Droplet microfluidics has revolutionized single-cell analysis across various omics levels, finding increasing use in diverse biomedical fields. While droplet-based assays are commercially successful (e.g., ddPCR, Chromium), current optical sensing methods predominantly rely on fluorescence microscopy. This approach presents significant limitations: high cost, complexity, limited flexibility, and restricted multiparametric capabilities. These limitations hinder widespread adoption of droplet microfluidics in both research and point-of-care diagnostics. Flow cytometry, known for its high-throughput multiparametric analysis, offers a promising alternative. However, adapting flow cytometry principles to analyze cells encapsulated within droplets remains challenging due to optical complexities introduced by the different refractive indices and droplet size. While some studies have demonstrated detection of scatter and fluorescence from droplets, they lack the sensitivity and multiparametric capabilities needed for optimal high-throughput analysis. On-chip optofluidics, integrating micro-optical components with microfluidic chips, offers a potential solution. In particular, optical fibers are attractive due to their cost-effectiveness, ease of integration, and compatibility with various wavelengths. This paper presents OptiDrop, a platform integrating optical fibers onto a microfluidic chip for multiparametric scatter and fluorescence analysis of droplet contents with single-cell resolution. The system uses a single laser and multiple photomultiplier tubes (PMTs) for signal detection, offering a customizable and cost-effective solution.

The introduction extensively reviews existing techniques for droplet-based single-cell analysis and their limitations, particularly concerning optical detection. It highlights the challenges of microscopy-based approaches and the potential of flow cytometry for high-throughput analysis. Existing attempts to miniaturize flow cytometry for on-chip applications are discussed, emphasizing the challenges of adapting these techniques to droplet-based systems. The literature review focuses on the advantages of using on-chip optical fibers for light collection and detection in microfluidic systems, setting the stage for the proposed OptiDrop platform.

The OptiDrop platform consists of a microfluidic chip with a flow-focusing junction for generating monodisperse droplets, optical fibers for laser illumination and signal collection, PMTs for signal detection, and a pulse counter for data acquisition. The microfluidic chip incorporates coplanar grooves to house optical fibers at specific angles (45°) to the incident laser fiber. A 488 nm laser provides illumination, and scattered and fluorescent light is collected by the fibers and directed to PMTs equipped with appropriate filters. A pulse counter records the PMT signals, generating data for real-time visualization and analysis. The system is compact and avoids free-space optical components, reducing cost and complexity. The platform's performance was characterized using standard dyes and fluorescent intensity beads. A biological application was demonstrated by analyzing the expression of MHC I and II proteins on the surface of mouse embryonic fibroblast (MEF) cells in response to interferon-gamma (IFNγ) stimulation. The refractive index of the oil was manipulated by adding 3-bromobenzotrifluoride to control the strength of the scatter signal.

OptiDrop successfully detected both scattered and multiplexed fluorescence signals from individual droplets with single-cell resolution. The droplet scatter signal showed a characteristic two-peak pattern, with the distance between peaks (dwell time) being determined by the oil flow rate and independent of the aqueous flow rate. The inter-droplet distance was primarily influenced by the aqueous flow rate. Modifying the oil's refractive index using 3-bromobenzotrifluoride enabled control over the scatter signal intensity. The fluorescence signal showed a linear relationship with dye concentration and was affected by the oil flow rate (dwell time). The platform achieved high sensitivity, enabling the detection of differences in MHC I and MHC II expression on MEF cells in response to IFNγ stimulation. The cost of the entire setup is estimated at approximately USD 12,500, with potential for significant cost reduction in larger-scale production.

The OptiDrop platform successfully addresses the limitations of existing methods for optical detection in droplet microfluidics. Its on-chip fiber optic design enables simplified, miniaturized, and cost-effective multiparametric analysis with single-cell resolution. The successful detection of MHC protein expression demonstrates the platform's biological relevance and potential for high-throughput single-cell analysis. The platform's flexibility and customizability make it suitable for a broad range of applications in research and diagnostics. The demonstrated cost reduction compared to microscopy-based methods could significantly broaden the accessibility of sophisticated single-cell analysis techniques.

OptiDrop presents a significant advancement in droplet microfluidic optical sensing. Its combination of on-chip fiber optics, multiplexed detection, and single-cell resolution offers a powerful and cost-effective tool for various applications. Future work could focus on integrating additional functionalities, such as droplet sorting or on-chip sample preparation, and further reducing the system's cost and size for widespread adoption.

The current prototype is limited by the fluidics and the larger droplet sizes, affecting the maximum achievable droplet frequency. Further optimization of the microfluidic chip design could potentially increase this frequency. The study's biological application focused on a specific example of MHC protein expression; further validation with different cell types and biomarkers is warranted.