Development of a PMMA-based Droplet microfluidic device for High-throughput Screening in Health care Applications

Development of a PMMA-based Droplet microfluidic device for High-throughput Screening in Health care Applications | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development of a PMMA-based Droplet microfluidic device for High-throughput Screening in Health care Applications Kaavya Purushothaman, Ashwin Kumar Narasimhan, Gnanavel S This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6102019/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Jun, 2025 Read the published version in Microfluidics and Nanofluidics → Version 1 posted 6 You are reading this latest preprint version Abstract The research focuses on the development of a novel, cost-effective, three-layer droplet microfluidic device fabricated using Polymethyl methacrylate (PMMA) engineered for high-throughput screening in healthcare applications. PMMA offered improved optical transparency, chemical resistance, low absorption, and high scalability. Here, we evolved a T-junction integrated microchannel with a squeezer mechanism for consistent monodisperse droplet generation. Device fabrication was achieved via a laser ablation technique followed by an ethanol-enhanced UV-irradiation method for strong and leak-free bonding between the PMMA layers. The surface properties of the PMMA layer revealed an increased surface energy and uniform wettability. The tensile strength of fabricated PMMA microfluidic devices demonstrated superior bonding strength and structural integrity compared to the existing fabrication methods. The device reliably generated uniform monodisperse droplets up to a 100 ml/hr flow rate, confirming its robustness and suitability for high-throughput screening. Overall, this PMMA-based Microfluidics platform offers a scalable and reliable solution for droplet generation suitable for applications such as drug delivery, single-cell analysis, and diagnostic assays. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Microfluidic technology impacts various applications for analyzing fluid behavior within microscale channels. The ability to manipulate microscale volumes of fluids has revolutionized these fields, enabling high precision, miniaturization, and automation (Nan et al. 2024 ). Between 2018 and x, innovations in Point-of-Care (POC) testing drove an 11.9% compound annual growth rate (CAGR) worldwide (Konwar, 2020). Among the diverse subfields of microfluidics, droplet-based microfluidics has gained significant attention due to its capacity to compartmentalize and manipulate fluids into discrete droplet volumes within the microscale (Bakhshi et al. 2024 ; Abalde-Cela et al. 2018 ). Droplet microfluidics offers a controlled, contamination-free environment, minimizing sample volume and enhancing sustainability, allowing for high-throughput screening (Sözmen, 2021). Droplets are commonly generated using configurations such as T-junctions, flow focusing, and co-flow junctions, each influencing flow behavior and mixing efficiency under controlled flow rates (Elvira et al. 2022 ; Tirandazi and Hidrovo 2017 ; Bardin and Lee 2014 ; Hettiarachchi et al. 2021 ). Xiaoping Li et al. reported a micropump-driven flow-focusing junction for monodispersed droplet generation; however, the minimum droplet size achieved was 360 µm, with diameters deviating from expected values. Islam et al. explored PMMA-based droplet microfluidics using a flow-focusing design to observe DNA-coated particles, achieving droplet sizes of 10–200 µm(Islam, 2018). The results indicate that the design produced polydisperse droplets, reflecting uneven compartmentalization. According to the literature, T-junction microfluidic devices generate droplets in an unsteady manner, often resulting in polydispersity due to inconsistent droplet generation. The design and selection of materials are critical for device performance, reliability, and scalability. While existing designs and fabrication techniques have improved droplet formation, challenges such as device instability, material incompatibility, limited scalability, and reliance on flow-focusing configurations restrict the complexity and multifunctionality of microfluidic platforms. Optimizing design parameters can help produce monodisperse droplets with more effective and precise size control, which is essential for reproducibility and accuracy (Li et al. 2016 ). Traditionally, polymethyl siloxane (PDMS), glass, and silicon are the three primary materials used for microfluidic fabrication. Each material offers various advantages in terms of flexibility, optical clarity, and biocompatibility. However, these materials are unsuitable for large-scale manufacturing due to limitations, including high fabrication costs, limited long-term durability, long processing times, and complex fabrication processes (Sanjay et al. 2020 ; Amirifar et al. 2022 ; Mashiyama et al. 2024 ; Wu et al. 2009). The PDMS material provides compatibility with various solvents and flexibility. However, PDMS faces restrictions in certain applications, particularly in droplet generation, due to its gas permeability, solvent swelling, absorption of small hydrophobic molecules, and poor scalability (Katare, 2020). In contrast, devices made from silicon or glass offer superior mechanical stability and chemical resistance. However, they necessitate sophisticated fabrication techniques such as cleanroom facilities, high temperatures, and complex etching processes, which increase costs and limit scalability, especially for high-throughput screening (Vogt, 2022; Trantidou, 2017). Thermoplastics have recently emerged as an alternative to PDMS and other conventional materials, providing improved functionality, reliability, and scalability (Tan, Loke, and Nguyen 2010). Common thermoplastics used in microfluidic systems include poly(methyl methacrylate) (PMMA), cyclic olefin polymer (COP), polycarbonate (PC), and polystyrene (PS). PMMA is gaining popularity due to its optical clarity, biocompatibility, chemical resistance, hydrophobic surface, and cost-effectiveness. PMMA substrates also facilitate rapid prototyping and large-scale production (Kotz et al. 2020; Ma et al. 2019 ; Shakeri et al. 2022 ; Ng, Tjeung, and Wang 2006). PMMA exhibits low permeability to organic solvents, allows for controllable wettability, and provides greater mechanical robustness, making it suitable for long-term use and ensuring better droplet integrity. Various microfabrication techniques, including hot embossing, injection molding, 3D printing, and laser ablation, are employed to create customized microchannels and patterns in thermoplastic materials (Nasser et al. 2019 ). Hot embossing involves pressing a master mold to fabricate microchannels but necessitates high temperatures above the material’s glass transition temperature, leading to channel deformation and high processing costs, which render it unsuitable for droplet microfluidics (Chen, Li, and Gao 2019 ; Huang et al. 2020 ; Xue et al. 2020 ). CO2 laser ablation employs a high-intensity laser beam to create microchannels and patterns, offering rapid prototyping, low cost, and large-scale production. However, thermal-based ablation can result in heat-affected zones around the channel edges, though this can be mitigated through adjustments to laser power and speed (Fan et al. 2018 ; Perez-Sosa et al. 2022 ; Trinh, Chae, and Lee 2021 ). The selection of bonding methods also plays a significant role in microfluidic device fabrication. Popular methods include thermal fusion bonding, adhesive layer bonding, chemical bonding, microwave bonding, and solvent bonding (Padilha, Giacon, and Bartoli 2017; Xiang, 2023 ). Recent studies indicate that thermal fusion bonding, while effective, requires high temperatures and pressure, which may distort or shrink the channels (Liu et al. 2024 ). Solvent-mediated bonding has been widely utilized, as it employs organic solvents like ethanol, isopropanol, and acetic acid for bonding (Trantidou et al. 2017 ; Carneiro, Campos, and Miranda 2019 ; Ali, Karim, and Buang 2015). Nevertheless, solvent-based bonding can lead to undesirable effects, such as excessive sample deposition causing channel clogging, high pressure leading to channel breakage and air bubble formation, and uneven bonding resulting in increased hydrophobicity. These factors negatively affect droplet generation and may lead to channel blockage. Furthermore, solvent bonding can be time-consuming, and the vapor process may result in unwanted surface properties. This research investigates a PMMA-based three-layer microfluidic device fabricated using laser engraving for high-resolution channels and UV-assisted solvent-mediated bonding. Key characterization studies, including leakage tests and bonding strength analysis, were conducted to ensure the device’s integrity under varying bonding concentrations. Contact angle and surface energy analysis were also performed to optimize wettability and fluid interaction. The device geometry was optimized to address flow distortions and enhance droplet uniformity. Systematically, the effects of flow rates, fabrication technique, and material interaction on droplet generation and size tuning for high-throughput screening were analysed. The study tackles the challenges in device performance by utilizing high-speed imaging and volumetric analysis to demonstrate real-time monitoring. Materials and methodology 2.1. Materials PMMA substrates with a thickness of 1 mm were sourced from MG Polyplast (Ambattur, India). Ethanol (99%) and mineral oil were purchased from Hayman and Sigma-Aldrich (SISCON, India). Commercially available blunt needles of size 19G were purchased with tubing fitted over these needles. High-precision infusion pump can drive the fluid from 0.1 ml/hr to 100 ml/hr based on the syringe size used. A diluted ethanol-based solution was used for chip bonding application. 2.2. Proposed Design for Droplet Microfluidic The proposed microfluidic design allows the production of surfactant-free monodisperse droplet function for high throughput assay. The design integrates three core functions for droplet generator optimization: First, the geometry initiates with a Y-shaped microchannel for controlled laminar flow in Fig. 1 (a). The fluid streams merge into the central mixing region, promoting diffusion. The main inlets and branches, each 600 µm wide, ensure a stable laminar flow regime up to the diffusion interface. The length of the branch is 5mm, which is optimized to prevent turbulence while maximizing the controlled interfacial mixing between the fluids. Second, in T-junction with shear-driven droplet formation, the droplet regime was integrated with a squeezer mechanism for precise droplet generation through shear-induced droplet break-up shown in Fig. 1 (b). The squeezer channel is 250 µm wide and 2mm long at this junction. The interface of shear force induces monodisperse droplet formation. Third, a square serpentine mixer channel (700 µm width, 1 cm height) was introduced to enhance droplet mixing, which improves mixing efficiency shown in Fig. 1 (c). The serpentine structure induces rotational flow and flow-driven convective rapid homogenous mixing within the droplets. The optimized flow dynamics of the channel structure and its dimensions ensure the consistent generation of droplets. 2.3. Fabrication of Thermoplastic-Based Microfluidic Device The microchannel fabrication was achieved using CO2 laser ablation with a wavelength of 1064 nm, which operates in two different modes: vector and radar. To optimize the channel width and depth, the laser power was adjusted, and a focal length of 6 cm was set. The channel depth, in conjunction with the width, stabilizes the droplets, controls the cross-sectional area, and minimizes the risk of clogging. The deeper the channel, the less interaction there is between droplets and the top and bottom surfaces. To achieve a depth of 1mm, the laser power was selected between 20 and 70 W, while the speed was adjusted from 5 to 30 mm/s. To measure the channel width and the structure formation, the substrates were imaged under a microscope. All channels were measured three times at different locations to confirm structural consistency. However, the obtained microscopic images showed varied channel widths. To observe the channel width and the structure, microscopic images of the PMMA engraving were taken after CO 2 laser ablation. For the microfabrication process, the laser power was tuned between 20 to 70W, while the speed was adjusted from 5 mm/s to 30 mm/s with a fixed laser focal length of 6cm. Based on the trials conducted, the channel width and surface quality were confirmed using optical microscopy. The microscopic image shows channel widths ranging from 145 to 251 µm. The focused laser beam has a 6mm fixed distance. However, the obtained design channels were slightly increased than the given dimensions. Figure 2 (a) shows the bonding mechanism of a three-layer PMMA substrate using various concentrations of ethanolic solution, followed by UV irradiation (254 nm). First, the middle substrate was brushed and sonicated in distilled water for 5 minutes to clear the residual debris and surface contamination. An ethanolic solution was evenly applied to one PMMA substrate, which was then covered with another PMMA, gently pressed, and securely assembled into a three-layer structure. Then, the PMMA was exposed under 254 nm UV light for 4 mins front and back. Finally, permanent bonding was observed after the UV treatment. The bonding mechanism relies on the interacting effect of ethanol and UV irradiation Fig. 2 (b). The ethanol solvent temporarily softens the PMMA surface monomers to crosslink. Whereas exposure to UV-irradiation, the photoactivation generates free radicals within the softened PMMA surface, initiating a covalent crosslinking reaction that forms robust chemical bonds between the layers shown in Fig. 2 (c). This dual-action mechanism helps in permanent bonding with high structural integrity for stable droplet generation. Followed by 19G blunt needle insertion on the inlets and outlets given in the device. 2.4. Leakage Test: The leakage test was conducted to evaluate the sealing efficiency of the bonded PMMA microfluidic devices, using devices bonded with ethanol at three different concentrations: 70%, 80%, and 90% (v/v). For the test, a diluted blue dye solution was introduced into the microchannel through the straight inlet using a 1 ml syringe, while the remaining inlets were sealed with tape. The device was then monitored under a microscope to confirm the leakage for a set period. This leakage test setup allowed for visual assessment of any fluid escape. 2.5. Contact angle measurement The contact angle and surface energy were measured using distilled water for varied concentrations of PMMA-fabricated substrates. To determine the suitable concentration (70%, 80%, and 90%) ethanolic solvent treatment was performed for the microfluidic devices. After the fabrication, the substrates were peeled apart, and the bottom layer was used for contact angle and surface energy measurement. The surface wettability of the PMMA substrates was assessed using distilled water in a contact angle meter (DMe-211 Plus, KYOWA, Japan) to measure the angle of steady (2 µl) water droplet on the surface. 2.6. Bond strength measurement Bonding strength measurement was taken with PMMA-PMMA partial bonding characterized via tensile strength (UTM. Make UTN10, Omega, India). To determine the different concentrations (70, 80, and 90%) of ethanolic solution, it was treated on the substrate and exposed under UV light for 4 mins front and back. The sample was fixed in a tensile testing machine and pulled apart to measure the tensile strength. To measure the bonding reproducibility, the experiment was repeated thrice by measuring the maximum strength obtained during the separation. 2.7. Modelling of Droplet Generation Experiment The proposed droplet generator device was engineered to produce precise, consistent droplets using T-junction with a squeezer regime. The design comprises three inlets with a central straight channel for the continuous phase (Qc) and a Y-shaped channel for the dispersed phase (Qd), converging at the T-junction. A Squeezer regime was integrated at the intersection of continuous and dispersed phase channels to increase the shear force for discrete droplet formation. Droplet frequency and size are controlled by interfacial tension and viscous force. Downstream, the square serpentine channel induces fluid turbulence for rapid and homogeneous mixing without droplet deformation. The sample preparation was done with mineral oil selected as the continuous phase and diluted red ink solvent as the dispersed phase. High-end syringe pumps were used to regulate the independent flow rate of continuous and dispersed phases. The syringe pump was loaded with samples, and the PTFE tube was connected to the device. Then, the microfluidic device was mounted on a torch equipped with a high-speed camera to record video and capture the high throughput flow rates within the range of T-junction and the squeezer regime. The detailed observation of droplet generation is shown in Fig. 3 (a). The flow rates of the continuous phase were varied across a range of 10 to 100 ml/hr, and the dispersed phase was kept constant at 1 ml/hr to determine the effect of droplet formation, uniformity, size, and volume. Using the captured images, the average diameter of the droplet was estimated using Image J software. Results and Discussion 3.1. Optimizing CO 2 laser ablation parameters for fabricating microchannels The CO 2 laser micromachining depended on thermal diffusivity to ablate the surface of the PMMA substrate. Figure 4 (a) shows the effect of laser power on the line width, where the width increased proportionally with laser power and inversely to the line width. The parameter influences the energy delivered to the substrate. The higher the laser power, the wider the channel. At lower power, a narrower channel is produced. A change in laser power from 10 to 70 W resulted in an increase in channel width in the focused laser configuration. The power was fixed at 20, 30, and 40 W with laser speed; further width lines fall into the range of 251.20 to 144.45 µm. When laser power increases, more energy is contained in each pulse. A change in laser speed resulted in a decrease in channel width in the focused laser configuration shown in Fig. 4 (b). According to the study, the designs have different zones for droplet generation and mixing. A narrowed channel will help in droplet expansion by increasing shear at junctions. The observed variation with such a width difference remains within the acceptable functional range for stable operation. Figure 4 (c) showed the optimization, channel fabrication was finalized with fine precision engraving at laser power of 30W and a laser speed of 16 mm/s, which yielded smooth and consistent geometries. 3.2. Bonding performance of droplet microchannel The bonding performance of the PMMA substrate was systematically evaluated using Ethanolic Solvent-Enhanced UV Irradiation Bonding. Using a lower concentration of ethanol solvent (10–40%) was insufficient to achieve bonding. In contrast, moderate ethanol concentration (50–70%) resulted in partial bonding, indicating some surface interaction but not fully sealed. Notably, an ethanolic solution (80–100%) facilitated the complete bonding of the PMMA substrate with a uniform seal. Despite the successful bonding achieved at higher concentrations, several challenges were encountered during the optimization process. Excess solvent or the highest percent ethanol solvent (100%) led to the formation of a white appearance on the PMMA surface, which damaged transparency by compromising the optical clarity shown in Fig. 5 (c). The white cloudy appearance observed at higher ethanol concentrations is due to the deeper penetration of 100% ethanolic solvent into the PMMA substrate, causing uncontrolled and rapid swelling. Unlike diluted ethanol (70–90%), which contains water that moderates solvent power and diffuses, 100% ethanol lacks this buffering effect. During UV irradiation, the absence of water leads to excessive surface stress, resulting in microcracks and structural damage, particularly at the edges. Additionally, a higher concentrated ethanol solution causes channel clogging, rendering it unsuitable for fluidic operation Fig. 5 (d-f). Uneven or intense pressure during bonding resulted in incomplete sealing and channel breakage, while uneven solvent distribution produces partial sealing for microfluidic application Fig. 5 (g-i). After the optimization, 90% ethanolic solvent presented a complete bonding coverage and higher bonding strength with consistent and clear bonding performance. 3.3. Leakage test To further evaluate the performance of the fabricated PMMA microfluidic devices, we conducted a leakage test and analysed the results with previous studies involving PMMA-based bonding techniques. In this experiment, 50 µL of blue dye solution was introduced into microfluidic devices fabricated using different ethanol concentrations (70%, 80%, and 90%). Three trials were conducted for each device with different concentrations ethanol fabricated device was observed continuously at ambient room temperature for 60 minutes. Figure 6 shows that 70% of fabricated devices had slight leakage over the period. Whereas 80% and 90% fabrication showed no evidence of leakage observed during this period, indicating the robustness of the bonding technique and the structural integrity of the device. In comparison, similar studies, such as the work by Kieu et al., employed acetic-acid-based solvent-mediated bonding for PMMA microfluidic devices. Our results confirm that even partial leakage is unacceptance, as it can alter flow dynamics within the microchannel. Among the test bonding conditions, the device bonded with 90% ethanol-bonded microfluidic device exhibited complete leak-free performance, as verified through optical microscope observation (Trinh et al. 2020 ; Saadat et al. 2014 ). Kieu et al. reported that the pressurized bonding technique for stability leads to the deformation of the microscale channel. The absence of leakage in our device suggests a highly reliable interlayer adhesion between the substrates, even under high continuous fluid flow conditions. 3.4. Bonding strength Measurement The examined bonding strength measurement was assessed using the solvent-enhanced UV irradiation bonding, and the PMMA substrates that are partially bonded (30 x 20 mm area) were evaluated. As illustrated in Figure (7), the solvent-enhanced UV-irradiation technique exhibited higher bonding strength compared to the existing fabrication methods. Among all tested conditions, 90% ethanol bonded microfluidic device showed the highest bonding strength of 35.6 MPa, and no observable delamination was observed. The bonding strength measurement demonstrated high reproducibility, with a low error margin of 0.78% and a standard deviation of 0.595. In comparison, the conventional techniques like Trinh et al. reported an acetic acid-based PMMA microfluidic device fabrication, achieved a maximum bond strength of 11. 75 MPa. In this study, a similar approach was adopted. However, the optimized bonding area and enhanced bonding mechanism contributed to significant improvement in bonding strength. Devices that bonded using 70% and 80% ethanol-bonded microfluidics device yielded intermediate bonding strength of 13.06 MPa and 25.7 MPa, respectively. However, reproducibility was quiet challenging, and small cracks were observed near the edges. The study found that 90% ethanolic solution achieved complete bonding with strong adhesion, structural integrity, and bubble-free bonding, making it a stable microfluidics device operation. 3.5. Contact angle measurement Contact angle measurements were carried out to assess the wettability of PMMA-based microfluidics after solvent-enhanced UV-irradiation bonding. Contact angles were measured on both the original PMMA surface and the bonded surface under different bonding conditions. For the original PMMA surface, the contact angle was observed to be 93.0 ± 3.4, indicating a hydrophobic surface. After solvent-enhanced UV- irradiation bonding, the contact angle of the PMMA surface was taken with varied concentrations of 70 to 90% and decreased from 76.5 ± 0.4 to 69.8 ± 0.4 at higher concentrations, as shown in Fig. (8). This reduction in contact angle suggests an increase in surface energy due to chemical change induced by UV -irradiation and solvent interaction. To further understand the surface characteristics, surface energy was calculated using the Owens-Wendt method, which decomposes the total surface energy into polar and dispersive components. The surface energy of the original PMMA surface was calculated to be 34.5 mJ/m². After solvent-enhanced bonding, the surface energy of the PMMA increased significantly, measured to be 64.8 mJ/m². 90% ethanolic bonding enhanced surface energy has improved bonding potential for fluid interactions like droplet formation and flow. The result indicated that bonded PMMA significantly improves wetting properties, making it more suitable for high-precision droplet microfluidics. 3.6. T-junction droplet generator for high-throughput analysis Our proposed design utilizes a T-junction droplet generator with an integrated squeezer mechanism to produce monodisperse water-in-oil emulsion droplets. The system facilitates the formation of spherical droplets, with the dispersed phase (Qd) and continuous phase (Qc) flow rates carefully controlled to achieve a stable droplet generation regime. Several studies have explored T-junction droplet generators for water-in-oil emulsion formation, with an initial impact on flow rates and channel geometry on droplet size and uniformity. Pradeep et. Al studied droplet formation in T-junction structure using Multiphysics simulation, demonstrating a minimum droplet size of 490 µm. Yin et, al and Ahmadpour et. Al investigated droplet generation in a T-junction channel through finite element analysis, reporting a wide range of continuous and dispersed phases influencing the contact angle and droplet formation using image processing {Yin, 2022}{Ahmadpour, 2024}. In this study, we investigated the effect of varying Qc while maintaining a constant Qd to characterize the transition between different flow regimes. Droplet generation was examined over a broad range of continuous phase flow rates (Qc = 10–70 mL/hr), while the dispersed phase was fixed at Qd = 1 mL/hr. The channel depth was maintained at 1 mm, ensuring an equivalent flow velocity and promoting uniformity across Qc flow conditions. Figure (9) shows the flow conditions, distinct flow regimes including jetting, squeezing, and dripping were observed, dictated by the interplay between viscous forces, interfacial tension, and shear stress at the droplet formation site. At low flow rate ratios (Qc/Qd = 0.03–0.01), the system exhibited a jetting regime characterized by polydisperse droplet formation and the emergence of a double-helix flow pattern. In previous study, satellite droplets were challenging in jetting regime, resulting in unstable breakup of the dispersed phase. whereas our system showed an improved monodispersity by reducing unstable breakup. The transition to dripping occurred within a flow rate ratio range of 0.2–0.05, where the droplet interface experienced a balance between viscous drag and interfacial tension at the junction. The presence of the squeezer mechanism significantly influenced droplet detachment, stabilizing the system and promoting monodisperse droplet formation in high throughput screening. 3.7. Droplet size and volume measurement in the microfluidic system at varying flow rates The microfluidic device was optimized by varying flow rates to study their impact on droplet formation. As shown in Table 1 , droplet size exhibited a significant dependence on flow rates ranging from 0.01 to 0.2 variations, exerting a more substantial influence on droplet size than the channel dimensions. The droplet diameter ranged from 675 µm to 150 µm across different flow rates, reflecting the impact of flow rate adjustments within the experimental conditions. This demonstrates that droplet size is highly channel-dependent and can be effectively optimized by increasing the oil flow rate while maintaining a low aqueous flow rate. The outlet channel width of the device was 700 µm wide, and droplet diameters were calculated based on images captured at various flow rate ratios. A clear trend was observed, with droplet diameter decreasing as the flow rate ratio decreased. Specifically, a droplet diameter of 150 µm was achieved at a flow rate ratio of 0.01, as shown in Fig. (10). To further minimize the droplet size, channel dimensions can be optimized by adjusting the width, depth, and geometry to improve flow control and shear force. These findings highlight the critical role of flow rate ratios in controlling droplet size. The droplet diameter and the droplet volume exhibited a strong dependence on the flow rate ratio (FFR). At higher FFR, where larger droplet diameters were observed, the corresponding droplet volumes were significantly larger. At lower FFR conditions, the droplet volume decreased to approximately 0.00206 m³. This relationship between flow rate ratio and droplet volume highlights the capability to precisely tune droplet size and volume by adjusting the flow rates, which is essential for applications requiring specific droplet volumes in high-throughput assays. The observed variation in droplet volumes further underscores the effectiveness of the microfluidic device in achieving consistent and reproducible droplet production under different flow conditions. Table 1 The table presents flow rate optimization, droplet size, and Volume in the droplet generation microfluidic system. Qc (ml/hr) Qd (ml/hr) Flow rate ratio Droplet size (µm) Volume (m^3) 5 1 0.2 678 0.17 8 1 0.125 639 0.14 10 1 0.100 447 0.056 20 1 0.050 287 0.0101 30 1 0.033 262 0.00834 40 1 0.025 256 0.00648 50 1 0.020 174 0.00327 60 1 0.010 150 0.00206 Conclusion The fabricated PMMA-based three- layer microfluidic platform, developed via CO 2 laser engraving and followed by solvent-enhanced UV-irradiation bonding, demonstrated high structural integrity and functional reliability. A leakage test conducted with varying ethanol concentration confirmed that 90% ethanol concentration bonded device exhibited complete leak-free operation over 60 minutes. Compared to the previous method, bonding strategies include acetic acid or pressure-assisted bonding techniques (Trinh, 2020). Which showed deformation or instability, our approach provided superior sealing without compromising channel geometry. The surface wettability analysis showed a marked decrease in contact angle from 93.0° (untreated PMMA) to 69.8° after 90% ethanol bonding, indicating increased surface hydrophilicity. Correspondingly, the surface energy increased from 34.5 mJ/m² to 64.8 mJ/m², enhancing fluid-substrate interaction critical for precise droplet formation and flow control. Using a T-junction with a squeezer mechanism, controlled droplet formation was accomplished. By adjusting the continuous phase flow velocity, monodisperse droplets with a diameter of 675 µm to 150 µm were produced. The PMMA platform eliminated swelling and deformation problems and showed better mechanical robustness, optical clarity, and chemical resistance when compared to traditional PDMS devices. Since the UV-assisted approach avoids high temperatures, it can be used for rapid prototyping without the need for cleanroom facilities, unlike thermal or pressure-based bonding. These improvements driven by chemical modification from UV irradiation and solvent interaction enhance the performance of the microfluidic device in the high-throughput screening. Controlled droplet together, the leakage resistance, improved structural integrity, controlled surface characteristics, and ease of fabrication without thermal or pressure and cleanroom requirements underscore the PMMA-based microfluidics device potential as a scalable, cost-effective alternative for droplet microfluidic applications. Future work will focus on the integration of active elements and multiplex assays used in biomedical diagnostic applications. Declarations Author Contribution Dr. S. Gnanavel – Conceptualization, Methodology, Validation, Formal analysis, Supervision, Project administration. Kaavya P - Methodology, Investigation, Data Curation, Writing - Original Draft and VisualizationAshwin Kumar N - Investigation, Resources, Formal analysis, Data Curation, Validation Acknowledgement The author would like to thank Dr. Kannan, Assistant Professor, Department of Aerospace Engineering, for providing the High-speed camera used for imaging and recording the droplets in this study. Appreciation is also extended to Omega- Inspection and Analytical Laboratory, Maraimalar Nagar, for conducting the mechanical stability analysis. ANRF-SERB (CRG/2024/3902) for facilitating the contact angle measurement. References Abalde-Cela, S., P. Taladriz-Blanco, M. G. de Oliveira, and C. Abell. 2018. 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Neuzil. 2024. 'Microfluidics chips fabrication techniques comparison', Sci Rep , 14: 28793. Ma, Xiuqing, Rui Li, Zhiming Jin, Yiqiang Fan, Xuance Zhou, and Yajun Zhang. 2019. 'Injection molding and characterization of PMMA-based microfluidic devices', Microsystem Technologies , 26: 1317-24. Mashiyama, S., R. Hemmi, T. Sato, A. Kato, T. Taniguchi, and M. Yamada. 2024. 'Pushing the limits of microfluidic droplet production efficiency: engineering microchannels with seamlessly implemented 3D inverse colloidal crystals', Lab Chip , 24: 171-81. Nan, L., H. Zhang, D. A. Weitz, and H. C. Shum. 2024. 'Development and future of droplet microfluidics', Lab Chip , 24: 1135-53. Nasser, G. A., A. M. R. Fath El-Bab, A. L. Abdel-Mawgood, H. Mohamed, and A. M. Saleh. 2019. 'CO(2) Laser Fabrication of PMMA Microfluidic Double T-Junction Device with Modified Padilha, Giovana da Silva, Virginia Mansanares Giacon, and Julio Roberto Bartoli. 2017. 'Effect of solvents on the morphology of PMMA films fabricated by spin-coating', Polímeros , 27: 195-200. Perez-Sosa, C., A. B. Penaherrera-Pazmino, G. Rosero, N. Bourguignon, A. Aravelli, S. Bhansali, M. S. Perez, and B. Lerner. 2022. 'Novel Reproducible Manufacturing and Reversible Sealing Method for Microfluidic Devices', Micromachines (Basel) , 13. Saadat, Mozafar, Marie Taylor, Arran Hughes, and Amir M. Hajiyavand. 2014. 'Rapid prototyping method for 3D PDMS microfluidic devices using a red femtosecond laser', Advances in Mechanical Engineering , 12. Sanjay, S. T., M. Li, W. Zhou, X. Li, and X. Li. 2020. 'A reusable PMMA/paper hybrid plug-and-play microfluidic device for an ultrasensitive immunoassay with a wide dynamic range', Microsyst Nanoeng , 6: 28. Shakeri, A., N. A. Jarad, S. Khan, and F. Didar T. 2022. 'Bio-functionalization of microfluidic platforms made of thermoplastic materials: A review', Anal Chim Acta , 1209: 339283. Tan, His Yin, Weng Keong Loke, and Nam-Trung Nguyen. 2010. 'Integration of PDMS and PMMA for Batch Fabrication of Microfluidic Devices.' in, 6th World Congress of Biomechanics (WCB 2010). August 1-6, 2010 Singapore . Tirandazi, Pooyan, and Carlos H. Hidrovo. 2017. 'Liquid-in-gas droplet microfluidics; experimental characterization of droplet morphology, generation frequency, and monodispersity in a flow-focusing microfluidic device', Journal of Micromechanics and Microengineering , 27. Trantidou, T., Y. Elani, E. Parsons, and O. Ces. 2017. 'Hydrophilic surface modification of PDMS for droplet microfluidics using a simple, quick, and robust method via PVA deposition', Microsyst Nanoeng , 3: 16091. Trinh, K. T. L., W. R. Chae, and N. Y. Lee. 2021. 'Pressure-Free Assembling of Poly(methyl methacrylate) Microdevices via Microwave-Assisted Solvent Bonding and Its Biomedical Applications', Biosensors (Basel) , 11. Trinh, K. T. L., D. A. Thai, W. R. Chae, and N. Y. Lee. 2020. 'Rapid Fabrication of Poly(methyl methacrylate) Devices for Lab-on-a-Chip Applications Using Acetic Acid and UV Treatment', ACS Omega , 5: 17396-404. Wu, N., Y. Zhu, S. Brown, J. Oakeshott, T. S. Peat, R. Surjadi, C. Easton, P. W. Leech, and B. A. Sexton. 2009. 'A PMMA microfluidic droplet platform for in vitro protein expression using crude E. coli S30 extract', Lab Chip , 9: 3391-8. Xue, Bo, Yanquan Geng, Yongda Yan, Gaojie Ma, Dong Wang, and Yang He. 2020. 'Rapid prototyping of microfluidic chip with burr-free PMMA microchannel fabricated by revolving tip-based micro-cutting', Journal of Materials Processing Technology , 277. Xiang N, Ni Z. Microfluidics for biomedical applications. Biosensors. 2023 Jan 20;13(2):161. Additional Declarations No competing interests reported. Supplementary Files SupplementaryData.docx GraphicalAbstract.png Graphical Abstract Cite Share Download PDF Status: Published Journal Publication published 04 Jun, 2025 Read the published version in Microfluidics and Nanofluidics → Version 1 posted Editorial decision: Revision requested 27 Apr, 2025 Reviews received at journal 24 Apr, 2025 Reviewers agreed at journal 12 Apr, 2025 Reviewers invited by journal 11 Apr, 2025 Submission checks completed at journal 11 Apr, 2025 First submitted to journal 08 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6102019","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":448777602,"identity":"9716afad-4798-4fea-9181-01e8a31f46ce","order_by":0,"name":"Kaavya Purushothaman","email":"","orcid":"","institution":"SRM Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Kaavya","middleName":"","lastName":"Purushothaman","suffix":""},{"id":448777603,"identity":"196f08fd-08a2-4c53-a57e-6e045fc3237d","order_by":1,"name":"Ashwin Kumar Narasimhan","email":"","orcid":"","institution":"University of Wisconsin Milwaukee","correspondingAuthor":false,"prefix":"","firstName":"Ashwin","middleName":"Kumar","lastName":"Narasimhan","suffix":""},{"id":448777604,"identity":"22744b67-3b1c-4169-9d2a-90ff90d585af","order_by":2,"name":"Gnanavel S","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYDADfhCRwAAlE4jRItkAVmxAghaDAxCKgaB6+ejmZw9+ttnYG58/+3TDgz9/GPjZcwwYHu7ArcXwzjFzw962tMRtN9LNbiS2GTBI9rwxYEg8g0fLjAQzCZ4zhxPMbrCx3UhsMGAwuAG0JbENn5b0b5J/zvy3N+4/xnYj4Y8Bgz0hLfISOWbSPBUHGDcwpAG1sAFtkSCgBaigTFqmIjlxxg2glsQ2Yx6JM88KDuC1ZUb6Nsk3Bnb2/ECH3fzxR06Ovz1548Of+Gw5gCbAAyLQBVFtacAnOwpGwSgYBaMABABRFFEPdTbC3wAAAABJRU5ErkJggg==","orcid":"","institution":"SRM Institute of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Gnanavel","middleName":"","lastName":"S","suffix":""}],"badges":[],"createdAt":"2025-02-25 06:38:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6102019/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6102019/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10404-025-02816-5","type":"published","date":"2025-06-04T15:57:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81633218,"identity":"58f3fbff-094e-439a-b544-c3560e059110","added_by":"auto","created_at":"2025-04-29 11:53:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":255517,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the droplet microfluidic device design. (a) Y-shaped channel is designed to facilitate the diffusion process. (b)T-junction with squeezer mechanism for precise droplet formation. (c) square serpentine structure incorporated for droplet rotation and mixing.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6102019/v1/a370d7aa72b0d7e634a95d0e.png"},{"id":81632484,"identity":"a3537bc2-9e85-4ef6-95a8-821b73b111de","added_by":"auto","created_at":"2025-04-29 11:45:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":385875,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Schematic illustration of the fabrication procedure for three-layer PMMA substrates using ethanolic solvent-mediated UV-irradiation involving (b \u0026amp; c) representation of the chemical reactions occurring on the PMMA substrates surface after ethanol deposition and subsequent UV-irradiation leading to surface activation for enhanced bonding.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6102019/v1/b7c054d1dc75316dc05346b7.png"},{"id":81632887,"identity":"7979b3d1-e0c1-464f-a749-173e0cedea40","added_by":"auto","created_at":"2025-04-29 11:53:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2221626,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Schematic of the droplet microfluidic system experimental setup (b) Illustration of the droplet generator, showing the continuous phase (Qc: mineral oil) and dispersed phase (Qd: red ink dye) delivered by syringe pumps, with droplet formation.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6102019/v1/b3d056b3798643d7590138be.png"},{"id":81632888,"identity":"f3ff74d8-d863-4605-a86e-202f11eb54c9","added_by":"auto","created_at":"2025-04-29 11:53:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2250653,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of laser power and speed on laser engraving line width. (a) line width increases with laser power ranging from 10 to 70W, measured at a constant speed of 16 mm/s.(b) Both laser speed and laser power are adjusted, line width decreases with increasing speed at 20 – 40 W, line width decreases up to 146 µm at 25 mm/s. data represents mean values from three independent measurements. (c) Laser-engraved channel created using 30W laser power at a speed of 16 mm/s.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6102019/v1/35ce485f8cd8b07084e040ef.png"},{"id":81632483,"identity":"6fb78feb-10cf-40a8-a687-39108a0c8d57","added_by":"auto","created_at":"2025-04-29 11:45:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2361925,"visible":true,"origin":"","legend":"\u003cp\u003eThe image illustrates the challenges faced during the bonding process and bonding efficiency of PMMA substrates at different ratio of ethanolic solvents followed by UV irradiation. The image highlights the issues, including incomplete bonding, leakage, and channel breakage (a – f), as well as partial bonding with channel integrity (g – i). The white dash regions in each image emphasize the critical bonding area and outcome.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6102019/v1/9e70823715708f9e16ea2848.png"},{"id":81634000,"identity":"71aaa3b9-9c74-40ef-99c8-d7a869f461a2","added_by":"auto","created_at":"2025-04-29 12:01:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2589506,"visible":true,"origin":"","legend":"\u003cp\u003eThe figure illustrates the design of the microfluidic device used for the leakage test along with the result over a 0-60 mins observation period. The device bonded using 70% ethanol exhibit a mild leakage around the channel edges, indicated by the black squares. 90% ethanolic concentration showed a complete sealing with no visible leakage observed under optical microscopy throughout the duration of the test.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6102019/v1/73d08735e9fc57b6d14c695e.png"},{"id":81634852,"identity":"5e6df5cb-c198-41ca-b77c-86d196ae10e2","added_by":"auto","created_at":"2025-04-29 12:09:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":39849,"visible":true,"origin":"","legend":"\u003cp\u003eBond strength measurements of PMMA substrates. Graphical representation of the average bond strength for PMMA substrates bonded with an overlap length of 3 cm, using different ethanolic solvent concentrations.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6102019/v1/dd4027739e0a736db8e83258.png"},{"id":81632891,"identity":"8a62accc-93d3-4673-890f-485130a35e08","added_by":"auto","created_at":"2025-04-29 11:53:01","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":508021,"visible":true,"origin":"","legend":"\u003cp\u003eContact angle measurement of PMMA microfluidic substrate before and after solvent-enhanced UV-irradiation bonding.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6102019/v1/99084537db75ee5ae871b7fd.png"},{"id":81632501,"identity":"29786386-9a14-4b43-b1b6-4c76ae183185","added_by":"auto","created_at":"2025-04-29 11:45:01","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":957726,"visible":true,"origin":"","legend":"\u003cp\u003eThis illustration depicts an image sequence of the jetting and dripping regime effects on droplet generation in a W/O system. In the dripping regimes, which occur at lower dispersed phase flow rates, discrete and larger droplets are formed directly at the junction due to the detachment driven by interfacial tension. As the flow rate increases, the system transitions into jetting regimes, elongated fluid thread that subsequently breaks into smaller and uniform droplets. This is mainly characterized by higher shear force and inertial effect. Enabling of high throughput droplet generation with smaller size distribution.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6102019/v1/c5e73d2bfed7a1ecbf816fe0.png"},{"id":81632494,"identity":"885006e3-e7de-4118-8564-2a40339d763f","added_by":"auto","created_at":"2025-04-29 11:45:01","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":168087,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical Representation of droplet size measurement as a function of the flow rate ratio. The data confirm a clear inverse relationship between flow rate ratio and droplet size, indicating the effectiveness of the flow tuning for precise droplet size control.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6102019/v1/5e506cb9087bd8e574bf41c4.png"},{"id":84242712,"identity":"34e33a77-61a6-4c89-b0b4-3dfc33276a15","added_by":"auto","created_at":"2025-06-09 16:11:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11854996,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6102019/v1/3e2fb6a1-acc9-4d8b-aa5e-4c84803150f7.pdf"},{"id":81633999,"identity":"55430fa6-9e2f-4a37-bc09-5d55735406c6","added_by":"auto","created_at":"2025-04-29 12:01:01","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":32241,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData.docx","url":"https://assets-eu.researchsquare.com/files/rs-6102019/v1/ab93c527d79aa417acf65278.docx"},{"id":81633224,"identity":"8429f63c-4253-448f-91c8-d9d03c02e3e5","added_by":"auto","created_at":"2025-04-29 11:53:39","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":171689,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-6102019/v1/0db6cd8297f2c2fd29637647.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eDevelopment of a PMMA-based Droplet microfluidic device for High-throughput Screening in Health care Applications\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eMicrofluidic technology impacts various applications for analyzing fluid behavior within microscale channels. The ability to manipulate microscale volumes of fluids has revolutionized these fields, enabling high precision, miniaturization, and automation (Nan et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Between 2018 and x, innovations in Point-of-Care (POC) testing drove an 11.9% compound annual growth rate (CAGR) worldwide (Konwar, 2020). Among the diverse subfields of microfluidics, droplet-based microfluidics has gained significant attention due to its capacity to compartmentalize and manipulate fluids into discrete droplet volumes within the microscale (Bakhshi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Abalde-Cela et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Droplet microfluidics offers a controlled, contamination-free environment, minimizing sample volume and enhancing sustainability, allowing for high-throughput screening (S\u0026ouml;zmen, 2021). Droplets are commonly generated using configurations such as T-junctions, flow focusing, and co-flow junctions, each influencing flow behavior and mixing efficiency under controlled flow rates (Elvira et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Tirandazi and Hidrovo \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Bardin and Lee \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Hettiarachchi et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Xiaoping Li et al. reported a micropump-driven flow-focusing junction for monodispersed droplet generation; however, the minimum droplet size achieved was 360 \u0026micro;m, with diameters deviating from expected values. Islam et al. explored PMMA-based droplet microfluidics using a flow-focusing design to observe DNA-coated particles, achieving droplet sizes of 10\u0026ndash;200 \u0026micro;m(Islam, 2018). The results indicate that the design produced polydisperse droplets, reflecting uneven compartmentalization. According to the literature, T-junction microfluidic devices generate droplets in an unsteady manner, often resulting in polydispersity due to inconsistent droplet generation. The design and selection of materials are critical for device performance, reliability, and scalability. While existing designs and fabrication techniques have improved droplet formation, challenges such as device instability, material incompatibility, limited scalability, and reliance on flow-focusing configurations restrict the complexity and multifunctionality of microfluidic platforms. Optimizing design parameters can help produce monodisperse droplets with more effective and precise size control, which is essential for reproducibility and accuracy (Li et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Traditionally, polymethyl siloxane (PDMS), glass, and silicon are the three primary materials used for microfluidic fabrication. Each material offers various advantages in terms of flexibility, optical clarity, and biocompatibility. However, these materials are unsuitable for large-scale manufacturing due to limitations, including high fabrication costs, limited long-term durability, long processing times, and complex fabrication processes (Sanjay et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Amirifar et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mashiyama et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Wu et al. 2009). The PDMS material provides compatibility with various solvents and flexibility. However, PDMS faces restrictions in certain applications, particularly in droplet generation, due to its gas permeability, solvent swelling, absorption of small hydrophobic molecules, and poor scalability (Katare, 2020). In contrast, devices made from silicon or glass offer superior mechanical stability and chemical resistance. However, they necessitate sophisticated fabrication techniques such as cleanroom facilities, high temperatures, and complex etching processes, which increase costs and limit scalability, especially for high-throughput screening (Vogt, 2022; Trantidou, 2017). Thermoplastics have recently emerged as an alternative to PDMS and other conventional materials, providing improved functionality, reliability, and scalability (Tan, Loke, and Nguyen 2010). Common thermoplastics used in microfluidic systems include poly(methyl methacrylate) (PMMA), cyclic olefin polymer (COP), polycarbonate (PC), and polystyrene (PS). PMMA is gaining popularity due to its optical clarity, biocompatibility, chemical resistance, hydrophobic surface, and cost-effectiveness. PMMA substrates also facilitate rapid prototyping and large-scale production (Kotz et al. 2020; Ma et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Shakeri et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ng, Tjeung, and Wang 2006). PMMA exhibits low permeability to organic solvents, allows for controllable wettability, and provides greater mechanical robustness, making it suitable for long-term use and ensuring better droplet integrity. Various microfabrication techniques, including hot embossing, injection molding, 3D printing, and laser ablation, are employed to create customized microchannels and patterns in thermoplastic materials (Nasser et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Hot embossing involves pressing a master mold to fabricate microchannels but necessitates high temperatures above the material\u0026rsquo;s glass transition temperature, leading to channel deformation and high processing costs, which render it unsuitable for droplet microfluidics (Chen, Li, and Gao \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Xue et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). CO2 laser ablation employs a high-intensity laser beam to create microchannels and patterns, offering rapid prototyping, low cost, and large-scale production. However, thermal-based ablation can result in heat-affected zones around the channel edges, though this can be mitigated through adjustments to laser power and speed (Fan et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Perez-Sosa et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Trinh, Chae, and Lee \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The selection of bonding methods also plays a significant role in microfluidic device fabrication. Popular methods include thermal fusion bonding, adhesive layer bonding, chemical bonding, microwave bonding, and solvent bonding (Padilha, Giacon, and Bartoli 2017; Xiang, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Recent studies indicate that thermal fusion bonding, while effective, requires high temperatures and pressure, which may distort or shrink the channels (Liu et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Solvent-mediated bonding has been widely utilized, as it employs organic solvents like ethanol, isopropanol, and acetic acid for bonding (Trantidou et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Carneiro, Campos, and Miranda \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ali, Karim, and Buang 2015). Nevertheless, solvent-based bonding can lead to undesirable effects, such as excessive sample deposition causing channel clogging, high pressure leading to channel breakage and air bubble formation, and uneven bonding resulting in increased hydrophobicity. These factors negatively affect droplet generation and may lead to channel blockage. Furthermore, solvent bonding can be time-consuming, and the vapor process may result in unwanted surface properties. This research investigates a PMMA-based three-layer microfluidic device fabricated using laser engraving for high-resolution channels and UV-assisted solvent-mediated bonding. Key characterization studies, including leakage tests and bonding strength analysis, were conducted to ensure the device\u0026rsquo;s integrity under varying bonding concentrations. Contact angle and surface energy analysis were also performed to optimize wettability and fluid interaction. The device geometry was optimized to address flow distortions and enhance droplet uniformity. Systematically, the effects of flow rates, fabrication technique, and material interaction on droplet generation and size tuning for high-throughput screening were analysed. The study tackles the challenges in device performance by utilizing high-speed imaging and volumetric analysis to demonstrate real-time monitoring.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Materials and methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePMMA substrates with a thickness of 1 mm were sourced from MG Polyplast (Ambattur, India). Ethanol (99%) and mineral oil were purchased from Hayman and Sigma-Aldrich (SISCON, India). Commercially available blunt needles of size 19G were purchased with tubing fitted over these needles. High-precision infusion pump can drive the fluid from 0.1 ml/hr to 100 ml/hr based on the syringe size used. A diluted ethanol-based solution was used for chip bonding application.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2.2. Proposed Design for Droplet Microfluidic\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe proposed microfluidic design allows the production of surfactant-free monodisperse droplet function for high throughput assay. The design integrates three core functions for droplet generator optimization: First, the geometry initiates with a Y-shaped microchannel for controlled laminar flow in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (a). The fluid streams merge into the central mixing region, promoting diffusion. The main inlets and branches, each 600 \u0026micro;m wide, ensure a stable laminar flow regime up to the diffusion interface. The length of the branch is 5mm, which is optimized to prevent turbulence while maximizing the controlled interfacial mixing between the fluids. Second, in T-junction with shear-driven droplet formation, the droplet regime was integrated with a squeezer mechanism for precise droplet generation through shear-induced droplet break-up shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (b). The squeezer channel is 250 \u0026micro;m wide and 2mm long at this junction. The interface of shear force induces monodisperse droplet formation. Third, a square serpentine mixer channel (700 \u0026micro;m width, 1 cm height) was introduced to enhance droplet mixing, which improves mixing efficiency shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (c). The serpentine structure induces rotational flow and flow-driven convective rapid homogenous mixing within the droplets. The optimized flow dynamics of the channel structure and its dimensions ensure the consistent generation of droplets.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003e2.3. Fabrication of Thermoplastic-Based Microfluidic Device\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe microchannel fabrication was achieved using CO2 laser ablation with a wavelength of 1064 nm, which operates in two different modes: vector and radar. To optimize the channel width and depth, the laser power was adjusted, and a focal length of 6 cm was set. The channel depth, in conjunction with the width, stabilizes the droplets, controls the cross-sectional area, and minimizes the risk of clogging. The deeper the channel, the less interaction there is between droplets and the top and bottom surfaces. To achieve a depth of 1mm, the laser power was selected between 20 and 70 W, while the speed was adjusted from 5 to 30 mm/s. To measure the channel width and the structure formation, the substrates were imaged under a microscope. All channels were measured three times at different locations to confirm structural consistency. However, the obtained microscopic images showed varied channel widths.\u003c/p\u003e \u003cp\u003eTo observe the channel width and the structure, microscopic images of the PMMA engraving were taken after CO\u003csub\u003e2\u003c/sub\u003e laser ablation. For the microfabrication process, the laser power was tuned between 20 to 70W, while the speed was adjusted from 5 mm/s to 30 mm/s with a fixed laser focal length of 6cm. Based on the trials conducted, the channel width and surface quality were confirmed using optical microscopy. The microscopic image shows channel widths ranging from 145 to 251 \u0026micro;m. The focused laser beam has a 6mm fixed distance. However, the obtained design channels were slightly increased than the given dimensions.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (a) shows the bonding mechanism of a three-layer PMMA substrate using various concentrations of ethanolic solution, followed by UV irradiation (254 nm). First, the middle substrate was brushed and sonicated in distilled water for 5 minutes to clear the residual debris and surface contamination. An ethanolic solution was evenly applied to one PMMA substrate, which was then covered with another PMMA, gently pressed, and securely assembled into a three-layer structure. Then, the PMMA was exposed under 254 nm UV light for 4 mins front and back. Finally, permanent bonding was observed after the UV treatment. The bonding mechanism relies on the interacting effect of ethanol and UV irradiation Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (b). The ethanol solvent temporarily softens the PMMA surface monomers to crosslink. Whereas exposure to UV-irradiation, the photoactivation generates free radicals within the softened PMMA surface, initiating a covalent crosslinking reaction that forms robust chemical bonds between the layers shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (c). This dual-action mechanism helps in permanent bonding with high structural integrity for stable droplet generation. Followed by 19G blunt needle insertion on the inlets and outlets given in the device.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003e2.4. Leakage Test:\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe leakage test was conducted to evaluate the sealing efficiency of the bonded PMMA microfluidic devices, using devices bonded with ethanol at three different concentrations: 70%, 80%, and 90% (v/v). For the test, a diluted blue dye solution was introduced into the microchannel through the straight inlet using a 1 ml syringe, while the remaining inlets were sealed with tape. The device was then monitored under a microscope to confirm the leakage for a set period. This leakage test setup allowed for visual assessment of any fluid escape.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003e2.5. Contact angle measurement\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe contact angle and surface energy were measured using distilled water for varied concentrations of PMMA-fabricated substrates. To determine the suitable concentration (70%, 80%, and 90%) ethanolic solvent treatment was performed for the microfluidic devices. After the fabrication, the substrates were peeled apart, and the bottom layer was used for contact angle and surface energy measurement. The surface wettability of the PMMA substrates was assessed using distilled water in a contact angle meter (DMe-211 Plus, KYOWA, Japan) to measure the angle of steady (2 \u0026micro;l) water droplet on the surface.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Bond strength measurement\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBonding strength measurement was taken with PMMA-PMMA partial bonding characterized via tensile strength (UTM. Make UTN10, Omega, India). To determine the different concentrations (70, 80, and 90%) of ethanolic solution, it was treated on the substrate and exposed under UV light for 4 mins front and back. The sample was fixed in a tensile testing machine and pulled apart to measure the tensile strength. To measure the bonding reproducibility, the experiment was repeated thrice by measuring the maximum strength obtained during the separation.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2.7. Modelling of Droplet Generation Experiment\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe proposed droplet generator device was engineered to produce precise, consistent droplets using T-junction with a squeezer regime. The design comprises three inlets with a central straight channel for the continuous phase (Qc) and a Y-shaped channel for the dispersed phase (Qd), converging at the T-junction. A Squeezer regime was integrated at the intersection of continuous and dispersed phase channels to increase the shear force for discrete droplet formation. Droplet frequency and size are controlled by interfacial tension and viscous force. Downstream, the square serpentine channel induces fluid turbulence for rapid and homogeneous mixing without droplet deformation. The sample preparation was done with mineral oil selected as the continuous phase and diluted red ink solvent as the dispersed phase. High-end syringe pumps were used to regulate the independent flow rate of continuous and dispersed phases. The syringe pump was loaded with samples, and the PTFE tube was connected to the device. Then, the microfluidic device was mounted on a torch equipped with a high-speed camera to record video and capture the high throughput flow rates within the range of T-junction and the squeezer regime. The detailed observation of droplet generation is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (a). The flow rates of the continuous phase were varied across a range of 10 to 100 ml/hr, and the dispersed phase was kept constant at 1 ml/hr to determine the effect of droplet formation, uniformity, size, and volume. Using the captured images, the average diameter of the droplet was estimated using Image J software.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Optimizing CO\u003csub\u003e2\u003c/sub\u003e laser ablation parameters for fabricating microchannels\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe CO\u003csub\u003e2\u003c/sub\u003e laser micromachining depended on thermal diffusivity to ablate the surface of the PMMA substrate. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (a) shows the effect of laser power on the line width, where the width increased proportionally with laser power and inversely to the line width. The parameter influences the energy delivered to the substrate. The higher the laser power, the wider the channel. At lower power, a narrower channel is produced. A change in laser power from 10 to 70 W resulted in an increase in channel width in the focused laser configuration. The power was fixed at 20, 30, and 40 W with laser speed; further width lines fall into the range of 251.20 to 144.45 \u0026micro;m. When laser power increases, more energy is contained in each pulse. A change in laser speed resulted in a decrease in channel width in the focused laser configuration shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (b). According to the study, the designs have different zones for droplet generation and mixing. A narrowed channel will help in droplet expansion by increasing shear at junctions. The observed variation with such a width difference remains within the acceptable functional range for stable operation. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (c) showed the optimization, channel fabrication was finalized with fine precision engraving at laser power of 30W and a laser speed of 16 mm/s, which yielded smooth and consistent geometries.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Bonding performance of droplet microchannel\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe bonding performance of the PMMA substrate was systematically evaluated using Ethanolic Solvent-Enhanced UV Irradiation Bonding. Using a lower concentration of ethanol solvent (10\u0026ndash;40%) was insufficient to achieve bonding. In contrast, moderate ethanol concentration (50\u0026ndash;70%) resulted in partial bonding, indicating some surface interaction but not fully sealed. Notably, an ethanolic solution (80\u0026ndash;100%) facilitated the complete bonding of the PMMA substrate with a uniform seal. Despite the successful bonding achieved at higher concentrations, several challenges were encountered during the optimization process. Excess solvent or the highest percent ethanol solvent (100%) led to the formation of a white appearance on the PMMA surface, which damaged transparency by compromising the optical clarity shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (c). The white cloudy appearance observed at higher ethanol concentrations is due to the deeper penetration of 100% ethanolic solvent into the PMMA substrate, causing uncontrolled and rapid swelling. Unlike diluted ethanol (70\u0026ndash;90%), which contains water that moderates solvent power and diffuses, 100% ethanol lacks this buffering effect. During UV irradiation, the absence of water leads to excessive surface stress, resulting in microcracks and structural damage, particularly at the edges. Additionally, a higher concentrated ethanol solution causes channel clogging, rendering it unsuitable for fluidic operation Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (d-f). Uneven or intense pressure during bonding resulted in incomplete sealing and channel breakage, while uneven solvent distribution produces partial sealing for microfluidic application Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (g-i). After the optimization, 90% ethanolic solvent presented a complete bonding coverage and higher bonding strength with consistent and clear bonding performance.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Leakage test\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo further evaluate the performance of the fabricated PMMA microfluidic devices, we conducted a leakage test and analysed the results with previous studies involving PMMA-based bonding techniques. In this experiment, 50 \u0026micro;L of blue dye solution was introduced into microfluidic devices fabricated using different ethanol concentrations (70%, 80%, and 90%). Three trials were conducted for each device with different concentrations ethanol fabricated device was observed continuously at ambient room temperature for 60 minutes. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows that 70% of fabricated devices had slight leakage over the period. Whereas 80% and 90% fabrication showed no evidence of leakage observed during this period, indicating the robustness of the bonding technique and the structural integrity of the device. In comparison, similar studies, such as the work by Kieu et al., employed acetic-acid-based solvent-mediated bonding for PMMA microfluidic devices. Our results confirm that even partial leakage is unacceptance, as it can alter flow dynamics within the microchannel. Among the test bonding conditions, the device bonded with 90% ethanol-bonded microfluidic device exhibited complete leak-free performance, as verified through optical microscope observation (Trinh et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Saadat et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Kieu et al. reported that the pressurized bonding technique for stability leads to the deformation of the microscale channel. The absence of leakage in our device suggests a highly reliable interlayer adhesion between the substrates, even under high continuous fluid flow conditions.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Bonding strength Measurement\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe examined bonding strength measurement was assessed using the solvent-enhanced UV irradiation bonding, and the PMMA substrates that are partially bonded (30 x 20 mm area) were evaluated. As illustrated in Figure (7), the solvent-enhanced UV-irradiation technique exhibited higher bonding strength compared to the existing fabrication methods. Among all tested conditions, 90% ethanol bonded microfluidic device showed the highest bonding strength of 35.6 MPa, and no observable delamination was observed. The bonding strength measurement demonstrated high reproducibility, with a low error margin of 0.78% and a standard deviation of 0.595. In comparison, the conventional techniques like Trinh et al. reported an acetic acid-based PMMA microfluidic device fabrication, achieved a maximum bond strength of 11. 75 MPa. In this study, a similar approach was adopted. However, the optimized bonding area and enhanced bonding mechanism contributed to significant improvement in bonding strength. Devices that bonded using 70% and 80% ethanol-bonded microfluidics device yielded intermediate bonding strength of 13.06 MPa and 25.7 MPa, respectively. However, reproducibility was quiet challenging, and small cracks were observed near the edges. The study found that 90% ethanolic solution achieved complete bonding with strong adhesion, structural integrity, and bubble-free bonding, making it a stable microfluidics device operation.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Contact angle measurement\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eContact angle measurements were carried out to assess the wettability of PMMA-based microfluidics after solvent-enhanced UV-irradiation bonding. Contact angles were measured on both the original PMMA surface and the bonded surface under different bonding conditions. For the original PMMA surface, the contact angle was observed to be 93.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4, indicating a hydrophobic surface. After solvent-enhanced UV- irradiation bonding, the contact angle of the PMMA surface was taken with varied concentrations of 70 to 90% and decreased from 76.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 to 69.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 at higher concentrations, as shown in Fig.\u0026nbsp;(8). This reduction in contact angle suggests an increase in surface energy due to chemical change induced by UV -irradiation and solvent interaction. To further understand the surface characteristics, surface energy was calculated using the Owens-Wendt method, which decomposes the total surface energy into polar and dispersive components. The surface energy of the original PMMA surface was calculated to be 34.5 mJ/m\u0026sup2;. After solvent-enhanced bonding, the surface energy of the PMMA increased significantly, measured to be 64.8 mJ/m\u0026sup2;. 90% ethanolic bonding enhanced surface energy has improved bonding potential for fluid interactions like droplet formation and flow. The result indicated that bonded PMMA significantly improves wetting properties, making it more suitable for high-precision droplet microfluidics.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.6. T-junction droplet generator for high-throughput analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eOur proposed design utilizes a T-junction droplet generator with an integrated squeezer mechanism to produce monodisperse water-in-oil emulsion droplets. The system facilitates the formation of spherical droplets, with the dispersed phase (Qd) and continuous phase (Qc) flow rates carefully controlled to achieve a stable droplet generation regime. Several studies have explored T-junction droplet generators for water-in-oil emulsion formation, with an initial impact on flow rates and channel geometry on droplet size and uniformity. Pradeep et. Al studied droplet formation in T-junction structure using Multiphysics simulation, demonstrating a minimum droplet size of 490 \u0026micro;m. Yin et, al and Ahmadpour et. Al investigated droplet generation in a T-junction channel through finite element analysis, reporting a wide range of continuous and dispersed phases influencing the contact angle and droplet formation using image processing {Yin, 2022}{Ahmadpour, 2024}. In this study, we investigated the effect of varying Qc while maintaining a constant Qd to characterize the transition between different flow regimes. Droplet generation was examined over a broad range of continuous phase flow rates (Qc\u0026thinsp;=\u0026thinsp;10\u0026ndash;70 mL/hr), while the dispersed phase was fixed at Qd\u0026thinsp;=\u0026thinsp;1 mL/hr. The channel depth was maintained at 1 mm, ensuring an equivalent flow velocity and promoting uniformity across Qc flow conditions. Figure\u0026nbsp;(9) shows the flow conditions, distinct flow regimes including jetting, squeezing, and dripping were observed, dictated by the interplay between viscous forces, interfacial tension, and shear stress at the droplet formation site. At low flow rate ratios (Qc/Qd\u0026thinsp;=\u0026thinsp;0.03\u0026ndash;0.01), the system exhibited a jetting regime characterized by polydisperse droplet formation and the emergence of a double-helix flow pattern. In previous study, satellite droplets were challenging in jetting regime, resulting in unstable breakup of the dispersed phase. whereas our system showed an improved monodispersity by reducing unstable breakup. The transition to dripping occurred within a flow rate ratio range of 0.2\u0026ndash;0.05, where the droplet interface experienced a balance between viscous drag and interfacial tension at the junction. The presence of the squeezer mechanism significantly influenced droplet detachment, stabilizing the system and promoting monodisperse droplet formation in high throughput screening.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Droplet size and volume measurement in the microfluidic system at varying flow rates\u003c/h2\u003e \u003cp\u003eThe microfluidic device was optimized by varying flow rates to study their impact on droplet formation. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, droplet size exhibited a significant dependence on flow rates ranging from 0.01 to 0.2 variations, exerting a more substantial influence on droplet size than the channel dimensions. The droplet diameter ranged from 675 \u0026micro;m to 150 \u0026micro;m across different flow rates, reflecting the impact of flow rate adjustments within the experimental conditions. This demonstrates that droplet size is highly channel-dependent and can be effectively optimized by increasing the oil flow rate while maintaining a low aqueous flow rate. The outlet channel width of the device was 700 \u0026micro;m wide, and droplet diameters were calculated based on images captured at various flow rate ratios. A clear trend was observed, with droplet diameter decreasing as the flow rate ratio decreased. Specifically, a droplet diameter of 150 \u0026micro;m was achieved at a flow rate ratio of 0.01, as shown in Fig.\u0026nbsp;(10). To further minimize the droplet size, channel dimensions can be optimized by adjusting the width, depth, and geometry to improve flow control and shear force. These findings highlight the critical role of flow rate ratios in controlling droplet size. The droplet diameter and the droplet volume exhibited a strong dependence on the flow rate ratio (FFR). At higher FFR, where larger droplet diameters were observed, the corresponding droplet volumes were significantly larger. At lower FFR conditions, the droplet volume decreased to approximately 0.00206 m\u0026sup3;. This relationship between flow rate ratio and droplet volume highlights the capability to precisely tune droplet size and volume by adjusting the flow rates, which is essential for applications requiring specific droplet volumes in high-throughput assays. The observed variation in droplet volumes further underscores the effectiveness of the microfluidic device in achieving consistent and reproducible droplet production under different flow conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe table presents flow rate optimization, droplet size, and Volume in the droplet generation microfluidic system.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQc (ml/hr)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eQd (ml/hr)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFlow rate ratio\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDroplet size (\u0026micro;m)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eVolume (m^3)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e678\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e639\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e447\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.056\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.050\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e287\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0101\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.033\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e262\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.00834\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.00648\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e174\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.00327\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.00206\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe fabricated PMMA-based three- layer microfluidic platform, developed via CO\u003csub\u003e2\u003c/sub\u003e laser engraving and followed by solvent-enhanced UV-irradiation bonding, demonstrated high structural integrity and functional reliability. A leakage test conducted with varying ethanol concentration confirmed that 90% ethanol concentration bonded device exhibited complete leak-free operation over 60 minutes. Compared to the previous method, bonding strategies include acetic acid or pressure-assisted bonding techniques (Trinh, 2020). Which showed deformation or instability, our approach provided superior sealing without compromising channel geometry. The surface wettability analysis showed a marked decrease in contact angle from 93.0\u0026deg; (untreated PMMA) to 69.8\u0026deg; after 90% ethanol bonding, indicating increased surface hydrophilicity. Correspondingly, the surface energy increased from 34.5 mJ/m\u0026sup2; to 64.8 mJ/m\u0026sup2;, enhancing fluid-substrate interaction critical for precise droplet formation and flow control. Using a T-junction with a squeezer mechanism, controlled droplet formation was accomplished. By adjusting the continuous phase flow velocity, monodisperse droplets with a diameter of 675 \u0026micro;m to 150 \u0026micro;m were produced. The PMMA platform eliminated swelling and deformation problems and showed better mechanical robustness, optical clarity, and chemical resistance when compared to traditional PDMS devices. Since the UV-assisted approach avoids high temperatures, it can be used for rapid prototyping without the need for cleanroom facilities, unlike thermal or pressure-based bonding. These improvements driven by chemical modification from UV irradiation and solvent interaction enhance the performance of the microfluidic device in the high-throughput screening. Controlled droplet together, the leakage resistance, improved structural integrity, controlled surface characteristics, and ease of fabrication without thermal or pressure and cleanroom requirements underscore the PMMA-based microfluidics device potential as a scalable, cost-effective alternative for droplet microfluidic applications. Future work will focus on the integration of active elements and multiplex assays used in biomedical diagnostic applications.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eDr. S. Gnanavel \u0026ndash; Conceptualization, Methodology, Validation, Formal analysis, Supervision, Project administration. Kaavya P - Methodology, Investigation, Data Curation, Writing - Original Draft and VisualizationAshwin Kumar N - Investigation, Resources, Formal analysis, Data Curation, Validation\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eThe author would like to thank Dr. Kannan, Assistant Professor, Department of Aerospace Engineering, for providing the High-speed camera used for imaging and recording the droplets in this study. Appreciation is also extended to Omega- Inspection and Analytical Laboratory, Maraimalar Nagar, for conducting the mechanical stability analysis. ANRF-SERB (CRG/2024/3902) for facilitating the contact angle measurement.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbalde-Cela, S., P. Taladriz-Blanco, M. G. de Oliveira, and C. 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Biosensors. 2023 Jan 20;13(2):161.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microfluidics-and-nanofluidics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mano","sideBox":"Learn more about [Microfluidics and Nanofluidics](http://link.springer.com/journal/10404)","snPcode":"10404","submissionUrl":"https://submission.nature.com/new-submission/10404/3","title":"Microfluidics and Nanofluidics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6102019/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6102019/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe research focuses on the development of a novel, cost-effective, three-layer droplet microfluidic device fabricated using Polymethyl methacrylate (PMMA) engineered for high-throughput screening in healthcare applications. PMMA offered improved optical transparency, chemical resistance, low absorption, and high scalability. Here, we evolved a T-junction integrated microchannel with a squeezer mechanism for consistent monodisperse droplet generation. Device fabrication was achieved via a laser ablation technique followed by an ethanol-enhanced UV-irradiation method for strong and leak-free bonding between the PMMA layers. The surface properties of the PMMA layer revealed an increased surface energy and uniform wettability. The tensile strength of fabricated PMMA microfluidic devices demonstrated superior bonding strength and structural integrity compared to the existing fabrication methods. The device reliably generated uniform monodisperse droplets up to a 100 ml/hr flow rate, confirming its robustness and suitability for high-throughput screening. Overall, this PMMA-based Microfluidics platform offers a scalable and reliable solution for droplet generation suitable for applications such as drug delivery, single-cell analysis, and diagnostic assays.\u003c/p\u003e","manuscriptTitle":"Development of a PMMA-based Droplet microfluidic device for High-throughput Screening in Health care Applications","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-29 11:44:56","doi":"10.21203/rs.3.rs-6102019/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-28T02:42:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-24T12:20:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"238408705053823680212704079898464036866","date":"2025-04-12T10:42:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-11T12:39:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-11T12:23:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microfluidics and Nanofluidics","date":"2025-04-08T12:57:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microfluidics-and-nanofluidics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mano","sideBox":"Learn more about [Microfluidics and Nanofluidics](http://link.springer.com/journal/10404)","snPcode":"10404","submissionUrl":"https://submission.nature.com/new-submission/10404/3","title":"Microfluidics and Nanofluidics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"09e074c7-d78c-41d3-95cd-f849016415df","owner":[],"postedDate":"April 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-09T16:06:52+00:00","versionOfRecord":{"articleIdentity":"rs-6102019","link":"https://doi.org/10.1007/s10404-025-02816-5","journal":{"identity":"microfluidics-and-nanofluidics","isVorOnly":false,"title":"Microfluidics and Nanofluidics"},"publishedOn":"2025-06-04 15:57:01","publishedOnDateReadable":"June 4th, 2025"},"versionCreatedAt":"2025-04-29 11:44:56","video":"","vorDoi":"10.1007/s10404-025-02816-5","vorDoiUrl":"https://doi.org/10.1007/s10404-025-02816-5","workflowStages":[]},"version":"v1","identity":"rs-6102019","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6102019","identity":"rs-6102019","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}