Forensic case samples collected in sexual assaults typically contain DNA from multiple sources, which complicates short-tandem repeat (STR) profiling. These samples are typically sent to a laboratory to separate the DNA from sperm and non-sperm sources prior to analysis. Here, the automation and miniaturization of these steps using digital microfluidics (DMF) is reported, which may eventually enable processing sexual assault samples outside of the laboratory, at the point of need. When applied to vaginal swab samples collected up to 12 h post-coitus (PC), the new method identifies single-source (male) STR profiles. When applied to samples collected 24-72 h PC, the method identifies mixed STR profiles, suggesting room for improvement and/or potential for data deconvolution. In sum, an automated, miniaturized sample pre-processing method for separating the DNA contained in sexual assault samples is demonstrated. This type of automated processing using DMF, especially when combined with Rapid DNA Analysis, has the potential to be used for processing of sexual assault samples in hospitals, police offices, and other locations outside of the laboratory.

With the continuous expansion of pixel arrays in digital microfluidics (DMF) chips, precise droplet control has emerged as a critical issue requiring detailed consideration. This paper proposes a novel capacitance-based droplet sensing system for thin-film transistor DMF. The proposed circuit features a distinctive inner and outer dual-pixel electrode structure, integrating droplet driving and sensing functionalities. Discharge occurs exclusively at the inner electrode during droplet sensing, effectively addressing droplet perturbation in existing sensing circuits. The circuit employs a novel fan-shaped structure of thin-film transistors. Simulation results show that it can provide a 48 V pixel voltage and demonstrate a sensing voltage difference of over 10 V between deionized water and silicone oil, illustrating its proficiency in droplet driving and accurate sensing. The stability of threshold voltage drift and temperature was also verified for the circuit. The design is tailored for integration into active matrix electrowetting-on-dielectric (AM-EWOD) chips, offering a novel approach to achieve precise closed-loop control of droplets.

PD1/PD-L1 checkpoint inhibitors are at the forefront of cancer immunotherapies. However, the overall response rate remains only 10-30%. Even among initial responders, drug resistance often occurs, which can lead to prolonged use of a futile therapy in the race with the fatal disease. It would be ideal to closely monitor key indicators of patients\' immune responsiveness, such as circulating PD-L1 levels. Traditional PD-L1 detection methods, such as ELISA, are limited in sensitivity and rely on core lab facilities, preventing their use for the regular monitoring. Electrochemical sensors exist as an attractive candidate for point-of-care tool, yet, streamlining multiple processes in a single platform remains a challenge. To overcome this challenge, this work integrated electrochemical sensor arrays into a digital microfluidic device to combine their distinct merits, so that soluble PD-L1 (sPD-L1) molecules can be rapidly detected in a programmed and automated manner. This new platform featured microscale electrochemical sensor arrays modified with electrically conductive 3D matrix, and can detect as low as 1 pg/mL sPD-L1 with high specificity. The sensors also have desired repeatability and can obtain reproducible results on different days. To demonstrate the functionality of the device to process more complex biofluids, we used the device to detect sPD-L1 molecules secreted by human breast cancer cell line in culture media directly and observed 2X increase in signal compared with control experiment. This novel platform holds promise for the close monitoring of sPD-L1 level in human physiological fluids to evaluate the efficacy of PD-1/PD-L1 immunotherapy.

Accurate assessment of phenotypic and genotypic characteristics of bacteria can facilitate comprehensive cataloguing of all the resistance factors for better understanding of antibiotic resistance. However, current methods primarily focus on individual phenotypic or genotypic profiles across different colonies. Here, a Digital microfluidic-based automated assay for whole-genome sequencing of single-antibiotic-resistant bacteria is reported, enabling Genotypic and Phenotypic Analysis of antibiotic-resistant strains (Digital-GPA). Digital-GPA can efficiently isolate and sequence antibiotic-resistant bacteria illuminated by fluorescent D-amino acid (FDAA)-labeling, producing high-quality single-cell amplified genomes (SAGs). This enables identifications of both minor and major mutations, pinpointing substrains with distinctive resistance mechanisms. Digital-GPA can directly process clinical samples to detect and sequence resistant pathogens without bacterial culture, subsequently provide genetic profiles of antibiotic susceptibility, promising to expedite the analysis of hard-to-culture or slow-growing bacteria. Overall, Digital-GPA opens a new avenue for antibiotic resistance analysis by providing accurate and comprehensive molecular profiles of antibiotic resistance at single-cell resolution.

Early and rapid diagnostic of acute myocardial infarction (AMI) during its developing stage is crucial due to its high fatality rate. Heart-type fatty acid binding protein (h-FABP) is an ideal biomarker for the quantitative diagnosis of AMI, surpassing traditional markers such as myoglobin, creatine phosphokinase-MB, and troponin in terms of sensitivity, specificity, and prognostic value. To obtain diagnostic and prognostic information, a precise and fully quantitative measurement of h-FABP is essential, typically achieved through an immunosorbent assay like the enzyme-linked immunosorbent assay. Nevertheless, this method has several limitations, including extended detection time, complex assay procedures, the necessity for skilled technicians, and challenges in implementing automated detection. This research introduces a novel biosensor, utilizing aggregation-induced emission nanoparticles (AIENPs) and integrated with a digital microfluidic (DMF) workstation, designed for the sensitive, rapid, and automated detection of h-FABP in low-volume serum samples. AIENPs and magnetic beads in nanoscale were served as the capture particles and the fluorescent probe, which were linked covalently to anti-h-FABP antibodies respectively. The approach was based on a sandwich immunoassay and performed on a fully automated DMF workstation with assay time by 15 min. We demonstrated the determination of h-FABP in serum samples with detection limit of 0.14 ng/mL using this biosensor under optimal condition. Furthermore, excellent correlations (R2 = 0.9536, n = 50) were obtained between utilizing this biosensor and commercialized ELISA kits in clinical serum detecting. These results demonstrate that our flexible and reliable biosensor is suitable for direct integration into clinical diagnostics, and it is expected to be promising diagnostic tool for early detection and screening tests as well as prognosis evaluation for AMI patients.

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.

The ongoing viral outbreak labeled COVID-19 is spreading rapidly across states and is posing a great threat to public health. Rapid identification of the virus in the population plays a crucial role in isolating the individual and breaking the transmission chain, apart from initiating an appropriate treatment procedure. Here, we discuss an electrowetting-on-dielectric (EWOD) technology that uses a microprocessor-controlled electrode array to merge a possibly infected sample carried by a liquid drop with a drop of a reagent to carry out the testing process. Changes in color occurring during the mixing process of the drops are imaged using a camera.

Proteins as crucial components of cells are responsible for the majority of cellular processes. Sensitive and efficient protein detection enables a more accurate and comprehensive investigation of cellular phenotypes and life activities. Here, a protein sequencing method with high multiplexing, high throughput, high cell utilization, and integration based on digital microfluidics (DMF-Protein-seq) is proposed, which transforms protein information into DNA sequencing readout via DNA-tagged antibodies and labels single cells with unique cell barcodes. In a 184-electrode DMF-Protein-seq system, ≈1800 cells are simultaneously detected per experimental run. The digital microfluidics device harnessing low-adsorbed hydrophobic surface and contaminants-isolated reaction space supports high cell utilization (>90%) and high mapping reads (>90%) with the input cells ranging from 140 to 2000. This system leverages split&pool strategy on the DMF chip for the first time to overcome DMF platform restriction in cell analysis throughput and replace the traditionally tedious bench-top combinatorial barcoding. With the benefits of high efficiency and sensitivity in protein analysis, the system offers great potential for cell classification and drug monitoring based on protein expression at the single-cell level.

Electrowetting-on-dielectric (EWOD), recognized as the most successful electrical droplet actuation method, is essential in diverse applications, ranging from thermal management to microfluidics and water harvesting. Despite significant advances, it remains challenging to achieve repeatability, high speed, and simple circuitry in EWOD-based droplet manipulation on superhydrophobic surfaces. Moreover, its efficient operation typically requires electrode arrays and sophisticated circuit control. Here, a newly observed droplet manipulation phenomenon on superhydrophobic surfaces with orbital EWOD (OEW) is reported. Due to the asymmetric electrowetting force generated on the orbit, flexible and versatile droplet manipulation is facilitated with OEW. It is demonstrated that OEW droplet manipulation on superhydrophobic surfaces exhibits higher speed (up to 5 times faster), enhanced functionality (antigravity), and manipulation of diverse liquids (acid, base, salt, organic, e.g., methyl blue, artificial blood) without contamination, and good durability after 1000 tests. It is envisioned that this robust droplet manipulation strategy using OEW will provide a valuable platform for various processes involving droplets, spanning from microfluidic devices to controllable chemical reactions. The previously unreported droplet manipulation phenomenon and control strategy shown here can potentially upgrade EWOD-based microfluidics, antifogging, anti-icing, dust removal, and beyond.

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%.
CONCLUSIONS: 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.