Digital Microfluidic System Research Articles

An artificial intelligence-assisted digital microfluidic system for multistate droplet control

Digital microfluidics (DMF) is a versatile technique for parallel and field-programmable control of individual droplets. Given the high level of variability in droplet manipulation, it is essential to establish self-adaptive and intelligent control methods for DMF systems that are informed by the transient state of droplets and their interactions. However, most related studies focus on droplet localization and shape recognition. In this study, we develop the AI-assisted DMF framework μDropAI for multistate droplet control on the basis of droplet morphology. The semantic segmentation model is integrated into our custom-designed DMF system to recognize the droplet states and their interactions for feedback control with a state machine. The proposed model has strong flexibility and can recognize droplets of different colors and shapes with an error rate of less than 0.63%; it enables control of droplets without user intervention. The coefficient of variation (CV) of the volumes of split droplets can be limited to 2.74%, which is lower than the CV of traditional dispensed droplets, contributing to an improvement in the precision of volume control for droplet splitting. The proposed system inspires the development of semantic-driven DMF systems that can interface with multimodal large language models (MLLMs) for fully automatic control.

From Friction to Function: A High-Voltage Sliding Triboelectric Nanogenerator for Highly Efficient Energy Autonomous IoTs and Self-Powered Actuation.

An advanced energy autonomous system that simultaneously harnesses and stores energy on the same platform offers exciting opportunities for the near-future self-powered miniature electronics. However, achieving optimal synchronization between the power output of an energy harvester and the storage unit or integrating it seamlessly with real-time microelectronics to build a highly efficient energy autonomous system remains challenging. Herein, a unique bimetallic layered double hydroxide (LDH) based tribo-positive layer is introduced for a high-voltage sliding triboelectric nanogenerator (S-TENG) with an output voltage of ≈1485V and power output of 250 µW, respectively. To demonstrate the potential of a self-charging power system, S-TENG is integrated with on-chip micro-supercapacitors (MSCs) as a storage unit. The MSC array effectively self-charged up to 4.8V (within 220s), providing ample power to support micro-sensory systems. In addition, by utilizing the high-voltage output of the S-TENG, the efficient operation of electrostatic actuators and digital microfluidic (DMF) systems driven directly by simple mechanical motion is further demonstrated. Overall, this work can provide a solid foundation for the advancement of next-generation energy-autonomous systems.

Automated Droplet Ejection from a Digital Microfluidics Sample Pretreatment Device Enables Batch-Mode Chemiluminescence Immunoassay.

Digital microfluidics (DMF) features programmed manipulation of fluids in multiple steps, making it a valuable tool for sample pretreatment. However, the integration of sample pretreatment with its downstream reaction and detection requires transferring droplets from the DMF device to the outside world. To address this issue, the present study developed a modified DMF device that allows automated droplet ejection out of the chip, facilitated by a tailor-designed interface. A double-layered DMF microchip with an oil-filled medium was flipped over, with a liquid infusion port and a liquid expulsion port accommodated on the top working PCB plate and the bottom grounded ITO plate, respectively, to facilitate chip-to-world delivery of droplets. Using chemiluminescent immunoassay (CLIA) as an illustrative application, the sample pretreatment was programmed on the DMF device, and CLIA droplets were ejected from the chip for signal reading. In our workflow, CLIA droplets can be ejected from the DMF device through the chip-to-world interface, freeing up otherwise occupied electrodes for more sample pretreatment and enabling streamlined droplet microreactions and batch-mode operation for bioanalysis. Integrated with these interfacing portals, the DMF system achieved a single-channel throughput of 17 samples per hour, which can be further upscaled for more productive applications by parallelizing the DMF modules. The results of this study demonstrate that the droplet ejection function that is innovated in a DMF sample pretreatment microsystem can significantly improve analytical throughput, providing an approach to establishing an automated but decentralized biochemical sample preparation workstation for large-scale and continuous bioanalysis.

Drug screening on digital microfluidics for cancer precision medicine

Drug screening based on in-vitro primary tumor cell culture has demonstrated potential in personalized cancer diagnosis. However, the limited number of tumor cells, especially from patients with early stage cancer, has hindered the widespread application of this technique. Hence, we developed a digital microfluidic system for drug screening using primary tumor cells and established a working protocol for precision medicine. Smart control logic was developed to increase the throughput of the system and decrease its footprint to parallelly screen three drugs on a 4 × 4 cm2 chip in a device measuring 23 × 16 × 3.5 cm3. We validated this method in an MDA-MB-231 breast cancer xenograft mouse model and liver cancer specimens from patients, demonstrating tumor suppression in mice/patients treated with drugs that were screened to be effective on individual primary tumor cells. Mice treated with drugs screened on-chip as ineffective exhibited similar results to those in the control groups. The effective drug identified through on-chip screening demonstrated consistency with the absence of mutations in their related genes determined via exome sequencing of individual tumors, further validating this protocol. Therefore, this technique and system may promote advances in precision medicine for cancer treatment and, eventually, for any disease.

Rapid and multi-target genotyping of Helicobacter pylori with digital microfluidics

Helicobacter pylori (H. pylori) infection correlates closely with gastric diseases such as gastritis, ulcers, and cancer, influencing more than half of the world's population. Establishing a rapid, precise, and automated platform for H. pylori diagnosis is an urgent clinical need and would significantly benefit therapeutic intervention. Recombinase polymerase amplification (RPA)-CRISPR recently emerged as a promising molecular diagnostic assay due to its rapid detection capability, high specificity, and mild reaction conditions. In this work, we adapted the RPA-CRISPR assay on a digital microfluidics (DMF) system for automated H. pylori detection and genotyping. The system can achieve multi-target parallel detection of H. pylori nucleotide conservative genes (ureB) and virulence genes (cagA and vacA) across different samples within 30 min, exhibiting a detection limit of 10 copies/rxn and no false positives. We further conducted tests on 80 clinical saliva samples and compared the results with those derived from real-time quantitative polymerase chain reaction, demonstrating 100% diagnostic sensitivity and specificity for the RPA-CRISPR/DMF method. By automating the assay process on a single chip, the DMF system can significantly reduce the usage of reagents and samples, minimize the cross-contamination effect, and shorten the reaction time, with the additional benefit of losing the chance of experiment failure/inconsistency due to manual operations. The DMF system together with the RPA-CRISPR assay can be used for early detection and genotyping of H. pylori with high sensitivity and specificity, and has the potential to become a universal molecular diagnostic platform.

Active-matrix digital microfluidics design for field programmable high-throughput digitalized liquid handling

Digital liquid sample handling is an enabling tool for cutting-edge life-sciences research. We present here an active-matrix thin-film transistor (TFT) based digital microfluidics system, referred to as Field Programmable Droplet Array (FPDA). The system contains 256×256 pixels in an active area of 10.65cm2, which can manipulate thousands of addressable liquid droplets simultaneously. By leveraging a novel TFT device and circuits design solution, we manage to programmatically manipulate droplets at single-pixel level. The minimum achievable droplet volume is around 0.5 nL, which is two orders of magnitude smaller than the smallest droplet ever reported on active-matrix digital microfluidics. The movement of droplets can be either pre-programmed or controlled in real-time. The FPDA system shows great potential of the ubiquitous thin-film electronics technology in digital liquid handling. These efforts will make it possible to create a true programmable lab-on-a-chip device to enable great advances in life science research.

Open Thermal Control System for Stable Polymerase Chain Reaction on a Digital Microfluidic Chip.

In this paper, a digital microfluidic thermal control system was introduced for the stable polymerase chain reaction (PCR). The system consists of a thermoelectric cooler unit, a thermal control board, and graphical-user-interface software capable of simultaneously achieving temperature control and on-chip droplet observation. A fuzzy proportional-integral-derivative (PID) method was developed for this system. The simulation analysis was performed to evaluate the temperature of different reagents within the chip. Based on the results, applying fuzzy PID control for PCR will enhance the thermal stability by 67.8% and save the time by 1195 s, demonstrating excellent dynamic response capability and thermal robustness. The experimental results are consistent with the simulation results on the planar temperature distribution, with a data consistency rate of over 99%. The PCR validation was carried out on this system, successfully amplifying the rat GAPDH gene at a concentration of 193 copies/μL. This work has the potential to be useful in numerous existing lab-on-a-chip applications.

Transitional Electrodes in Electrowetting-Based Droplet Dispensing.

Digital microfluidic systems based on electrowetting-on-dielectric technology, particularly valuable in producing and manipulating microdroplets steadily and consistently, have experienced notable advancements in recent years. In this paper, experimental characterizations reveal that simply adding one transitional electrode between the reservoir and the splitting electrode improves the volumetric consistency and reproducibility for droplet dispensing. The volumetric coefficient variation of the consecutively dispensed droplets from a non-refilling reservoir decreases by 1% after the addition of one transitional electrode, with no extra external apparatus. This work provides a straightforward yet effective approach to the improvement of digital microfluidic systems and micro total analysis systems.

Droplet manipulation on an adjustable closed-open digital microfluidic system utilizing asymmetric EWOD.

The closed-open digital microfluidic (DMF) system offers a versatile and powerful platform for various applications by combining the advantages of both closed and open structures. The current closed-open DMF system faces challenges in scaling up due to electrode structural differences between closed and open regions. Here we developed an adjustable closed-open DMF platform by utilizing the modified slippery liquid-infused porous surfaces (SLIPS) with asymmetric electrowetting on dielectric (AEWOD) as a hydrophobic dielectric layer. The consistent electrode structures of the bottom printed circuit board (PCB) electrode array on both the closed and open regions, and the utilization of a transparent acrylic with floating potential as the top plate allow a low-cost and easily scalable closed-open DMF system to be achieved. The impacts of applied voltage, parallel plate spacing, electrode switching interval, and electrode driving strategies on various droplet manipulations were investigated. The results show that the optimal plate spacings range from 340-510 μm within the closed region. Meanwhile, we also studied the influence of the thickness, geometry, and position of the top plate on the droplet movement at the closed-open boundary. Through force analysis and experimentation, it is found that a thin top plate and a bevel of ∼4° can effectively facilitate the movement of droplets at the boundary. Finally, we successfully achieved protein staining experiments on this platform and developed a customized smartphone application for the accurate detection of protein concentration. This innovative closed-open DMF system provides new possibilities for future applications in real-time biological sample processing and detection.

Integration of complementary split-ring resonators into digital microfluidics for manipulation and direct sensing of droplet composition.

This paper demonstrates the integration of complementary split-ring resonators (CSSRs) with digital microfluidics (DMF) sample manipulation for passive, on-chip radio-frequency (RF) sensing. Integration is accomplished by having the DMF and the RF-sensing components share the same ground plane: by designing the RF-resonant openings directly into the ground plane of a DMF device, both droplet motion and sensing are achieved, adding a new on-board detection mode for use in DMF. The system was modelled to determine basic features and to balance various factors that need to be optimized to maintain both functionalities (DMF-enabled droplet movement and RF detection) on the same chip. Simulated and experimental results show good agreement. Using a portable measurement setup, the integrated CSSR sensor was used to effectively identify a series of DMF-generated drops of ethanol-water mixtures of different compositions by measuring the resonant frequency of the CSSR. In addition, we show that a binary solvent system (ethanol/water mixtures) results in consistent changes in the measured spectrum in response to changes in concentration, indicating that the sensor can distinguish not only between pure solvents from each other, but also between mixtures of varied compositions. We anticipate that this system can be refined further to enable additional applications and detection modes for DMF systems and other portable sensing platforms alike. This proof-of-principle study demonstrates that the integrated DMF-CSSR sensor provides a new platform for monitoring and characterization of liquids with high sensitivity and low consumption of materials, and opens the way for new and exciting applications of RF sensing in microfluidics.

Dual magnet solenoid actuator: Practical applications

We demonstrate several practical applications of a novel linear solenoid actuator design previously proposed to create a solenoid micropump which can be used in portable microfluidic systems. The proposed solenoid actuator design is extended to a system that can apply a constant force with a longer stroke and apply a higher force. It also demonstrates effective mini-train operation with easy and simple control, a spring-loaded linear servo system that solves the classic non-linear control problem, and droplet dispensing with accurate and fast control, showing its potential as a compact microfluidic solenoid micropump capable of precisely handling small volumes of liquid. Applications to digital microfluidic and flow-focusing microchannel systems have also been successfully demonstrated. We hope that this study will promote the development of various actuators for microfluidics and contribute to the broader application of microfluidics in more fields.

Sub-5-Minute Ultrafast PCR using Digital Microfluidics

The development of a rapid and reliable polymerase chain reaction (PCR) method for point-of-care (POC) diagnosis is crucial for the timely identification of pathogens. Microfluidics, which involves the manipulation of small volumes of fluidic samples, has been shown to be an ideal approach for POC analysis. Among the various microfluidic platforms available, digital microfluidics (DMF) offers high degree of configurability in manipulating μL/nL-scale liquid and achieving automation. However, the successful implementation of ultrafast PCR on DMF platforms presents challenges due to inherent system instability. In this study, we developed a robust and ultrafast PCR in 3.7–5 min with a detection sensitivity comparable to conventional PCR. Specifically, the implementation of the pincer heating scheme homogenises the temperature within a drop. The utilization of a μm-scale porous hydrophobic membrane suppresses the formation of bubbles under high temperatures. The design of a groove around the high-temperature zone effectively mitigates the temperature interference. The integration of a soluble sensor into the droplets provides an accurate and instant in-drop temperature sensing. We envision that the fast, robust, sensitive, and automatic DMF system will empower the POC testing for infectious diseases.

Magnetic digital microfluidic manipulation with mobile surface energy traps capable of releasable droplet dispensing

Magnetic digital microfluidics (MDM) is a technology that manipulates droplets on an open surface with magnetic force for lab-on-a-chip (LOC) applications. MDM is capable of dispensing droplets of desired volume using surface energy traps (SETs); however, the daughter droplets dispensed by this approach cannot be released from the SET for subsequent microfluidic manipulation. This is a major disadvantage compared to the electrowetting-on-dielectrics (EWOD)-based digital microfluidics which is capable of releasable droplet dispensing. In this work, a polydopamine-coated thin magnet is introduced to MDM as a mobile SET for droplet manipulation, and for the first time, releasable droplet dispensing is accomplished on the MDM platform. In addition, the mobile SET is capable of all-terrain droplet transport regardless of the surface condition and rapid droplet transport via a unique droplet slingshot mechanism. The mobile SET-enabled MDM platform is able to match the functionality of the EWOD-based digital microfluidic system in terms of droplet dispensing and release, which will significantly expand the applicability and acceptability of MDM technology in biosensing, in vitro diagnostics, and many other biomedical applications.

Probing the kinetics of chemical reactions in ultra-small droplet samples using digital microfluidic nuclear magnetic resonance spectroscopy

In this study, we present a digital microfluidic (DMF) system that connects analyte droplets with planar microcoils in a high-field nuclear magnetic resonance (NMR) spectrometer for in situ photoreaction analysis. Transparent indium tin oxide glass was employed in fabricating the DMF electrode chips. NMR spectroscopy was utilized for its superior sensitivity and ability to detect small changes in molecular structure, making it highly advantageous for chemical analysis. Utilizing Computer Simulation Technology Studio Suite, we designed a plane annular microstrip coil that accurately matches droplet shape to optimize detection sensitivity and increase filling factor. An advanced DMF control circuit remotely manages droplet samples and facilitates precise mixing for in situ reaction analysis. Our results demonstrate improved sensitivity in the in situ experimental analysis, such as the aldehyde amine reaction (sodium benzaldehyde-2-sulfonate solution and aniline solution) and photopolymerization reaction (polyethylene glycol ester). The integration of DMF and NMR technologies provides a promising approach for increasing efficiency and accuracy in chemical analysis.

A digital microfluidic single-cell manipulation system optimized by extending-depth-of-field device

Microfluidic systems have been widely utilized in high-throughput biology analysis, but the difficulties in liquid manipulation and cell cultivation limit its application. This work has developed a new digital microfluidic (DMF) system for on-demand droplet control. By adopting an extending-depth-of-field (EDoF) phase modulator to the optical system, the entire depth of the microfluidic channel can be covered in one image without any refocusing process, ensuring that 95% of the particles in the droplet are captured within three shots together with shaking processes. With this system, suspension droplets are generated and droplets containing only one yeast cell can be recognized, then each single cell is cultured in the array of the chip. By observing their growth in cell numbers and the green fluorescence protein (GFP) production via fluorescence imaging, the single cell with the highest production can be identified. The results have proved the heterogeneity of yeast cells, and showed that the combined system can be applied for rapid single-cell sorting, cultivation, and analysis.

Enhanced absorbance detection system for online bacterial monitoring in digital microfluidics.

We report a fully integrated digital microfluidic absorbance detection system with an enhanced sensitivity for online bacterial monitoring. Through a 100 μm gap in the chip, our optical detection system has a detection sensitivity for a BCA protein concentration of 0.1 mg mL-1. The absorbance detection limit of our system is 1.4 × 10-3 OD units, which is one order of magnitude better than that of the existing studies. The system's linear region is 0.1-7 mg mL-1, and the dynamic range is 0-25 mg mL-1. We measured the growth curves of wild-type and E. coli transformed with resistance plasmids and mixed at different ratios on chip. We sorted out the bacterial species including highly viable single cells based on the difference in absorbance data of growth curves. We explored the changes in the growth curves of E. coli under different concentrations of resistant media. In addition, we successfully screened for the optimal growth environment of the bacteria, in which the growth rate of PET30a-DH5α (in a medium with 33 μg mL-1 kanamycin resistance) was significantly higher than that of a 1 mg mL-1 resistance medium. In conclusion, the enhanced digital microfluidic absorbance detection system exhibits exceptional sensitivity, enabling precise bacterial monitoring and growth curve analysis, while also laying the foundation for DMF-based automated bioresearch platforms, thus advancing research in the life sciences.

Digital Microfluidics with an On-Chip Drug Dispenser for Single or Combinational Drug Screening.

Rapid and accurate cancer drug screening is of great importance in precision medicine. However, the limited amount of tumor biopsy samples has hindered the application of traditional drug screening methods with microwell plates for individual patients. A microfluidic system provides an ideal platform for handling trace amounts of samples. This emerging platform has a good role in nucleic acid-related and cell related assays. Nevertheless, convenient drug dispensing remains a challenge for clinical on-chip cancer drug screening. Similar sized droplets are merged to add drugs for a desired screened concentration which significantly complicated the on-chip drug dispensing protocols. Here, we introduce a novel digital microfluidic system with a specially structured electrode (a drug dispenser) to dispense drugs by droplet electro-ejection under a high-voltage actuation signal, which can be conveniently adjusted by external electric controls. With this system, the screened drug concentrations span up to four orders of magnitude with small sample consumption. Various amounts of drugs can be delivered to the cell sample with desired amount in a flexible electric control. Moreover, single drug or combinatorial multidrug on-chip screening can be readily achieved. The drug response of normal MCF-10A breast cells and MDA-MB-231 breast tumor cells to two chemotherapeutic substances, cisplatin (Cis) and epirubicin (EP), was tested individually and in combination for proof-of-principle verification. The comparable on-chip and off-chip results confirmed the feasibility of our innovative DMF system for cancer drug screening.

Combining sensors and actuators with electrowetting-on-dielectric (EWOD): advanced digital microfluidic systems for biomedical applications.

The concept of digital microfluidics (DMF) enables highly flexible and precise droplet manipulation at a picoliter scale, making DMF a promising approach to realize integrated, miniaturized "lab-on-a-chip" (LOC) systems for research and clinical purposes. Owing to its simplicity and effectiveness, electrowetting-on-dielectric (EWOD) is one of the most commonly studied and applied effects to implement DMF. However, complex biomedical assays usually require more sophisticated sample handling and detection capabilities than basic EWOD manipulation. Alternatively, combined systems integrating EWOD actuators and other fluidic handling techniques are essential for bringing DMF into practical use. In this paper, we briefly review the main approaches for the integration/combination of EWOD with other microfluidic manipulation methods or additional external fields for specified biomedical applications. The form of integration ranges from independently operating sub-systems to fully coupled hybrid actuators. The corresponding biomedical applications of these works are also summarized to illustrate the significance of these innovative combination attempts.

A low-temperature digital microfluidic system used for protein-protein interaction detection.

The occurrence, development and prediction of various biological processes and diseases are inseparable from the protein-protein interaction (PPI), so it is extremely meaningful to perfect PPI networks. However, shortcomings of traditional detection methods, such as protein degradation, long detection time, complex operation, poor automation and high cost, restrict the rapid development of PPI networks. Here, a low-temperature digital microfluidic (LTDMF) system-based PPI detection box (LTDMF-PPI-Box) was developed to achieve rapid, lossless and efficient PPI detection. It consists of a PMMA shell, LTDMF-PPI and an integrated temperature control system. LTDMF reduces the PPI detection time from tens of hours to 1.5 hours by programmatically controlling the movement of droplets. Moreover, an integrated thermoelectric cooler (TEC) ensures an operating temperature of 4 °C, resulting in a protein protection up to 90%. The interaction between RILP protein and Rab26 protein which has a close connection to insulin secretion was demonstrated as a prototype to illustrate the feasibility of the LTDMF-PPI-Box. LTDMF with automation characteristics is capable of meeting the requirement of high-throughput screening of interacting proteins; therefore, the LTDMF-PPI-Box is expected to accelerate the establishment of the PPI network in the future.

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, β-glucanases, and β-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.