Multiscale 2PP and LCD 3D Printing for High-Resolution Membrane-Integrated Microfluidic Chips

Abstract

This study presents a microfluidic chip platform designed using a multiscale 3D printing strategy for fabricating microfluidic chips with integrated, high-resolution, and customizable membrane structures. By combining two-photon polymerization (2PP) for submicron membrane fabrication with liquid crystal display printing for rapid production of larger components, this approach addresses key challenges in membrane integration, including sealing reliability and the use of transparent materials. Compared to fully 2PP-based fabrication, the multiscale method achieved a 56-fold reduction in production time, reducing total fabrication time to approximately 7.2 h per chip and offering a highly efficient solution for integrating complex structures into fluidic chips. The fabricated chips demonstrated excellent mechanical integrity. Burst pressure testing showed that all samples withstood internal pressures averaging 1.27 ± 0.099 MPa, with some reaching up to 1.4 MPa. Flow testing from ~35 $\mathsf{\mu }$L/min to ~345 $\mathsf{\mu }$L/min confirmed stable operation in 75 $\mathsf{\mu }$m square channels, with no leakage and minimal flow resistance up to ~175 $\mathsf{\mu }$L/min without deviation from the predicted behavior in the 75 $\mathsf{\mu }$m. Membrane-integrated chips exhibited outlet flow asymmetries greater than 10%, indicating active fluid transfer across the membrane and highlighting flow-dependent permeability. Overall, this multiscale 3D printing approach offers a scalable and versatile solution for microfluidic device manufacturing. The method’s ability to integrate precise membrane structures enable advanced functionalities such as diffusion-driven particle sorting and molecular filtration, supporting a wide range of biomedical, environmental, and industrial lab-on-a-chip applications.

1. Introduction

Microfluidic systems are essential tools for the precise manipulation and analysis of small fluid volumes, enabling advances across biomedical diagnostics, environmental monitoring, and pharmaceutical development [1,2,3]. Among various operational strategies, passive sorting is particularly attractive, as it enhances separation efficiency without external energy input [4]. Passive sorting is widely used in biomedical diagnostics, environmental monitoring, pharmaceutical development, and many other fields [5,6]. Various techniques can be utilized for passive sorting; for instance, inertial methods rely on balancing lift and drag forces in microchannels to direct particles of different sizes or densities along distinct paths [7,8,9]. However, inertial methods face challenges related to material handling and exhibit high sensitivity to changes in flow rates during testing [10]. Adsorption methods exploit the affinity of certain molecules or particles for specific surfaces, facilitating selective capture and separation [11]. However, concerns regarding the non-specific binding of molecules limit the applicability of adsorption methods [12,13], and complications with fouling further limit the use of this technology [14].

Membrane-based separation has long been established in large-scale manufacturing and remains an attractive solution for microfluidics due to it’s low energy requirements and ability to acieve high selectivity [15,16,17,18]. Crossflow filtration offers further advantages over dead end filtration by recucing clogging and improving throughput with complex samples [19,20,21]. Despite this, integrating membranes directly into microfluidic and lab-on-a-chip (LOC) systems remains challenging. Conventional membrane incorporation methods often suffer from sealing issues, incompatibility with optical imaging, and limited geometric customizations [22,23,24,25,26,27,28,29,30]. Furthermore, traditional microfabrication techniques, such as soft lithography and hot embossing, struggle to achieve the sub-micron precision needed for advanced membrane applications [31,32,33].

The use of 3D printing in microfluidics opens new possibilities for the rapid prototyping and fabrication of intricate structures that are crucial for advanced device functionalities [34]. Despite these possibilities, the application of 3D printing for producing membranes that combine both high porosity and sub-micron pore sizes has been limited [35,36,37]. This limitation is significant because pore size and porosity are fundamental parameters in membrane technology applications [38,39,40,41]. One challenge is that typical 3D printing methods often rely on the inherent porosity of the printed material or produce pore sizes in the range of microns [42]. For example, fused deposition modeling (FDM) typically produces features in the range of 100–200 $\mathsf{\mu }$m, while stereolithography and digital light processing methods can achieve resolutions down to 10–100 $\mathsf{\mu }$m [43,44]. In contrast, two-photon polymerization (2PP) offers exceptional resolution in the hundreds of nanometers and has been employed for the fabrication of porous membranes and intricate microfluidic structures [45,46,47,48,49]. 2PP’s ability to fabricate transparent resins at sub-micron scales also makes it well-suited for the use of embedded optical and sensing components within microfluidic devices [50,51]. However, 2PP is limited by slow print speeds (typically ranging from a few millimeters per second up to several hundred), resin removal challenges, and difficulties in printing directly onto larger or prefabricated substrates [52,53,54].

Recent approaches have explored combining 2PP with other fabrication methods, such as printing directly onto PDMS or thermoplastics [55,56,57,58,59], and integrating 2PP features within DLP-printed microfluidic chips [60,61,62]. Nevertheless, these methods face alignment issues, material incompatibility, and difficulties in printing complex features within enclosed microchannels [63,64,65].

Building on these advancements and addressing current limitations, this work introduces a multiscale 3D printing strategy that combines the precision of 2PP with the scalability and speed of liquid crystal display (LCD) printing for microfluidic chip fabrication. As neither 2PP nor LCD printing alone satisfies the combined requirements of membrane-level resolution and large-scale component fabrication, this approach uses 2PP to fabricate integrated sub-micron membrane structures and small channel features, while LCD printing rapidly produces larger chip components. Post-print assembly simplifies integration and avoids alignment challenges associated with printing directly onto prefabricated structures.

The goal of this work is to describe a practical fabrication method and demonstrate its effectiveness for future LOC applications requiring integrated membranes. This method enables the fabrication of customizable, membrane-integrated microfluidic chips with strong sealing and mechanical integrity, offering a practical solution for advanced LOC applications requiring precise flow control and separation.

2. Materials and Methods

This section describes not only standard materials and testing procedures but also introduces the novel multiscale fabrication methods developed in this study, which are central to the contributions of this work.

2.1. Microfluidic Chip Design

The workflow for the combined 2PP and LCD printing approach begins with the design and preparation of digital files for both the 2PP and LCD components. Figure 1 provides computer designed and schematic views of the resulting integrated system.

2.1.1. 2PP-Printed Membranes and Channel Design  

The 2PP-printed membranes allow fluid passage in the vertical direction through the membrane, which facilitates cross-flow filtration. To support this functionality, the microfluidic system design included two parallel channels, each 3.05 mm in length, as depicted in Figure 2. The 3 mm long membrane was centrally printed within the channel configuration, with one channel positioned below the membrane and the other above it.

Each channel included a 25 $\mathsf{\mu }$m solid boundary at each end of the membrane to stabilize flow before and after the porous section (Figure 2d), creating an effective cross-flow filtration length of 3 mm. The cross-sectional dimensions of each channel in the 2PP component measured 75 by 75 $\mathsf{\mu }$m (Figure 2e). Flow directions for the cross-flow setup are indicated in Figure 2b,c, highlighting the fluid paths through these channels.

2.1.2. LCD-Printed Tubing Adaptors  

The LCD-printed tubing adaptors, presented in Figure 3, were designed to accommodate fluorinated ethylene propylene (FEP) tubing, which has an inner diameter of 120 $\mathsf{\mu }$m and an outer diameter of 650 $\mathsf{\mu }$m, to enable compatibility with the microfluidic testing setup. A key feature of the adaptors was a 5.02 mm diameter cylindrical void at the rear, which serves as a magnet attachment site to be used for alignment with the 2PP component. To ensure a secure seal at the 2PP-LCD interface, resin is applied at a designated point (marked by a red arrow in Figure 3a) so that it will flow around the hatched red area in Figure 3b. A tolerance of 0.05 mm at the 2PP-LCD component interfaces provides space for easy alignment and effective resin distribution to ensure a robust and leak-proof connection.

2.2. Microfluidic Chip Fabrication

To fabricate an integrated microfluidic chip with precision membranes, we utilize a multiscale 3D printing approach, combining 2PP and LCD printing techniques. This method was developed to allow for the integration of high-resolution membrane structures within a scalable microfluidic system.

2.2.1. Membrane and Channel Fabrication Using 2PP Printing  

2PP printing operates on the principle of two-photon absorption, where two photons of lower energy are absorbed simultaneously by a photopolymer material to induce localized polymerization. This enables the fabrication of structures with resolutions in the hundreds of nanometers. In our previous work, we have shown the diverse membrane geometries 2PP can produce by manipulating printer-slicing software and carefully tailoring print properties. This technique enabled the creation of membranes with pore diameters as small as 0.57 $\mathsf{\mu }$m and porosities up to 60%, facilitating precise control over fluid dynamics and filtration processes within LOC devices [47]. Figure 4 shows the slicing paths and resulting printed structures of various 2PP-printed membranes.

The 2PP-printed component of the chip was produced using the Nanoscribe 2PP 3D Printer (Photonic Professional GT System, Nanoscribe GmbH, Karlsruhe, Germany) and Ormocomp (Micro Resist Technology GmbH, Berlin, Germany), chosen for its excellent mechanical properties, transparency easy flow visualization, and ease of printing. The properties of Ormocomp are shown in

Table A1. Key chemical and mechanical properties of resins used in this study, including suppliers, viscosity, curing wavelengths, and applications.

Resin Type	Supplier Name	Viscosity (cP)	Curing Wavelength (nm)	Application
2PP	micro resist technology GmbH	100	780	High Resolution Membrane and Channel
LCD	Anycubic	500–600	405	Channel Adaptors

in Appendix A. The overall process for fabrication of the 2PP-printed component is shown in Figure 5.

The fabrication begins with the preparation of an ITO-coated glass substrate (Figure 5a). As shown in Figure 5b, the slide is first coated with Ormoprime (Micro Resist Technology GmbH, Berlin, Germany) to promote adhesion. Liquid Ormocomp is then applied to the surface (Figure 5c) to serve as the photopolymer material. Following preparation, the 2PP component is printed directly onto the substrate (Figure 5d). Post-processing involves a 2.5 h development in OrmoDev solution (Figure 5e), followed by an in-bath flood exposure to stabilize the structure (Figure 5f) and a secondary exposure as OrmoDev evaporates from the printed component (Figure 5g). These steps ensure complete removal of unpolymerized resin and enhance the mechanical stability of the final structures.

Key printing parameters were optimized for different components to ensure functionality and structural integrity. Non-membrane components were fabricated at a laser power of 100% and a scanning speed of 80 mm/s, while the membranes, were printed with a designed thickness of 10 $\mathsf{\mu }$m, a hatching pitch of 4 $\mathsf{\mu }$m, and a hatching angle of 90°, using a laser power of 100% and a scanning speed of 45 mm/s.

2.2.2. Tubing Adaptor Fabrication with LCD Printing  

LCD printing uses an LCD screen as a mask to selectively block or allow light to pass through pixel by pixel. Behind the LCD screen is a 405 nm LED light source, which shines through the LCD pixels to cure the resin in the desired pattern. The process repeats for each subsequent layer to form the 3D object. LCD printing excels in speed compared to point-by-point additive manufacturing techniques. However, the resolution is dependent on the pixel density of the LCD screen, which limits its ability to create features at sub-micron scales. Despite this limitation, LCD is an efficient solution for constructing larger structural elements for microfluidic components where only moderate resolution is needed.

The overall fabrication process for the LCD printed components is shown in Figure 6. The Anycubic Mono 4K LCD printer (Anycubic Technology Co., Ltd, Shenzhen, China) and Anycubic Clear Standard Resin (Anycubic Technology Co., Ltd, Shenzhen, China) were used to print the tubing adaptor components of this design. The properties of the Anycubic resin are shown in Table A1 in Appendix A. After printing, the adaptors undergo development in two isopropyl alcohol (IPA) baths: a 5 min immersion in the initial bath, followed by an additional 5 min in a fresh IPA bath. After these steps, the still-wet adaptors are purged with $N_2$ to remove any residual resin or IPA from the channels, ensuring the channels are clear of any partially developed resin that could obstruct flow. A final 2 min UV exposure in the printer’s curing station provides complete crosslinking for durable component performance.

2.2.3. Chip Assembly  

Upon completing the 2PP and LCD printing of the microfluidic chip, assembly begins. Figure 7 illustrates the alignment steps for combining the printed components. The process begins with the 2PP printed component on a glass slide (Figure 7a). Neodymium magnets with a 5 mm diameter and 0.9 mm thickness are positioned at either end of the channel. The magnets act as passive guides, allowing for easier manual alignment of features across the two print types. The magnets remain in place throughout the sealing process but do not contribute any clamping or mechanical force. Their purpose is purely positional, assisting with consistent and repeatable component placement. One magnet is placed beneath the slide on the non-component side, while four magnets are stacked on top to hold the system in place (Figure 7b). The glass slide and magnets are placed into a custom LCD-printed alignment tray, and the LCD-printed channel components are positioned onto the alignment magnets as shown in Figure 7c. Manual adjustments are made to align each LCD component with its corresponding 2PP interface (Figure 7d). Alignment of the system can be carried out under a microscope if additional visual precision is needed, but a 0.05 mm tolerance between the 2PP and LCD component interfaces facilitates manual alignment, allowing for precise fitting with minimal resistance. When properly aligned, the LCD component is flush with the surface of the glass slide, as shown in Figure 7e. Once both LCD-printed components are aligned (Figure 7f), the system is ready for compression and resin sealing.

In Figure 8, the compression and sealing process for the chip is shown in detail. The setup consists of a compression system primarily made from FDM-printed components, as well as a metal base plate at the bottom, and a 0.75-inch thick, 1.5-inch diameter quartz lens is used to compress and seal the chip. The metal plate and quartz lens provide rigid, non-pliable surfaces for compression and the lens allows sufficient UV transmission for resin curing.

To begin, the aligned system from Figure 7f is placed onto the metal base plate within the compression system, with a thin support structure around the chip (Figure 8a). The support structure stabilizes the quartz lens on top of the taller sections of the LCD-printed chip components (Figure 8b), and the support structure is shorter than the LCD component height to avoid interfering with compression. Next, the lens collar is inserted into the system (Figure 8c) to secure the quartz lens. This is followed by the top piece, which is screwed in to apply even pressure to the lens collar (Figure 8d). This downward force compresses the LCD components down onto the 2PP component as illustrated in Figure 8e, ensuring a secure interface. Ormocomp resin is then applied to designated reservoirs using a syringe (Figure 8f). A 5 min waiting period allows resin to flow around the 2PP and LCD interface (Figure 8g). The system is then exposed to ~80 mW/cm² UV light for 5 min to cure the resin (Figure 8h). Once removed from the compression system, the sealed chip (Figure 8i) is ready for testing and performance evaluation.

2.3. Burst Pressure Testing

Burst pressure testing was performed using the NE-1000 One Channel Programmable Syringe Pump (New Era Pump Systems Inc., Farmingdale, NY, USA) equipped with a 2.25 mL glass syringe. This pump was chosen for pressure testing because it was the most robust pumping system available. Dyed water was infused into the microfluidic chip at a rate of 100 $\mathsf{\mu }$L/min. A Measureman Digital Industrial Pressure Gauge (Measureman Direct, Nimo Harmony Sarl, Levallois-Perret, France) was integrated into the setup to monitor internal pressure in real time.

Three of the four inlet/outlet ports on the chip are sealed by applying a drop of Ormocomp resin over each port, followed by UV curing (approximately 80 mW/cm² for 5 min) to ensure a robust and leak-free seal. FEP tubing was used to connect the syringe, pressure gauge, and chip. Pressure was increased in the system until visible failure or a sudden pressure drop indicated structural failure of the chip, or until the maximum force capacity of the syringe was reached. The burst pressure was recorded as the maximum pressure sustained immediately before failure. If the syringe pump max pressure was reached without chip failure, each chip was tested in triplicate to assess the variability and reproducibility of the results.

2.4. Fluidic Testing

To evaluate the performance of the microfluidic chip, fluidic testing was conducted using a BASi Baby Bee Syringe Drive (BASi Research Products, West Lafayette, IN, USA) with two 2.25 mL glass syringes mounted on the pump. The use of these different volume syringes with the BASi pump requires calibration via a gravimetric analysis described below. Flow rates tested ranged from approximately 35 $\mathsf{\mu }$L/min to 345 $\mathsf{\mu }$L/min. The schematic diagram for the cross-flow testing setup is shown in Figure 9a with a lengthwise cross-sectional diagram of the chip’s internal structure for cross-flow filtration shown in Figure 9b.

Flow rates were measured gravimetrically over six 1 min intervals by collecting fluid at the outlet and calculating output rates. To assess differences in flow characteristics and membrane functionality, we tested flow both through the tubing directly connected to the syringe pumps (without the microfluidic chip) and through the microfluidic chip system. The chip featured channels with either completely solid walls or membranes printed between two parallel 75 $\mathsf{\mu }$m square channels. Because direct pressure measurement was not available during the initial fluidic testing and would have required significant modifications to the microfluidic setup, pressure drops across the channels were instead calculated from measured volumetric flow rates using the Hagen–Poiseuille equation. This approach provided a reliable and non-invasive method for estimating pressure while maintaining the integrity and simplicity of the testing configuration. The Hagen–Poiseuille equation (Equation (1)) describes the pressure drop $(\Delta P)$ for laminar flow through a long, narrow channel with a square cross-section:

$$\Delta P=0.63{\displaystyle \frac{8\eta QL}{\pi {\left(\frac{a}{2}\right)}^4}}$$

(1)

where $\Delta P$ is the pressure difference between the two ends of the channel, ($\eta$) is the dynamic viscosity of the fluid (Pa·s), Q is the volumetric flow rate (m³/s), L is the length of the channel (m), and a is the height and width of the square channel (m). For this work, water was used as the fluid and has an approximate viscosity of 1 mPa·s. The height and width of the channels are 75 $\mathsf{\mu }$m and the channel length is 3 mm. This equation is simplified with known constants to result in an output $\Delta P$ in kPa:

$$\Delta P=0.041\ast Q$$

(2)

where Q is the volumetric flow rate in $\mathsf{\mu }$L/min. This equation allows for the calculation of the pressure drop across the channel for each flow rate that is used during testing. This is essential for determining the fabrication method’s robustness, as higher-pressure tolerances allow for diverse testing and potentially high-throughput applications to be studied [66]. Additionally, the flow can be characterized using linear flow velocity (v) as calculated using Equation (3):

$$v={\displaystyle \frac{Q}{a^2}}$$

(3)

Equation (3) can be simplified to determine linear velocity in m/s from volumetric flow rate (Q) in $\mathsf{\mu }$L/min:

$$v=0.003\ast Q$$

(4)

From linear velocity the Reynolds number (Re) for each test condition was calculated using

$$Re={\displaystyle \frac{\rho v{D}_h}{\eta }}$$

(5)

where $\rho$ is the density of water (997 kg/m³), v is the linear velocity in the channel, D_h is the hydraulic diameter of the square channel (in this case 75 $\mathsf{\mu }$m), and $\eta$ is the dynamic viscosity of the fluid. These characterizations of pressure drop, flow velocity, and Reynolds number are crucial to understanding the chip’s behavior under varying flow rates, verifying the channel’s tolerance, and understanding the potential applications for which this fabrication method could be utilized.

3. Results and Discussion

3.1. Membrane Integration and Chip Fabrication

3.1.1. Challenges and Solutions in Membrane Incorporation

During preliminary testing, membranes were visible during the 2PP printing phase (Figure 10a). However, it was determined that membranes were tearing away from the edges of the microchannels as the developer evaporated, as shown in Figure 10b. Although 10 $\mathsf{\mu }$m wide support beams were added to the channel structure with 100 $\mathsf{\mu }$m and 75 $\mathsf{\mu }$m spacing to investigate the level of support needed to secure the membrane in place, even with the addition of support structures, membrane tearing persisted. This is shown in Figure 10d,f where the support structures slowed the tearing and deformation but did not fully prevent it. Further observation revealed that tearing was primarily occurring at the fluid–air interface during evaporation after the developer bath.

To address this challenge, structures printed with integrated membranes were produced using Ormocomp, soaked in developer for 2.5 h, and subsequently flood-exposed to 80 mW/cm² of UV light for 10 min while still immersed in the developer bath. Following this initial bath exposure, the structures were removed from the bath and immediately subjected to a second UV exposure for an additional 10 min at 80 mW/cm² as the developer evaporated. The in-bath exposure serves two purposes: the fluid developer surrounds and supports the membranes in place and preliminary crosslinking of the Ormocomp resin strengthens the material before evaporation. The secondary exposure further crosslinks the material and stabilizes the structure as the developer evaporates. SEM imaging in Figure 10g,h confirms the membrane’s placement across the channel width. Despite the lengthy 2.5 h development process required for these narrow, elongated channels, the combination of 100 $\mathsf{\mu }$m spaced, 10 $\mathsf{\mu }$m wide support beams and the wet–dry UV exposure method effectively prevented membrane detachment and deformation. This approach addressed the challenges observed in preliminary testing, ensuring reliable membrane integration throughout the development process.

3.1.2. 2PP and LCD Component Alignment

The assembly process demonstrated a high degree of precision and reliability in aligning and sealing the 2PP- and LCD-printed components. During development, the 2PP component interfaces with LCD components tend to lift slightly from the print substrate, as shown in Figure 11a. However, this lifting did not significantly affect the alignment of the 2PP interfaces with their corresponding LCD components. With minimal practice, manual alignment of the LCD components was consistently achieved within approximately 2 min. Additionally, as shown in Figure 11b,c, the LCD components compress the 2PP parts back into position on the surface. This compression step further seats the 2PP components, and the subsequent resin sealing ensures stability.

Figure 12 illustrates the alignment and sealing process of one print interface up close. SEM images in Figure 12a–d provide both isometric and head-on perspectives of the structure of the 2PP and LCD component interfaces. Microscope images in Figure 12e,f further show the precise alignment and effective sealing of the components. Figure 12e shows the underside of the glass slide when the 2PP and LCD components are aligned. This alignment shows that the 2PP and LCD channel’s fluid flow pathways are unobstructed in alignment and that there are small gaps surrounding the sides of the 2PP component to allow the resin to flow around the interfaces.

Figure 12f shows the completed resin seal, which not only effectively seals the channel, preventing potential fluid leakage, but also provides additional structural stability. Additionally, in Figure 12f, arrows highlight bubbles formed during the resin sealing process when displaced air escapes through the resin vat or becomes trapped in smaller areas. These bubbles are not a concern because the outside air does not connect to the 2PP-LCD interface and no leakage is observed.

In Figure 12f, additional validation of the chip’s sealing quality is provided. Blue-dyed water was manually injected into two inlets using a syringe, allowing for a full fill of the chip. Under microscope observation, the chip displayed no visible leaks, highlighting the effectiveness of the resin seal and the robustness of the alignment process. Together, these observations confirm the successful integration of 2PP and LCD components, as well as the quality of the resin sealing. The seamless interface and effective seal are crucial for ensuring stable and leak-proof fluidic channels, which are necessary for reliable microfluidic testing and analysis.

3.1.3. Chip Fabrication Time

The fabrication process using both 2PP and LCD printing offers significant time savings over fully 2PP-fabricated chips. According to the 2PP printer’s slicing software, the estimated time to print a single LCD tubing adaptor structure as designed using only 2PP is approximately 605.5 h, meaning a full system would require 1214.5 h to complete the channels, membranes, inlets, and alignment components. If the LCD component design is simplified by removing alignment features and printing only the channels and inlets via 2PP, the projected print time reduces to 201 h per side, which is still a total projected 405.5 h of print time for a system with a robust tubing inlet.

In contrast, the multiscale printing process reduces the total fabrication time for one chip to 7.2 h of equipment and labor time. This is over 56 times faster than the reduced tubing connector design print time. Additionally, there are only about 30 min of active labor per chip with the dual printing method. The primary time-consuming steps are the 2PP printing (3.5 h) and post-processing (2.7 h), while LCD components require only about 20 min for printing, 12 min for post-processing, and an estimated 30 min for assembly and sealing.

This process can be further optimized by producing multiple chips in parallel. The LCD printer can produce 80 adapter components in a single run, bringing the average LCD print time per part to about 15 s. Additionally, multiple 2PP chips can be developed simultaneously, which similarly reduces the impact of the 2PP post-processing time. For example, if four chips are printed in a day before post-processing, the time per chip is reduced to 40 min. Although the 2PP printing step cannot be shortened by grouping, the 3.5-h print duration makes this method suitable for rapid prototyping in research contexts, even if it falls short of the efficiency needed for mass production. Overall, this approach reduces active labor and equipment time, which offers a more efficient workflow compared to fully 2PP printing chips, making it a viable solution for research applications requiring fast turnaround and flexibility.

3.2. Burst Pressure Testing

Pressure testing was conducted on eight microfluidic chips to evaluate the structural robustness of the fabricated devices. Each chip was tested in triplicate to assess performance consistency. As shown in Figure 13a, steady input flow rates produced an exponential increase in pressure over time, which was ultimately limited by the mechanical capacity of the syringe pump. Maximum recorded pressures for each test are summarized in Figure 13b.

Across all tests, the chips demonstrated excellent durability. On average, chips withstood pressures of 1.27 ± 0.099 MPa, with some samples enduring pressures as high as 1.4 MPa before the pump’s maximum force capacity was reached. Notably, no chip failure was observed in any trial, and testing was only concluded upon reaching the upper pressure limit of the pumping system. Minor variations in maximum recorded pressures were observed across tests. These differences are likely attributable to variability in the syringe pump’s mechanical drive system, slight differences in chip sealing, or the compressibility of the fluid under elevated pressures, rather than to inconsistencies in chip fabrication or bonding.

The ability of all chips to withstand pressures exceeding 1 MPa highlights the robustness of the bonding and structural integrity of the assembled devices. This performance compares favorably to existing bonding and integration methods reported in the literature. For example, cycloolefin polymer (COP)-COP bonding via vapor-phase cyclohexane typically achieves burst pressures near 500 kPa [54], and surface-treated COP systems have reported burst pressure ranges between 0.4 MPa and 4 MPa, depending on treatment protocols and channel geometries [63]. Recent reports on 2PP-printed membranes integrated into LCD-printed PDMS chips have demonstrated survival only up to approximately 100 kPa without failure [59]. In comparison, the chips developed in this study substantially exceed the performance of similar 2PP-based integration approaches. These findings confirm the viability of the multiscale printing and assembly method for pressure-intensive microfluidic applications, where reliability under high-pressure conditions is essential.

3.3. Fluidic Testing

The flow rates tested, especially those over ~100 $\mathsf{\mu }$L/min, while higher than typical values for microchannels with dimensions in the tens of micrometers, were intentionally selected to evaluate the robustness of the integrated membrane under elevated pressure and flow conditions. No visible damage to the membranes or leakage was observed during testing, suggesting that the 2PP-fabricated membranes and channels maintained mechanical integrity at these elevated flow rates. The fluidic testing results, including flow rate measurements, as well as the corresponding calculated pressure drops, linear flow velocities, and Reynolds numbers at various flow rates for both the solid wall and membrane-integrated chips, provide valuable insights into chip performance. For instance, the Reynolds numbers calculated across all measured volumetric flow rates (32.6 $\mathsf{\mu }$L/min–346 $\mathsf{\mu }$L/min) range from approximately 7 to 76. These low Reynolds numbers indicate that the flow remains within the laminar regime throughout all experiments. Figure 14 compares the syringe pump outlet to the outlet of the solid wall chips.

Figure 14a compares the approximate flow rate from the syringe (measured without the chip) to the outlet flow rates for the solid wall chip. At the same pump conditions that lead to syringe flow rates up to 175 $\mathsf{\mu }$L/min, the solid wall chips outlet flow rates closely match the syringe values, indicating minimal resistance in the solid wall configuration. However, as the syringe flow rate increases to 250 $\mathsf{\mu }$L/min and 345 $\mathsf{\mu }$L/min, the solid wall chip outlet flow rates drop to 218 $\mathsf{\mu }$L/min and 270 $\mathsf{\mu }$L/min, respectively. Based on ANOVA statistical analysis of the data at a syringe flow rate of 250 $\mathsf{\mu }$L/min, the outlet flow rates were significantly lower, with Syringe 1 to Solid Wall Outlet 1 (p = 0.0099) and Syringe 2 to Solid Wall Outlet 2 (p = 0.0050) showing measurable differences. Similarly, at 345 $\mathsf{\mu }$L/min, Syringe 1 to Solid Wall Outlet 1 (p = 0.0015) and Syringe 2 to Solid Wall Outlet 2 (p = 0.0014) exhibited significant reductions in outlet flow rates compared to the syringe flow rates. While no leakage was observed during these higher flow rate tests, a noticeable pressure buildup was evident when the inlet tubing was disconnected, causing water to exit rapidly due to accumulated pressure. This pressure buildup likely represents the limitations of the syringe pump as it reached its maximum output.

The pressure buildup observed at high flow rates is consistent with the calculated pressure drop presented in Figure 14b. The relationship between inlet flow rate and pressure drop results follows the Hagen–Poiseuille equation, which predicts a linear trend between pressure drop and volumetric flow rate. The trendline for inlet flow rate is given by $y=0.0406Q$, where Q is the flow rate in $\mathsf{\mu }$L/min. This trendline is similar to the projected equation (Equation (2)), providing an analytical model that is consistent with the theoretical model to compare chip results to. However, for the solid wall chip’s outlet flow rate, a quadratic function provides a better fit. These results indicate a measurable increase in resistance and flow imbalance at higher flow rates, which could be attributed to internal channel design or wall friction that is more pronounced at the upper limits of pump performance.

Figure 15 compares the performance of solid wall and membrane-integrated chips. At the lowest flow rate of 35 $\mathsf{\mu }$L/min, ANOVA analysis indicates that no statistically significant differences exist between the solid wall and membrane chips due to minimal flow variation. However, as the flow rate increases, differences can be observed. At a flow rate of 70 $\mathsf{\mu }$L/min the most significant difference in flow rates observed is between Membrane Outlet 1 (70.9 $\mathsf{\mu }$L/min) and Membrane Outlet 2 (63.7 $\mathsf{\mu }$L/min), representing a 10.7% difference with p = 1.746 × 10⁻⁵. At 175 $\mathsf{\mu }$L/min, the differences become even more pronounced, with a statistically significant difference between Solid Wall Outlet 1 and Membrane Outlet 1 (p = 6.56 × 10⁻⁶), and between Solid Wall Outlet 2 and Membrane Outlet 2 (p = 4.12 × 10⁻⁶). Additionally, a p-value of 0 between Membrane Outlet 1 and Membrane Outlet 2 confirms a significant imbalance. Membrane Outlet 1 has a flow rate of 160.3 $\mathsf{\mu }$L/min, while Membrane Outlet 2 reaches 184.1 $\mathsf{\mu }$L/min, indicating a 13.8% preferential flow from Channel 1 to Channel 2 across the membrane. The 3.1% increase in flow asymmetry at a higher volumetric flow rate suggests that the membrane permeability is flow-dependent, highlighting the influence of operational conditions on membrane performance.

At the highest flow rate tested (~345 $\mathsf{\mu }$L/min), both chip designs exhibit considerable variations in outlet flow rates, as evidenced by large standard deviation bars. These fluctuations are likely due to a large buildup in the system, leading to periods of increased flow when pressure temporarily overcomes resistance, followed by slower flow as pressure buildup decreases in the syringe.

The Hagen–Poiseuille pressure–flow relationship for membrane-integrated chips, shown in Figure 15b, reveals how membrane incorporation affects flow behavior. The membrane chip’s trendline has a slightly lower intercept and a higher coefficient for the linear term compared to the solid wall chip, indicating an additional resistance component introduced by the membrane. This increase in resistance in the membrane chip, while subtle, is consistent with the membrane’s role in creating a controlled barrier for diffusion or particle filtration applications. Similarly to the solid wall testing, no leaking was observed for the membrane chip, implying that the additional pressure build-up is occurring within the syringe pump and system tubing.

Linear flow velocity trends (Figure 15c) mirror the trends in the Hagen–Poiseuille pressure drop results. The linear components of the trendlines close to the projected model (Equation (4)), indicating that both chip designs maintain consistent behavior up to a threshold flow rate, beyond which system resistance is too high to consistently overcome. Additionally, in the membrane chip flow velocity and pressure drop plots, there is a noticeable spread in the data points around the projected trendline compared to the solid wall chip data, reinforcing the idea that fluid transport across the membrane introduces subtle variations in flow dynamics. Although some deviation in outlet flow rates could stem from minor hydrodynamic resistance introduced by the integrated membrane, the imbalance was more strongly linked to external setup factors. Flow distribution shifted depending on pump positioning and tubing lengths, even when using the same chip, suggesting that these variations dominate over internal effects.

Maintaining a steady linear flow velocity is essential for experiments requiring controlled diffusion or particle sorting. Given the observed flow variations, a pull component in the fluidic setup would be beneficial. Introducing a downstream pull could help maintain consistent linear velocity across the membrane surface, which is crucial for precise control in applications needing stable fluid dynamics.

4. Conclusions

This study demonstrates a multiscale 3D printing strategy that integrates 2PP and LCD printing to fabricate microfluidic chips with customizable, high-resolution membrane structures. This approach substantially improves manufacturing efficiency, reducing total fabrication time from an estimated 400 h for a similar fully 2PP-printed channel system to approximately 7 h per chip, an over 56-fold decrease, while maintaining precision and structural integrity.

The printed chips exhibited excellent mechanical robustness, sustaining pressures up to 1.4 MPa without failure. Flow testing further confirmed stable operation across a wide range of flow rates, with membrane integration introducing minor variations in outlet distribution that can be controlled with a pump at each outlet of the chip, highlighting the system’s potential for tunable flow regulation.

Overall, the combined 2PP and LCD approach offers a scalable and adaptable platform for microfluidic device manufacturing. The ability to integrate precise membrane structures into robust chip designs expands the capabilities of LOC systems and supports applications in biomedical diagnostics, environmental analysis, and industrial processing. Additionally, the spatial precision of 2PP enables the fabrication of more complex architectures, including but not limited to chips with vertically integrated membranes, multiple membrane units, and serpentine or branched channel layouts. These structures can be tailored within the 2PP section without a need to change the overall 2PP-LCD approach.

The demonstrated balance of precision, reliability, and manufacturing efficiency positions this approach as a valuable contribution to the advancement of micro- and nanoscale manufacturing technologies. Looking forward, combining complementary 3D printing technologies holds significant promise for advancing microfluidic device fabrication. As these methods continue to mature, we anticipate increased integration with emerging application areas such as organ-on-chip systems, point-of-care diagnostics, and filtration-driven biochemical assays. Furthermore, innovations in printable materials and automated assembly may enable more sophisticated, multifunctional LOC platforms.