Advances in High-Accuracy, High-Throughput Droplet Microfluidic Sorting Using Dual Fluorescence and Size-Based Selection

In droplet microfluidics, high-throughput screening is critical for analyzing large cellular or molecular libraries at single-cell resolution. However, achieving high sorting accuracy remains a challenge, especially with the droplet size variability inherent in multi-step assays or extended incubations. Conventional microfluidic sorting methods, such as those relying on dielectrophoresis (DEP), can result in high error rates—an issue that intensifies when large-scale sorting is required. False positives and negatives resulting from size inconsistencies impose a substantial validation burden, especially in assays involving millions of droplets.

“Here, we present NOVAsort (Next-generation Opto-Volume-based Accurate droplet sorter), a device capable of discerning droplets based on both size and fluorescence intensity. With a 1000- and 10,000-fold reduction in false positives and false negatives, respectively. NOVAsort addresses the challenges of conventional droplet sorting approaches and sets standards for accuracy and throughput in droplet microfluidic assays.“, the authors explained.

To address these limitations, researchers developed NOVAsort, an innovative microfluidic device for opto-volume-based droplet sorting designed to accurately distinguish droplets based on both size and fluorescence. By targeting droplets that meet both parameters, the proposed microfluidic chip achieves a reduction in sorting errors, boasting a 1000-fold decrease in false positives and a 10,000-fold decrease in false negatives. This approach marks an improvement over traditional sorting techniques that can struggle with the polydispersity of droplet populations.

The NOVAsort system operates using interdigitated electrodes (IDEs) situated at the channel base, generating highly localized electric fields that manipulate droplets within a specified size range. The system uses a stepwise separation strategy, employing multiple size-based droplet filtering stages to remove large and small non-target droplets. Fluorescence detection further refines sorting, with IDE activation triggered only for fluorescent droplets of the correct size. Additionally, a feedback loop ensures IDE activation is precisely timed, guiding the droplet smoothly to the appropriate outlet without unwanted droplet disruption. This technique enables the microfluidic device to handle a range of droplet sizes while maintaining high sorting fidelity even under continuous, high-throughput conditions.

The proposed microfluidic platform was reported to demonstrate high sorting accuracy across various tests, achieving over 99% accuracy and a throughput of 30 Hz. The system successfully separated target droplets from a highly polydisperse input population, maintaining this accuracy despite changes in droplet-to-droplet spacing and even when subjected to different droplet sizes. In an in-droplet IVTT workflow, NOVAsort’s accuracy greatly enhanced assay results, improving the yield of correctly sorted target droplets by 217% over conventional methods. The system was further tested against the conventional linear sorter in negative-sorting scenarios, where NOVAsort maintained high accuracy (0.8% error rate), significantly outperforming the linear sorter.

This microfluidic technology represents an advance in droplet microfluidic sorting, overcoming long-standing limitations associated with droplet size variability and sorting accuracy. Its dual-parameter approach allows NOVAsort to achieve unparalleled accuracy and throughput, making it a powerful tool for high-throughput applications such as in vitro transcription/translation assays and other screening protocols that require precise sorting of large droplet libraries. This platform paves the way for reliable and efficient droplet-based analysis, offering substantial improvements in experimental accuracy and throughput for complex biological studies.

“In summary, NOVAsort allows the recovery of target-size, fluorescence-positive droplets from either an ideal monodisperse or a highly polydisperse droplet library. Even in the case of high input droplet polydispersity, the accuracy of the system was greater than 99% at throughputs up to 235 Hz. The sorter also shows good tolerance to insufficient droplet-to-droplet spacing. The accuracy remained high (96.6%) even when the spacing was very small (200 µm).“, the authors concluded.

Figures are reproduced from Zhang, H., Gupte, R., Li, Y. et al. NOVAsort for error-free droplet microfluidics. Nat Commun 15, 9444 (2024). https://doi.org/10.1038/s41467-024-52932-z  under a CC BY 4.0 Attribution 4.0 International license.

Read the original article: NOVAsort for error-free droplet microfluidics

For more insights into the world of microfluidics and its burgeoning applications in biomedical research, stay tuned to our blog and explore the limitless possibilities that this technology unfolds. If you need high quality microfluidics chip for your experiments, do not hesitate to contact us.