through seamless interaction between control software, an off-the-shelf microcontroller and a frequency divider implemented on an FPGA. Experimental results are reported for a fabricated silicon device and links to videos are provided for the first-ever experimental demonstration of cyberphysical coupling and dynamic error recovery in digital microfluidic biochips.