Although digital detection of nucleic acids has been achieved by amplification of single templates in uniform microfluidic droplets and widely used for genetic analysis, droplet-based digital detection of proteins has rarely been reported, largely due to the lack of an efficient target amplification method for protein in droplets. Here, we report a key step towards digital detection of proteins using a highly parallel microfluidic droplet approach for single enzyme molecule detection in picoliter droplets via enzyme catalyzed signal amplification. An integrated microfluidic chip was designed for high throughput uniform droplet generation, monolayer droplet collection, incubation, detection, and release. Single β-galatosidase (β-Gal) molecules and the fluorogenic substrate fluorescein di-β-D-galactopyranoside were injected from two separated inlets to form uniform 20 μm droplets in fluorinated oil at a frequency of 6.6 kHz. About 200 000 droplets were captured as a monolayer in a capture well on-chip for subsequent imaging detection. A series of β-Gal solutions at different concentrations were analyzed at the single-molecule level. With no enzyme present, no droplets were found to fluoresce, while brightly fluorescent droplets were observed under single-enzyme molecule conditions. Droplet fluorescence intensity distribution analysis showed that the distribution of enzyme molecules under single-molecule conditions matched well with theoretical prediction, further proving the feasibility of detecting single enzyme molecules in emulsion droplets. Moreover, the population of fluorescent droplets increased as the β-Gal concentration increased. Based on a digital counting method, the measured concentrations of the enzyme were found to match well with input enzyme concentration, establishing the accuracy of the digital detection method for the quantification of β-Gal enzyme molecules. The capability of highly parallel detection of single enzyme molecules in uniform picoliter droplets paves the way to microdroplet based digital detection of proteins.

Single-cell copy number variations (CNVs), major dynamic changes in humans, result in differential levels of gene expression and account for adaptive traits or underlying disease. Single-cell sequencing is needed to reveal these CNVs but has been hindered by single-cell whole-genome amplification (scWGA) bias, leading to inaccurate gene copy number counting. In addition, most of the current scWGA methods are labor intensive, time-consuming, and expensive with limited wide application. Here, we report a unique single-cell whole-genome library preparation approach based on d igital microfluidics for d igital counting of s ingle- c ell C opy N umber V ariation (dd-scCNV Seq). dd-scCNV Seq directly fragments the original single-cell DNA and uses these fragments as templates for amplification. These reduplicative fragments can be filtered computationally to generate the original partitioned unique identified fragments, thereby enabling digital counting of copy number variation. dd-scCNV Seq showed an increase in uniformity in the single-molecule data, leading to more accurate CNV patterns compared to other methods with low-depth sequencing. Benefiting from digital microfluidics, dd-scCNV Seq allows automated liquid handling, precise single-cell isolation, and high-efficiency and low-cost genome library preparation. dd-scCNV Seq will accelerate biological discovery by enabling accurate profiling of copy number variations at single-cell resolution.

Technologies based on digital microfluidics (DMF) have made significant advancements in the automated manipulation of microscale liquids and complex multistep processes. Due to their numerous benefits, such as automation, speed, cost-effectiveness, and minimal sample volume requirements, these systems are particularly well suited for immunoassays. In this review, an overview is provided of diverse DMF manipulation platforms and their applications in immunological analysis. Initially, droplet-driven DMF platforms based on electrowetting on dielectric (EWOD), magnetic manipulation, surface acoustic wave (SAW), and other related technologies are briefly introduced. The preparation of DMF is then described, including material selection, fabrication techniques and droplet generation. Subsequently, a comprehensive account of advancements in the integration of DMF with various immunoassay techniques is offered, encompassing colorimetric, direct chemiluminescence, enzymatic chemiluminescence, electrosensory, and other immunoassays. Ultimately, the potential challenges and future perspectives in this burgeoning field are delved into.

The manipulation of droplets plays a vital role in biomedicine, chemistry, and hydromechanics, especially in microfluidics. Magnetic droplet manipulation has emerged as a prominent and advanced technique in comparison to other modes such as dielectric infiltration, optical radiation, and surface acoustic waves. Its notable progress is attributed to several advantages, including excellent biocompatibility, remote and non-contact control, and instantaneous response. This review provides a comprehensive overview of recent developments in magnetic droplet manipulation and its applications within the biomedical field. Firstly, the discussion involves an examination of the distinctive features associated with droplet manipulation based on both permanent magnet and electromagnet principles, along with a thorough exploration of the influencing factors impacting magnetic droplet manipulation. Additionally, an in-depth review of magnetic actuation mechanisms and various droplet manipulation methods is presented. Furthermore, the article elucidates the biomedical applications of magnetic droplet manipulation, particularly its role in diagnostic assays, drug discovery, and cell culture. Finally, the highlights and challenges of magnetic droplet manipulation in biomedical applications are described in detail.