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 10.65 cm2, 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.

AM-DMF-SCP: Integrated Single-Cell Proteomics Analysis on an Active Matrix Digital Microfluidic Chip.

Single-cell proteomics offers unparalleled insights into cellular diversity and molecular mechanisms, enabling a deeper understanding of complex biological processes at the individual cell level. Here, we develop an integrated sample processing on an active-matrix digital microfluidic chip for single-cell proteomics (AM-DMF-SCP). Employing the AM-DMF-SCP approach and data-independent acquisition (DIA), we identify an average of 2258 protein groups in single HeLa cells within 15 min of the liquid chromatography gradient. We performed comparative analyses of three tumor cell lines: HeLa, A549, and HepG2, and machine learning was utilized to identify the unique features of these cell lines. Applying the AM-DMF-SCP to characterize the proteomes of a third-generation EGFR inhibitor, ASK120067-resistant cells (67R) and their parental NCI-H1975 cells, we observed a potential correlation between elevated VIM expression and 67R resistance, which is consistent with the findings from bulk sample analyses. These results suggest that AM-DMF-SCP is an automated, robust, and sensitive platform for single-cell proteomics and demonstrate the potential for providing valuable insights into cellular mechanisms.

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.

Polar coordinate active-matrix digital microfluidics for high-resolution concentration gradient generation.

Automated concentration gradient generation is one of the most important applications of lab-on-a-chip devices. Digital microfluidics is a unique platform that can effectively achieve digitalized gradient concentration preparation. However, the dynamic range and concentration resolution of the prepared samples heavily rely on the size and the number of effective electrodes. In this work, we report an active-matrix digital microfluidic device with polar coordinate electrode arrangement. The device contains 33 different electrode sizes, generating digital droplets of different volumes. To compare with the conventional rectangular coordinate arrangement with a similar electrode number, this work shows an approximately 19 times resolution enhancement for the achievable concentration gradient. We characterized the stability and uniformity of droplets generated by electrodes of different sizes, and the coefficient of variation of stable droplets was less than 3%. The fluorescent nanomaterial's concentration quantification and glucose concentration characterization experiments were also conducted, and the correlation coefficients for the linearities were all above 0.99.

AIEgens-enhanced rapid sensitive immunofluorescent assay for SARS-CoV-2 with digital microfluidics.

BACKGROUND: Sensitive and rapid antigen detection is critical for the diagnosis and treatment of infectious diseases, but conventional ELISAs including chemiluminescence-based assays are limited in sensitivity and require many operation steps. Fluorescence immunoassays are fast and convenient but often show limited sensitivity and dynamic range. RESULTS: To address the need, an aggregation-induced emission fluorgens (AIEgens) enhanced immunofluorescent assay with beads-based quantification on the digital microfluidic (DMF) platform was developed. Portable DMF devices and chips with small electrodes were fabricated, capable of manipulating droplets within 100 nL and boosting the reaction efficiency. AIEgen nanoparticles (NPs) with high fluorescence and photostability were synthesized to enhance the test sensitivity and detection range. The integration of AIEgen probes, transparent DMF chip design, and the large magnetic beads (10 µm) as capture agents enabled rapid and direct image-taking and signal calculation of the test result. The performance of this platform was demonstrated by point-of-care quantification of SARS-CoV-2 nucleocapsid (N) protein. Within 25 min, a limit of detection of 5.08 pg mL-1 and a limit of quantification of 8.91 pg mL-1 can be achieved using <1 µL sample. The system showed high reproducibility across the wide dynamic range (10-105 pg mL-1), with the coefficient of variance ranging from 2.6% to 9.8%. SIGNIFICANCE: This rapid, sensitive AIEgens-enhanced immunofluorescent assay on the DMF platform showed simplified reaction steps and improved performance, providing insight into the small-volume point-of-care testing of different biomarkers in research and clinical applications.

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.

Advanced design and applications of digital microfluidics in biomedical fields: An update of recent progress.

Significant breakthroughs have been made in digital microfluidic (DMF)-based technologies over the past decades. DMF technology has attracted great interest in bioassays depending on automatic microscale liquid manipulations and complicated multi-step processing. In this review, the recent advances of DMF platforms in the biomedical field were summarized, focusing on the integrated design and applications of the DMF system. Firstly, the electrowetting-on-dielectric principle, fabrication of DMF chips, and commercialization of the DMF system were elaborated. Then, the updated droplets and magnetic beads manipulation strategies with DMF were explored. DMF-based biomedical applications were comprehensively discussed, including automated sample preparation strategies, immunoassays, molecular diagnosis, blood processing/testing, and microbe analysis. Emerging applications such as enzyme activity assessment and DNA storage were also explored. The performance of each bioassay was compared and discussed, providing insight into the novel design and applications of the DMF technology. Finally, the advantages, challenges, and future trends of DMF systems were systematically summarized, demonstrating new perspectives on the extensive applications of DMF in basic research and commercialization.

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.

Large-Area Electronics-Enabled High-Resolution Digital Microfluidics for Parallel Single-Cell Manipulation.

Large-area electronics as switching elements are an ideal option for electrode-array-based digital microfluidics. With support of highly scalable thin-film semiconductor technology, high-resolution digital droplets (diameter around 100 µm) containing single-cell samples can be manipulated freely on a two-dimensional plane with programmable addressing logic. In addition, single-cell generation and manipulation as foundations for single-cell research demand ease of operation, multifunctionality, and accurate tools. In this work, we reported an active-matrix digital microfluidic platform for single-cell generation and manipulation. The active device contained 26,368 electrodes that could be independently addressed to perform parallel and simultaneous droplet generation and achieved single-cell manipulation. We demonstrate a high-resolution digital droplet generation with a droplet volume limit of 500 pL and show the continuous and stable movement of droplet-contained cells for over 1 h. Furthermore, the success rate of single droplet formation was higher than 98%, generating tens of single cells within 10 s. In addition, a pristine single-cell generation rate of 29% was achieved without further selection procedures, and the droplets containing single cells could then be tested for on-chip cell culturing. After 20 h of culturing, about 12.5% of the single cells showed cell proliferation.

All-in-One Digital Microfluidics System for Molecular Diagnosis with Loop-Mediated Isothermal Amplification.

In this study, an "all-in-one" digital microfluidics (DMF) system was developed for automatic and rapid molecular diagnosis and integrated with magnetic bead-based nucleic acid extraction, loop-mediated isothermal amplification (LAMP), and real-time optical signal monitoring. First, we performed on- and off-chip comparison experiments for the magnetic bead nucleic acid extraction module and LAMP amplification function. The extraction efficiency for the on-chip test was comparable to that of conventional off-chip methods. The processing time for the automatic on-chip workflow was only 23 min, which was less than that of the conventional methods of 28 min 45 s. Meanwhile, the number of samples used in on-chip experiments was significantly smaller than that used in off-chip experiments; only 5 µL of E. coli samples was required for nucleic acid extraction, and 1 µL of the nucleic acid template was needed for the amplification reaction. In addition, we selected SARS-CoV-2 nucleic acid reference materials for the nucleic acid detection experiment, demonstrating a limit of detection of 10 copies/µL. The proposed "all-in-one" DMF system provides an on-site "sample to answer" time of approximately 60 min, which can be a powerful tool for point-of-care molecular diagnostics.

A Review of Optical Imaging Technologies for Microfluidics.

Microfluidics can precisely control and manipulate micro-scale fluids, and are also known as lab-on-a-chip or micro total analysis systems. Microfluidics have huge application potential in biology, chemistry, and medicine, among other fields. Coupled with a suitable detection system, the detection and analysis of small-volume and low-concentration samples can be completed. This paper reviews an optical imaging system combined with microfluidics, including bright-field microscopy, chemiluminescence imaging, spectrum-based microscopy imaging, and fluorescence-based microscopy imaging. At the end of the article, we summarize the advantages and disadvantages of each imaging technology.

"One-to-three" droplet generation in digital microfluidics for parallel chemiluminescence immunoassays.

In digital microfluidics, droplet generation is a fundamental operation for quantitative liquid manipulation. The generation of well-defined micro-droplets on a chip with restricted device geometries has become a real obstacle for digital microfluidics platforms to be used in parallel for in vitro diagnostic applications. Here, we propose a "one-to-three" droplet splitting technique that is able to generate sub-microlitre droplets beyond the "well-known" geometry limit in electrowetting-on-dielectric digital microfluidics. Accordingly, we realized an on-chip magnetic bead chemiluminescence immunoassay for parallel detection with the "one-to-three" technique. With the help of the generated micro droplets, we were able to retain the magnetic beads by a significantly reduced magnetic force. We have shown the detection of five B-type natriuretic peptide analyte samples on a single chip for around 10 minutes. The correlation coefficient of the calibration curve was 0.9942, and the detection limit was lower than 5 pg mL-1.

Trends and recent development of the microelectrode arrays (MEAs).

In this paper, we reviewed the history of microelectrode arrays (MEAs), compared different microfabrication techniques applied to modern MEAs in terms of their material characters, device properties and application scenarios. Then we discussed the biocompatibility of different MEAs as well as corresponding strategy of improvement. At last, we analyzed the growing trend of MEAs' technical route, expected application of MEAs in the field of Electrical impedance tomography (EIT).

Microfluidic chip enabled one-step synthesis of biofunctionalized CuInS2/ZnS quantum dots.

Biofunctionalized quantum dots (QDs) are effective target fluorescent labels for bioimaging. However, conventional synthesis of biofunctionalized I-III-VI core-shell CuInS2/ZnS QDs requires complex bench-top operations, resulting in limited product performance and variety, and is not amenable to a 'one-step' approach. In this work, we have successfully demonstrated a fully automated method for preparing denatured bovine serum albumin (dBSA)-CuInS2/ZnS QDs by introducing microfluidic (MF) chips to synthesize biofunctionalized QDs, hence establishing a 'one-step' procedure. We have also studied and optimized the reaction synthesis parameters. The emission wavelength of the dBSA-CuInS2/ZnS QDs is located in the near-infrared range and can be tuned from 650 to 750 nm by simply varying the reaction parameters. In addition, the 'one-step'-synthesized dBSA-CuInS2/ZnS QDs have a long average fluorescence lifetime of 153.76 ns and a small particle size of 5 ± 2 nm. To demonstrate the applicability of the 'one-step'-synthesized dBSA-CuInS2/ZnS QDs in bioimaging studies, we modified the QDs with folic acid and hyaluronic acid, and then performed target bioimaging and cytotoxicity tests on macrophages, liver cancer cells and pancreatic cancer cells. The cell images show that the red emission signals originate from the QDs, which indicates that the dBSA-CuInS2/ZnS QDs prepared by the MF approach are suitable optical contrast agents for target bioimaging. This 'one-step' MF-based QD synthesis approach could serve as a rapid, cost-effective, and small-scale nanocrystal production platform for complex QD formulations for a wide range of bioapplications.

An Impedance Sensing Platform for Monitoring Heterogeneous Connectivity and Diagnostics in Lab-on-a-Chip Systems.

Reliable hardware connectivity is vital in heterogeneous integrated systems. For example, in digital microfluidics lab-on-a-chip systems, there are hundreds of physical connections required between a microelectromechanical fabricated device and the driving system that can be remotely located on a printed circuit board. Unfortunately, the connection reliability cannot be checked or monitored by vision-based detection methods that are commonly used in the semiconductor industry. Therefore, a sensing platform that can be seamlessly integrated into existing digital microfluidics systems and provide real-time monitoring of multiconnectivity is highly desired. Here, we report an impedance sensing platform that can provide fast detection of a single physical connection in timescales of milliseconds. Once connectivity is established, the same setup can be used to determine the droplet location. The sensing system can be scaled up to support multiple channels or applied to other heterogeneously integrated systems that require real-time monitoring and diagnostics of multiconnectivity systems.

A floating top-electrode electrowetting-on-dielectric system.

Herein, we describe a novel device configuration for a double-plate electrowetting-on-dielectric system with a floating top-electrode. As a conventional double-plate EWOD device requires a grounded electrode on the top-plate, it will cause additional fabrication complicity and cost during the encapsulation process. In this work, we found that by carefully designing the electrode arrangement and configuring the driving electronic circuit, the droplet driving force can be maintained with a floating electrode on the top-plate. This can provide the possibilities to integrate additional electrical or electrochemical sensing functions on the top-plate. We use both finite element analysis and the fabricated system to validate the theory, and the results indicate that floating top-electrode EWOD systems are highly reliable and reproducible once the design considerations are fully met.

Cigarette Smoke Induced Lung Barrier Dysfunction, EMT, and Tissue Remodeling: A Possible Link between COPD and Lung Cancer.

Chronic obstructive pulmonary disease (COPD) and lung cancer, closely related to smoking, are major lung diseases affecting millions of individuals worldwide. The generated gas mixture of smoking is proved to contain about 4,500 components such as carbon monoxide, nicotine, oxidants, fine particulate matter, and aldehydes. These components were considered to be the principle factor driving the pathogenesis and progression of pulmonary disease. A large proportion of lung cancer patients showed a history of COPD, which demonstrated that there might be a close relationship between COPD and lung cancer. In the early stages of smoking, lung barrier provoked protective response and DNA repair are likely to suppress these changes to a certain extent. In the presence of long-term smoking exposure, these mechanisms seem to be malfunctioned and lead to disease progression. The infiltration of inflammatory cells to mucosa, submucosa, and glandular tissue caused by inhaled cigarette smoke is responsible for the destruction of matrix, blood supply shortage, and epithelial cell death. Conversely, cancer cells have the capacity to modulate the proliferation of epithelial cells and produce of new vascular networks. Comprehension understanding of mechanisms responsible for both pathologies is necessary for the prevention and treatment of COPD and lung cancer. In this review, we will summarize related articles and give a glance of possible mechanism between cigarette smoking induced COPD and lung cancer.

Disposable impedance-based immunosensor array with direct-laser writing platform.

Immunoassay is a powerful technique to identify and quantify biological molecules, which base on the specificity and selectivity of antigen-antibody interaction. Impedance-based immunosensor has recently shown a great potential to provide rapid and label-free detections. However, the conventional impedance-based immunosensors rely on dedicated electrochemical measurement interface which involves expensive fabrication procedures such as gold deposition and photolithography. In this work, we propose an ultra-low-cost and high processing efficiency platform for impedance-based immunosensing. With effortless operations of direct-laser-writing, an impedance-based immunoassay can be fabricated within 5 min in standard laboratories. The as-fabricated devices have shown great stability and a high device-to-device uniformity. In order to further validate impedance sensing system's performance, finite element analysis and impedance equivalent model analysis were performed. The measured data was consistent with the simulation results. With the standard gold electrodes surface bio-functionalization procedures, the disposable immunoassay can detect anti-IgG down to 10 ng/ml.

Printed subthreshold organic transistors operating at high gain and ultralow power.

Overcoming the trade-offs among power consumption, fabrication cost, and signal amplification has been a long-standing issue for wearable electronics. We report a high-gain, fully inkjet-printed Schottky barrier organic thin-film transistor amplifier circuit. The transistor signal amplification efficiency is 38.2 siemens per ampere, which is near the theoretical thermionic limit, with an ultralow power consumption of <1 nanowatt. The use of a Schottky barrier for the source gave the transistor geometry-independent electrical characteristics and accommodated the large dimensional variation in inkjet-printed features. These transistors exhibited good reliability with negligible threshold-voltage shift. We demonstrated this capability with an ultralow-power high-gain amplifier for the detection of electrophysiological signals and showed a signal-to-noise ratio of >60 decibels and noise voltage of <0.3 microvolt per hertz1/2 at 100 hertz.