Efficient and cost-effective rapid prototyping method for accelerated production of thermoplastic micro- and milli-fluidic devices

Abstract

A method of fabricating a microfluidic device is disclosed, including creating a device template which may include one or more features on a surface of the device template, casting an inverse replica cavity mold of the device template, casting an embossing stamp by pouring a stamp material into the inverse replica cavity mold of the device template, forming the embossing stamp, and pressing the embossing stamp into a thermoplastic material to form a microfluidic device. The microfluidic device may include one or more channels having a cross-sectional dimension of from about 50 pm to about 1000 pm.

Description

Efficient and Cost-Effective Rapid Prototyping Method for Accelerated Production of Thermoplastic Micro- and Milli-fluidic DevicesREFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 538,207, filed on September 13, 2023, which is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0002] The present teachings relate generally to rapid prototyping of devices and, more particularly, to scalable production of microfluidic devices.BACKGROUND

[0003] The development of microfluidic devices faces the challenge of balancing ease of use and speed during prototyping with the ability to achieve scalable mass production with affordable and rapid fabrication processes for commercialization. Traditionally, to prototype microfluidic devices, molds are first fabricated via photolithography, utilizing spin coating and photopatteming of light sensitive resins. Subsequently, flexible polydimethylsiloxane (PDMS) replicas are created through casting. While this workflow is effective in research settings, it becomes cumbersome and time sensitive when attempting to scale up production capabilities.

[0004] Specifically, creating multilayer molds with features of different heights poses challenges and incurs high material and equipment costs. Additionally, PDMS has limitations as a device substrate due to poor chemical compatibility, biomolecule bioabsorption, batch consistency and swelling. In contrast, thermoplastics, such as poly(methyl methacrylate) (PMMA), offer a significant advantage for mass production of devices due to their compatibility with industrial manufacturing standards. Moreover, they possess a range of desirable characteristics that make them well-suited for use as biomedical devices / consumables, including optical clarity, rigid structures, and biocompatibility. Thermoforming methods widely adopted in industrial scale manufacturing, such as hot embossing, injection molding, or roller imprinting, enable quick and efficient fabrication with thermoplastics.1SUBSTITUTE SHEET (RULE 26)

[0005] However, transitioning from PDMS devices into PMMA for commercialization and mass production often requires re -optimization, which prolongs the time for technology translation. Aside from soft lithography from microfabricated molds, microfluidic devices have also been prototyped using emerging additive manufacturing technologies, but the throughput of these methods remains low. Others have proposed using microfabricated molds and a series of intermediates before ultimately molding thermoplastics, but this workflow still requires costly mold microfabrication.

[0006] Therefore, it is desirable to fabricate molds on a milli- or micro- scale devices utilizing a rapid, reliable, and repeatable method.SUMMARY

[0007] The following presents a simplified summary in order to provide a basic understanding of some aspects of one or more embodiments of the present teachings. This summary is not an extensive overview, nor is it intended to identify key or critical elements of the present teachings, nor to delineate the scope of the disclosure. Rather, its primary purpose is merely to present one or more concepts in simplified form as a prelude to the detailed description presented later.

[0008] A method of fabricating a microfluidic device is disclosed. The method of fabricating a microfluidic device includes creating a device template which may include one or more features on a surface of the device template, casting an inverse replica cavity mold of the device template by immersing the surface of the device template in a molding composition, casting an embossing stamp by pouring a stamp material into the inverse replica cavity mold of the device template, forming the embossing stamp, and pressing the embossing stamp into a thermoplastic material to form a microfluidic device.

[0009] Implementations of the method of fabricating a microfluidic device can include where the template is created using a three-dimensional printing method. The three-dimensional printing method further may include stereolithography (SLA). The microfluidic device may include one or more channels including a cross-sectional dimension of from about 50 pm to about 1000 pm. The one or more channels may include a cross-sectional dimension of from about 150 pm to about 600 pm. The thermoplastic material may include a polyacrylate, an acrylonitrile butadiene styrene, a polyamide, a poly(amide-imide), a polylactic acid, a polycarbonate, or a combination thereof. The thermoplastic material may include poly(methyl methacrylate). The molding composition may include a silicone or polydimethylsiloxane (PDMS). The method of fabricating a microfluidic device may include designing the device template with the use of computer aided design (CAD). Forming the embossing stamp further may include heating the stamp material. The method of fabricating a microfluidic device may2SUBSTITUTE SHEET (RULE 26)include heating the embossing stamp subsequent to casting the embossing stamp. The embossing stamp is heated to a temperature from about 120°C to about 160°C. The method of fabricating a microfluidic device may include heating the thermoplastic material prior to pressing the embossing stamp into a thermoplastic material. The method of fabricating the microfluidic device is conducted in an uncontrolled environment. The embossing stamp material may include a thermosetting resin. The embossing stamp material may include a non-metal.

[0010] Another method of fabricating a microfluidic device is disclosed. The method of fabricating a microfluidic device can include creating a device template may include one or more features on a surface of the device template, casting an inverse replica cavity mold of the device template by immersing the surface of the device template in a molding composition, casting an embossing stamp by pouring a thermosetting resin into inverse replica cavity mold of the device template, curing the thermosetting resin to fully form the embossing stamp, and pressing the embossing stamp into a thermoplastic material to form a microfluidic device.

[0011] Implementations of the method of fabricating a microfluidic device can include where the microfluidic device may include one or more channels may include a cross-sectional dimension of from about 50 pm to about 1000 pm. The method of fabricating the microfluidic device is conducted in an uncontrolled environment. The method of fabricating a microfluidic device may include heating the embossing stamp subsequent to casting the embossing stamp to a temperature from about 120°C to about 160°C.

[0012] The features, functions, and advantages that have been discussed can be achieved independently in various implementations or can be combined in yet other implementations further details of which can be seen with reference to the following description.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the present teachings and together with the description, serve to explain the principles of the disclosure. In the figures:

[0014] FIG. 1 is a schematic overview of a rapid fabrication process, in accordance with the present proposal.

[0015] FIG. 2 is a representative circuit diagram to represent different CC components, showing resistances and capillary pressures of relevant components that were optimized with this model to approximate channel geometries required to achieve desired flow patterns, in accordance with the present disclosure.3SUBSTITUTE SHEET (RULE 26)

[0016] FIGS. 3A-3G depict illustrations of the rapid fabrication process including FIG. 3 A, which is a schematic (Process Step and Final Product) and pictorial (Top View) representation of each fabrication step, FIG. 3B, which is a comparison of measured vertical and horizontal dimensions of 3D printed and PMMA features,. The entire workflow was replicated 3xto assess process variability. The line represents the 1: 1 dimension match between substrates, with the shaded region representing 10% deviation for a given dimension. FIG. 3C is a Vertical (V) and Horizontal (H) dimension comparison between PMMA Replicates 5 and 10, normalized to Replicate 1 using the same epoxy mold. FIG. 3D is atop view schematic of an exemplary design for use in a method for mass production of thermoplastic micro- and milli-fluidic devices, in accordance with the present disclosure. FIG. 3E is a series of SEM images of a staircase feature on the 3D print template, PDMS mold and PMMA replicate. FIG. 3F is a comparison of the PDMS cross section of a 3D printed 150pm and 1000pm square channel to contextualize dimension measurements. FIG. 3G shows a vertical and horizontal dimension comparison of PMMA features produced from a Form3 printed, PolyJet printed, photolithographic silicon mold, and High Temperature (HT) resin templates to demonstrate the versatility of the fabrication workflow with different molds and the ability to produce features smaller than those shown in FIG. 3B, in accordance with the present disclosure.

[0017] FIG. 4A is a schematic of a single device fully loaded with diluted food dye at the instance when flow out of Branch 1 was initiated. FIG. 4B is a plot of branch depletion time (n=18) after all the solutions were loaded, prior to depletion of Branch 1.

[0018] FIG. 5 A is a schematic representation of different reagents loaded into the CC, in accordance with the present disclosure. FIG. 5B is a plot comparing the mean fluorescence intensity of pre -determined regions of interest across different devices testing various BSA- FITC concentrations, in accordance with the present disclosure.

[0019] It should be noted that some details of the figures have been simplified and are drawn to facilitate understanding of the present teachings rather than to maintain strict structural accuracy, detail, and scale.DETAILED DESCRIPTION

[0020] Reference will now be made in detail to exemplary embodiments of the present teachings, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same, similar, or like parts.

[0021] Rapid prototyping comprises a set of techniques used to fabricate physical objects or components of a system directly from computer-aided design (CAD) data before the final4SUBSTITUTE SHEET (RULE 26)product is manufactured. This process significantly enhances overall product development by enabling the prediction, evaluation, and iteration of design features, concepts, and operational functionality and performance. The efficiency gained through rapid prototyping accelerates the product development timeline and cost. In the field of microfluidics, the significance of rapid prototyping is prominently exemplified by the advent of soft lithography using polydimethylsiloxane (PDMS). By leveraging the inherent benefits of miniaturization, the field of microfluidics has demonstrated extensive applications in sample preparation, biosensors, diagnostics, and other interdisciplinary fields within recent decades. The ability to allow convenient and cost-effective fabrication of microfluidic systems using polymers has elevated these systems from being marginally impactful in research settings, when manufactured using fabrication steps derived from microelectronics, to being actively explored for their full potentials.

[0022] Not surprisingly, there has been a continuous effort to expand the use of microfluidics beyond research settings for a broader audience. In addition to enabling previously-not-possible sophisticated assays in the highly well-structured research setting, the microfluidic community has started translating assays from bench to bedside by simplifying workflows for untrained users. For example, paper-based microfluidics and capillaric circuit (CC) technologies permit low cost, automated operational capability without the need for auxiliary equipment, highly skilled personnel, or benchtop footprint. CCs, in particular, excel in performing decentralized assays due to their ability to achieve complex deterministic, pre-programmed flows of different reagents into a reaction microchannel. Notably, this is accomplished in CCs without external pumps or pressure sources for on-chip enzyme-linked immunosorbent assays (ELISAs), detecting a variety of antibodies, proteins, drugs, anions, and bacteria.

[0023] The translation of these technologies necessitates a new rapid prototyping process facilitating uninterrupted transitions to mass production of systems with microscale features. Soft lithography using PDMS, the current gold standard rapid prototyping method for microfluidics, seems inadequate to meet the demands of the new designs in the field of of microfluidic commercialization due to the high requirements involved in scaling up its production, as increasing manufacturing volume does not significantly reduce individual unit costs due to long processing time. Additive manufacturing with resin-based 3D printing is emerging as an alternative prototyping methodology for microfluidic devices, but the process is still relatively low throughput due to its layer-by-layer methodology. Nevertheless, it is possible to achieve low-cost, high-volume production of microfluidic devices by using thermoplastics. In industry, microfluidic bioassay cartridges are often molded with thermoplastics using, for example, poly(methyl methacrylate) (PMMA), through methods such as hot embossing, injection molding and roll-to-roll imprinting. The tradeoff for these5SUBSTITUTE SHEET (RULE 26)fabrication techniques is their expensive upfront costs in fabricating molds, which is infeasible when device designs are still being iterated upon. Moreover, the translation of prototyped features from PDMS or 3D printed microfluidic devices into thermoplastic is not seamless. Materials used in microfluidic rapid prototyping exhibit drastically different material and surface properties than rigid thermoplastics. As a result, not all existing geometries and chemistries validated for prototypes in research settings are necessarily transferrable. Lengthy re -optimization processes are unavoidable, inevitably adding to development cost and deployment time. An optimal prototyping method for microfluidic devices with scalable appeal utilizes accessible, low-cost equipment with rapid processing time to produce thermoplastic devices.

[0024] The present disclosure provides an efficient, robust, and inexpensive protocol to rapidly transfer multi -height microscale features from stereolithography (SLA) 3D printed molds to hot embossed thermoplastics through a series of intermediate replicates, as shown in the overview of FIG. 1. The established protocol only utilizes materials that are common in research settings or commercially available at low cost. The fabrication pipeline offers the potential to move from device ideation to final product in less than 48 hours, with low total material costs. The proposed fabrication protocol serves as a rapid and practical prototyping strategy, while also considering scalability for downstream production.

[0025] The method, workflow, and devices of the present teachings addresses the challenges of utilizing a rapid, reliable, and repeatable method and aims to enable facile, rapid, and cost- effective fabrication of plastic devices with microscale features by leveraging the benefits of additive manufacturing and thermoplastic-based fabrication. The process of the present disclosure involves converting stereolithography (SLA) 3D printed molds into polymethylmethacrylate (PMMA) devices through a series of fast and robust intermediate steps. An initial SLA 3D printed mold serves as a template to create an engraved PDMS replica. This intermediate replica is then cast with a high-temperature-resistant thermosetting resin, i.e. epoxy, forming a second embossed mold. The epoxy features are used as a hot embossing mold to press PMMA into the final desired geometry.

[0026] Presently available prototyping methods for similar microfluidic devices can include soft lithography, complete additive manufacturing, or thermoplastic molding. Soft lithography involves the casting of an elastomeric material, such as PDMS, onto a template to produce negative replicates of the mold. Replica molding with the use of a silicone-based stamp can provide submicron features. Soft lithography typically requires a high photolithographic mold cost, slow fabrication speed and relatively poor industry translation. Additive manufacturing is a technique typically involving three-dimensional printing or forming of prototypes.Methods only using additive manufacturing techniques result in a low mold cost with multi-6SUBSTITUTE SHEET (RULE 26)height feature capability, but also result in poor industry translation. Thermoplastic molding to create microfluidic devices can provide faster fabrication speed and good industry translation, but at the expense of very high mold costs. The present teachings provide methods and device that are advantageous in all categories of mold cost, fabrication speed, and industry translation.

[0027] The workflow described in the present teachings offers several advantages. By eliminating the need of labor-intensive multilayer microfabrication, the desirable device geometries can be easily tuned by modifying computer-aided design (CAD) fdes, facilitating rapid engineering iterations. The integration of SLA 3D printing, or other 3D printing methods or techniques, into the mold fabrication process reduces turnaround time from concept ideation to production and expands the customizability of feature heights or other device dimensions. SLA and other 3D printers are commercially available and more commonly found in research settings than areas requiring cleanroom access, which aids in simplifying workflows and reducing costs. Additionally, raw material costs of thermoplastics and thermosetting resin materials are relatively low. Moreover, these processes can be carried out without requiring specialized facilities or relying on commercial providers for materials such as custom photomasks for photopatteming. As a result, the proposed workflow enables a remarkably short turnaround time from ideation to the final product, completing the entire process in less than 48 hours. For the purposes of the present teachings, a microfluidic device can be or include a device having features on a micro-scale or milli-scale as described herein in regard to cross-sectional dimensional features. It should be noted that a microfluidic device can include features having dimensions ranging from a micro-scale or a milli-scale, or a combination thereof.

[0028] This efficient and cost-effective rapid prototyping protocol for thermoplastic microfluidic devices offers a streamlined approach to device fabrication, significantly reducing fiscal and time costs. This methodology provides a highly customizable and scalable solution, accelerating the development and translation of microfluidic technologies for various applications. Exemplary processes as described herein have been optimized for the fabrication of dimensional features from about 50 pm to about 1000 pm. For applications outside of microfluidic devices, additional efforts in designing computer aided design (CAD) files can transfer the process to specific design considerations.

[0029] FIG. 1 is a schematic overview of a rapid fabrication process, in accordance with the present proposal. The method of fabricating a microfluidic device 100 as shown in FIG. 1 includes the creation or conceptualization of a new channel design 102, followed by the creation of a device template 104 including one or more dimensional features on a surface of the device template, for example, where the microscale, multi-height features are produced using an SLA7SUBSTITUTE SHEET (RULE 26)3D printer. The creation of the device template 104 can include the use of designing the device template with the use of computer aided design (CAD). Next, an inverse replica cavity mold of the device template is cast 106, such as with the use of sequential PDMS and epoxy casting, followed by casting an embossing stamp 108 by pouring a stamp material into the inverse replica cavity mold of the device template. Alternatively the surface of the device template can be immersed in a molding composition. The forming of the embossing stamp 108 can further include heating the stamp material, for example, to cure or solidify or anneal the embossing stamp. In other examples, heating the embossing stamp can be accomplished subsequent to casting the embossing stamp. In certain examples, the embossing stamp is heated to a temperature from about 120°C to about 160°C, and the embossing stamp material includes a thermosetting resin, such as, for example, an epoxy resin. The embossing stamp material may include a non-metal composition or material. Next, the embossing stamp is pressed into a thermoplastic material to form the microfluidic device 110. The thermoplastic material can be heated prior to pressing the embossing stamp into the thermoplastic material or the embossing stamp can be heated. The thermoplastic material may be in the form of a slab or sheet prior to being pressed with the embossing stamp. This process 100 can be iterative and repeated to continue to refine features or configurations of the microfluidic device. Geometries could be iterated quickly based on experimental outcomes. This workflow can be iterated and used to manufacture and validate capillaric circuits (CCs) 112 or other fine-featured microfluidic devices of the present disclosure.

[0030] This exemplary method is non-limiting in nature and may include variations in processing details as well as material considerations. The method of fabricating a microfluidic device 100 can include where the template is created using a three-dimensional printing method. An exemplary example of a three-dimensional printing method further includes stereolithography (SLA), yet the three-dimensional printing method can include additive manufacturing methods such as but not limited to fusion deposition modeling, drop-on- demand printing, selective laser sintering (SLS) or others known to those skilled in the art. In examples, the microfluidic device includes one or more channels which can include one or more cross-sectional dimensions of from about 50 pm to about 1000 pm, or from about 150 pm to about 600 pm, or in certain examples, dimensions of features can range from about 1 pm to about 1 or 2 mm. In examples, the cross-sectional dimensions can include dimensions of various orientations, height, width, diameter, or other lateral dimensions of channels, fluid paths, size-selective structural filters, capillaric valves (e.g., retention valves, burst valves, trigger valves), capillary pumps, hydraulic resistors, microfluidic mixers, or other features. In examples, the cross-sectional shapes can include any variable cross-sectional extrusion shapes, as long as they are convex in nature.8SUBSTITUTE SHEET (RULE 26)

[0031] In certain examples, the application of a release layer may be included in the method of fabricating a microfluidic device 100. The release layer can be in sheet, liquid, aerosol spray, or other form. A release layer can be or include a composition including low molecular silicones or silicone precursors. Moreover, a bubble removing step may also be optionally practiced. For example, when poring a thermosetting resin, such as an epoxy resin, the epoxy formulation can be centrifuged, the PDMS mold is desiccated, and a needle is used to agitate bubbles that may be trapped or formed in features after initially pouring an initial portion of the epoxy resin material.

[0032] In illustrative examples, the thermoplastic material forming the device can include a polyacrylate, an acrylonitrile butadiene styrene, a polyamide, a poly(amide -imide), a polylactic acid, a polycarbonate, or a combination thereof. In an exemplary example, the thermoplastic material is poly(methyl methacrylate). In examples, the molding composition can be or include a silicone or polydimethylsiloxane (PDMS). It should be noted that for the purposes of this disclosure, silicone is considered as a general material category, which can be used interchangeably with polydimethylsiloxane (PDMS), as would be understood by one skilled in the art.

[0033] The present method of fabricating a microfluidic device 100 can be conducted in an uncontrolled environment without compromising functional or dimensional features. For the purposes of this disclosure, an uncontrolled environment can be considered as an ambient laboratory location without specialized ventilation, humidity, temperature, or air filtration measures necessary for performance or use of the method 100. Furthermore, for practicing the methods described herein, no cleanroom facility or equipment is needed, thus enabling a rapid prototyping of plastic devices, including deploying multiple devices or cartridges having micro-sized features as described herein. Prototyping and testing can be tested promptly, without the need for cumbersome industry partnerships, and final features of fabricated devices can be tested in the lab inexpensively with the use of readily available materials. Aside from raw materials, requisite equipment may be found in standard laboratory environments, including electrical hot plates, laboratory balances, vacuum lines and centrifuges. The 3D printer and embossing rig are relatively low cost implements that are required for the proposed method.

[0034] Capillaric Circuit Design

[0035] CCs are passive microfluidic devices that enable pre-programmed, time -controlled fluid flow sequences with multiple reagents to perform complex assays on-chip. CCs are composed of multiple components, including retention burst valves (RBVs) and trigger valves (TVs), organized to form branches that facilitate a defined order of fluid flow. Branch depletion order and duration are geometry-sensitive, as CC components are organized to balance capillary9SUBSTITUTE SHEET (RULE 26)pressure (Pc) and hydraulic resistances (Rh), which are reflected by the following governing equations. Pccan be determined by the Young -Laplace equation,

[0036] where y is the surface tension, 0 is the contact angle between the fluid and the material of the associated microchannel wall, h is the height of the microchannel and w is the width of the microchannel. Rh of a rectangular channel is represented by

[0037] where p represents the fluid viscosity and L represents the microchannel length.

[0038] Traditional microfabrication methods historically hindered geometric innovation in CCs due to limited throughput and financial viability, particularly for producing multi-height microscale features using conventional microfabrication methods in microfluidic manufacturing. Recent advancements of CCs leverage additive manufacturing, allowing reliable 3D printing of features with previously unrealizable geometries that enhance CC functionalities without significant additional fabrication complexity. This flexibility enables a wider range of Rh and Pcwithin the same device footprint. As such, devices have been fabricated using PDMS cast from 3D printed templates or additive manufacturing directly.

[0039] Despite significant advancements, the full potential of CCs as powerful, inexpensive and decentralized biological assay kits remains unrealized due to the lack of high-volume production using thermoplastics. One of the limiting factors is that the structural and functional features, currently optimized for the selected substrate materials, must undergo inevitable reoptimization when transitioning from PDMS or 3D printed resins to thermoplastics due to the variations in contact angles, 0 (Eq. 1). In this study, the rapid prototyping method was validated for thermoplastic CC development, aiming for expedited mass production of CCs as decentralized diagnostic devices.

[0040] FIG. 2 is a representative circuit diagram to represent different CC components, showing resistances and capillary pressures of relevant components that were optimized with this model to approximate channel geometries required to achieve desired flow patterns, in accordance with the present disclosure.Materials and MethodsCapillaric Circuit Design

[0041] The CC device that was designed and constructed consists of multiple CC components, including RBVs, TVs, resistors, and a capillary pump. A circuit diagram 200 was devised for10SUBSTITUTE SHEET (RULE 26)the device layout and a Python script was written to calculate the hydraulic resistance and capillary pressure of each component. Shown are inputs 216 and outputs 214 of the circuit diagram, and the various resistances associated with the circuit components, such as the PC.RBV, 202, Rh,RBV 204, Rh, Reservoir 206, Rh, TV 208, Rh, Resistor 210, and P c, capillary pump 212. The sessile drop method was used to estimate the contact angle of water with plasma-treated PMMA (8560K257, McMaster Carr, USA) and pressure-sensitive tape (ARcare 90445Q, Adhesive Research, USA), with image analysis performed using the DropSnake plugin on FIJI. Measured contact angles, along with the fluid viscosity of water, were set as constants in Eq. 1 to estimate capillary pressures for each CC component. Feature dimensions were selected that aligned with the 3D printer capabilities (25 pm XY resolution and layer height).

[0042] Rapid Prototyping Protocol

[0043] A streamlined rapid prototyping process for constructing thermoplastic devices with microscale features is described in the present disclosure. This method utilized multiple casting steps and materials commonly found in research settings. The sequential processing steps comprise (i) 3D printed template design and construction, (ii) PDMS mold casting, (iii) high- temperature epoxy replication, and (iv) hot embossing thermoplastics. The materials employed in each step are detailed below, along with brief descriptions of their respective roles.

[0044] 3D Printed Template'. CCs were designed using CAD software (AutoCAD, Autodesk Inc., USA) (Table 1). The CAD design was exported as an STU file and uploaded in the PreForm software to be printed using the Form3 SEA 3D printer (FormLabs, USA) with commercially available, general purpose Clear v4 resin (RS-F2-GPCL-04, FormLabs, USA). After printing, the printed object was sonicated in fresh isopropanol for 5 minutes to wash away uncured resin and dried. Subsequently, the 3D printed template was baked for 1 hour at 120°C in an oven (Quincy Labs, USA). The 3D printed object was cooled to 20°C.11SUBSTITUTE SHEET (RULE 26)Table 1: Dimensions of all features on the designed CC. Branch 1, with the largest cross section RBV, has the lowest capillary pressure and depletes first.

[0045] PDMS Mold'. The 3D printed template was placed in a petri dish and lightly sprayed with an epoxy release spray (Ease Release 200, Mann Release Technologies, USA). It was left to dry for 5 minutes. Meanwhile, PDMS (Sylgard 184, Dow Coming, USA) was mixed thoroughly at a 10: 1 ratio. The PDMS mixture was poured onto the mold and degassed in a vacuum chamber before being cured at 85°C for 1 hour. After cooling to room temperature, the cured PDMS was cut to create a positive device mold.

[0046] Epoxy Cast'. The PDMS positive mold was placed in a petri dish and the feature side was sprayed lightly with an epoxy release spray. It was left to dry for 5 minutes, followed by an additional 5 minutes of degassing in a vacuum chamber. A two-part, high-temperature epoxy (EpoxAcast 670 HT, Smooth-On, USA) was mixed according to the manufacturer’s instructions (100: 16 weight ratio of Part A to Part B). Bubbles that formed from mixing were removed via centrifugation (5 minutes at 1000 RPM) and manual agitation with a 20G needle tip (Instech Laboratories, USA) after casting. The epoxy-filled mold was then cured according to the manufacturer’s instructions: 24 hours at room temperature (20°C), followed by 2 hours at 80°C, and 3 hours at 150°C. The epoxy replicate was removed from the PDMS mold after curing for 24 hour at 20°C. If needed, the base of the epoxy was smoothed using sandpaper (80 grit, Fandeli, Mexico).

[0047] Hot Embossed PMMA: 1 / 8” thick acrylic sheets (McMaster Carr, USA) were laser cut (VLS6.60, Universal Laser Systems, USA) into square pieces that were smaller than the epoxy mold dimensions. Of note, the laser cutting can be replaced with a manual handsaw without adversely impacting the manufacturability. The cut PMMA piece was placed on top of the epoxy mold, surrounded by an off-ratio PDMS (20: 1) spacer and placed on the lower platen (VEVOR, USA) of a hydraulic shop press (Strongway, USA). The upper platen was then lowered and heated to 140°C, and then approximately 500 lb of force was applied for 5 minutes. The substrate was allowed to cool down to 90°C before the pressure was released. The PMMA was subsequently separated from the epoxy mold using tweezers. Hot embossed PMMA devices were cleaned using tape (Scotch, USA) to remove dust before being O2 plasma treated (Technics MicroRIE, USA) at 0.3 mbar for 75 seconds. The device was sealed using precisely cut sealing tape.Feature Dimension Measurements

[0048] The feature dimensions on the manufactured substrates at various molding steps were characterized using a surface profilometer (VK-X100, Keyence, Japan). Vertical dimensions were measured by determining the vertical distance between a randomly selected location on12SUBSTITUTE SHEET (RULE 26)the base and embossed feature. Horizontal dimensions were measured by focusing the image along the base of each feature and measuring the lateral length at lOx magnification.

[0049] The manufactured features were visually inspected using images taken using a JSM- IT700HR Scanning Electron Microscope (SEM) (Jeol Ltd., USA) at a 15° angle at 45x magnification. Prior to imaging, substrates were sputtered with a lOnm thick platinum layer (EM ACE 600, Leica, Germany).Pre-Programmed Flow Sequence Validation

[0050] To evaluate the enclosed CC functionality, 1: 10 diluted blue food dye (McCormick, USA) in DI water was prepared. The volume of food dye, corresponding to the sum of the associated RBV, reservoir, and TV, was pipetted into branches 1-4 to sufficiently fill each branch. To trigger the reagent flow, 30 pL of diluted food dye (i.e., the wetting solution) was added to the wetting channel inlet. Once the injected wetting solution reached the capillary pump outlet, a 1 cm wide strip of filter paper (Qualitative filter paper, Grade 1, Whatman, USA) was inserted with a 90° angled tip along the designed groove. This action initiated fluid wicking, leading to the activation of the CC. A video was taken of the entire fluid flow process using a smartphone camera (iPhone 14 Pro, Apple Inc., USA).Immunofluorescence Protein Quantification Assay

[0051] Quantification of bovine serum albumin (BSA) was performed to validate this proof- of-concept CC device using anti-BSA antibody-coated polydisperse agarose beads prepared off-chip as a protein-detection packed bead column. Briefly, 40 pL of streptavidin- functionalized agarose beads (Streptavidin Agarose, Millipore Sigma, USA) (diameter 40-165 pm) were diluted in 320 pL PBS (14190250, ThermoFisher Scientific, USA), centrifuged and washed with PBS. Subsequently, beads were incubated in 100 pg / mL biotinylated anti-BSA antibody (A10-113B, ThermoFisher Scientific, USA) for 1 hour at 20°C. Beads were once again washed with PBS and resuspended in 400 pL PBS. Subsequently, serial dilutions (100 ng / mL - 1 mg / mL) of BSA-FITC (A23015, ThermoFisher Scientific, USA) were prepared in PBS to assess the limit of detection.

[0052] Branches 1-4 of the CC devices were loaded with a dilution of BSA-FITC or PBS for protein quantification and washing, respectively. The antibody-coated bead solution was resuspended and added to the wetting branch to trigger device flow. A video was taken of the entire flow sequence using a smartphone camera. This video was time stamped to determine the flow time of each branch.

[0053] After the remaining 10 pL of the wetting solution flown through the main channel, the device was protected from light and the bead column was imaged on an inverted microscope (Eclipse Ti2, Nikon Inc., Japan) with a light source (SOLA Light Engine, Lumencor, USA), fluorescent filter cubes, and a CCD camera (CoolSNAP DYNO, Photometries, USA).13SUBSTITUTE SHEET (RULE 26)

[0054] Each fluorescence image of the agarose bead detection column was analyzed using ImageJ. Three preset regions of interest (ROI, 300x300 px2) of each bead column were selected at the center of the channel. The mean intensity of each ROI was determined, and the average of these three values comprised one data point. Triplicate experiments at each protein concentration were performed. The same ROIs were used for each image. A four-parameter logistic regression was used to fit an S curve using the generated fluorescence data from the serial dilutions. The limit of detection (LOD) was calculated and matched with the S curve to determine the minimum detection concentration.Results and DiscussionEstablishment and Optimization of the Rapid Fabrication Protocol

[0055] Low-cost fabrication and design iteration of thermoplastic devices help expedite the deployment of microfluidic devices for impactful applications. There have been notable recent works expanding the scope of research beyond PDMS prototypes of microfluidic devices. For example, thermoplastic features have been embossed from photolithographic molds via reinforced PDMS molds or epoxy. Direct laser ablation onto surfaces has been proposed for quick design iteration by tuning laser power to achieve different engraving depths. Recent works have begun to utilize additive manufacturing for complex feature design, such as three- dimensional printing of a metal mold for direct hot embossing of thermoplastics. A PolyJet 3D printed mold to thermoplastic fabrication workflow was also recently published. These recent advances were built upon to leverage the benefits of designing diverse, multi-height features with additive manufacturing with the fabrication scalability of thermoplastic devices, while further reducing financial burdens, manufacturing time and complexity. The rapid, affordable fabrication protocol was developed by converting SLA 3D printed templates with microscale multi -height features into planar PMMA replicates (FIG. 3A). Fabrication steps are accomplished using materials and equipment readily available in microfluidic research labs or that can be acquired for low costs. Each stage is optimized to reduce the manual labor and overall processing time.

[0056] FIGS. 3A-3F depict illustrations of the rapid fabrication process including FIG. 3A, which is a schematic (Process Step and Final Product) and pictorial (Top View) representation of each fabrication step, FIG. 3B, which is a comparison of measured vertical and horizontal dimensions of 3D printed and PMMA features,. The entire workflow was replicated 3xto assess process variability. The line represents the 1: 1 dimension match between substrates, with the shaded region representing 10% deviation for a given dimension. FIG. 3C is a Vertical (V) and Horizontal (H) dimension comparison between PMMA Replicates 5 and 10, normalized to Replicate 1 using the same epoxy mold. FIG. 3D is atop view schematic of an exemplary design for use in a method for mass production of thermoplastic micro- and milli-fluidic devices, in14SUBSTITUTE SHEET (RULE 26)accordance with the present disclosure. FIG. 3E is a series of SEM images of a staircase feature on the 3D print template, PDMS mold and PMMA replicate. FIG. 3F is a comparison of the PDMS cross section of a 3D printed 150pm and 1000pm square channel to contextualize dimension measurements. FIG. 3G shows a vertical and horizontal dimension comparison of PMMA features produced from a Form3 printed, PolyJet printed, photolithographic silicon mold, and High Temperature (HT) resin templates to demonstrate the versatility of the fabrication workflow with different molds and the ability to produce features smaller than those shown in FIG. 3B, in accordance with the present disclosure.

[0057] FIG. 3 A shows a schematic, including process steps and final product, along with a pictorial top view, representation of each fabrication step. FIG. 3B is a plot comparison of measured vertical and horizontal dimensions of 3D printed and PMMA features, with replications to assess process variability. FIG. 3C is a plot showing vertical (V) and horizontal (H) dimension comparison between PMMA Replicates 5 and 10, normalized to Replicate 1 using the same epoxy mold. From a single epoxy stamp, multiple replicates of thermoplastic features may be reproduced without significant deviation in feature dimensions.

[0058] FIG. 3D is a top view schematic of an exemplary design for use in a method for mass production of thermoplastic micro- and milli-fluidic devices, in accordance with the present disclosure. In the top view of the microfluidic device design 300, several features and interconnected sets of features are shown. In a first set of interconnected features 310 a staircase 302 feature, which includes several steps or a stepped profile is shown. The staircase feature demonstrates the ability to vary cross-sectional shapes in the vertical direction, create devices with multi-heights (i.e., variable convex cross-section extrusions) in a single mold, and transfer micro- to -millimeter sized features. Also within the first set of interconnected features 310 is a first channel 304 having a 600 pm by 600 pm dimension. In examples, this first channel 304 or similar feature can have the same vertical and / or lateral (horizontal) dimensions or alternatively different vertical and / or lateral dimensions. Also shown in the first set of interconnected features 310 is a second channel 306 having a 1000 pm by 1000 pm dimension. In alternate examples, this second channel 306 can have the same vertical and / or lateral dimensions or alternatively different vertical and / or lateral dimensions. Also shown in the first set of interconnected features 310 is a third channel 308 having a 150 pm by 150 pm dimension. It should be noted that this third channel 310 can also have the same vertical and / or lateral dimensions or alternatively different vertical and / or lateral dimensions. Also shown in FIG. 3D is a second set of interconnected features 312, which can be same similar or different as compared to the first set of interconnected features 310, depending on the goals of the microfluidic device design 300. While the dimensions of these channels of the microfluidic device design 300 can range from about 50 pm to about 1000 pm, or from about15SUBSTITUTE SHEET (RULE 26)150 pm to about 1000 pm, or from about 150 pm to about 600 pm, it should be noted that design parameters outside limitations set by digital model designs or additive manufacturing capability can also be realized with methods of the present teachings. Various capillaric and microfluidic elements, including but not limited to fluidic paths with various cross-sectional sizes, fluidic mixers (e.g., serpentine conduits), size-selective structural fdters, capillaric valves (e.g., retention valves, burst valves, trigger valves), capillary pumps, hydraulic resistors, can be fabricated. In examples, features of from about 75 pm to about 1000 pm were evaluated, but theoretically could go smaller or larger, for example, from about 30 pm to about 2000 pm with different molding process technologies, such as photolithography, as shown herein, or other additive manufacturing techniques.Feature Resolution and Reproducibility

[0059] FIG. 3E depicts SEM images of a staircase feature on the 3D print template 314, PDMS mold 316, and PMMA replicate 318, in accordance with the present disclosure. The images show a portion or feature of a design feature, or staircase fabricated using the process shown in FIG. 3A. The 3D printed template was produced using a Form3 printer with Clear v4 resin and a 25pm layer thickness. The features depicted guide a 1000pm by 1000pm channel into a 500pm by 500pm channel in 50pm increments.

[0060] FIG. 3F depicts microscopic images comparing the PDMS cross section of a 3D printed 150pm and 1000pm square channel to contextualize dimension measurements. Profdometer readings were taken with the focal plane at the base of the channel, yielding the dimension measurements denoted with the top lines. Scale bar = 500pm. A cross-section 320 of 3D printed 150 pm square channel 324 and a cross-section 322 of 3D printed 1000 pm square channel 326 are shown. It was determined that the replication resolution limit and reproducibility of the multi-height microscale features produced using the rapid prototyping method through systematic and quantitative measurements and analyses of dimensions. The horizontal and vertical dimensions of various features were measured throughout the fabrication process to assess deviations from the expected feature size and output. Microchannels with square crosssections (150 pm, 600 pm, 1000 pm) in the designed CC were characterized using an optical profdometer. FIG. 3B and FIG. 3E illustrate the variations in feature dimensions as the replication process proceeds from the 3D printed template to the final PMMA product. Due to the method of measuring dimensions, there were initial deviations between the CAD and 3D printed dimensions (FIG. 3F). However, the relative change in feature dimensions from the 3D printed template to the hot embossed PMMA were consistent. Across all three PMMA channels, there was a -10% reduction in height and -7% increase in width relative to the 3D printed feature (FIG. 3B). To determine whether the dimension changes would be further exacerbated with repeated hot embossing steps, ten replicate devices were fabricated from the16SUBSTITUTE SHEET (RULE 26)same epoxy mold. Replicates 1, 5 and 10 were assessed to identify feature dimension changes (FIG. 3C). There were minimal changes in both horizontal (<5%) and vertical (<3%) dimensions in Replicates 5 and 10 compared to Replicate 1. Thus, as geometries are finalized and multiple device replicates are needed for downstream experiments, the same epoxy mold may be used for multiple fabrication cycles instead of repeating the entire process. Notably, others have demonstrated the ability to hot emboss thermoplastics using epoxy molds derived from photolithographic molds. As a demonstration, additional fabrication cycles were performed using smaller features produced with an expensive PolyJet 3D printed and photolithographic templates, along with PMMA features replicated directly from Form3 printed templates using high temperature resin (FIG. 3G). The smaller features were reproduced in PMMA with similar deviations (<10%) to those produced from the Form3 printed template. As 3D printing resolution improves and becomes more affordable, the method should be generalizable and smaller features should be properly transferred.

[0061] As shown in FIG. 3G, vertical and horizontal dimension comparisons of PMMA features produced from a Form3 printed, PolyJet printed, photolithographic silicon mold, and High Temperature (HT) resin templates are shown to demonstrate the versatility of the fabrication workflow with different molds and the ability to produce features smaller than those shown in FIG. 3B. Almost identical fabrication steps to the manuscript were performed for both the PolyJet and photolithographic mold templates. The one exception was an extended cooling time (30 minutes) for the PDMS mold of the PolyJet print after curing due to the lower heat deflection temperature (50°C) of the print. The HT resin template was directly hot embossed into PMMA. Aside from the Form3 results replicated from the manuscript, the other fabrication workflows were completed once and characterized at three separate points per feature. The shaded region represents a 10% deviation from the 1: 1 dimension match between substrates.

[0062] The feature transferability of the fabrication process, in accordance with the present disclosure is demonstrated by the plot, which depicts data reflecting expected dimensions vs. measured dimensions in a comparison of both lateral features and vertical features for devices and methods of the present teachings. The data shown demonstrates a <10% dimension change from 3D printed features to final device as produced by methods described herein. This shows that 3D printed features as designed for microfluidic devices can be readily transferred into thermoplastic replicates to create and produce microfluidic devices for multiple iterations. This further demonstrates viability of the present teachings, as state-of-the-art microfluidic device prototyping methods are currently expensive, slow or not suited for mass production. The present fabrication protocol can effectively translate ideas or concepts for microfluidic devices into thermoplastic prototypes or finished devices a low cost and efficient process. The fabrication protocol can be validated by such measurements, as well as evidenced by the17SUBSTITUTE SHEET (RULE 26)preceding images. The present methods and devices can be implemented in designing capillary circuits for passive, autonomous flow.

[0063] 3D Printed Template: An SLA 3D printer with a low capital-expenditure (<$2500) was utilized to produce complex, multi-height features. SLA 3D printing has emerged as a widespread engineering tool for in-house prototyping before the product development completion. A range of low-cost, easy-to-use options, with resolution down to the tens of microns, are now commercially available. The reliable and autonomous nature of 3D printers ensures high repeatability. Additionally, multiple different geometries can be printed on the same print bed for parallelized testing. Features were designed atop a 4 mm base, which was sufficiently thick to minimize print warping from thin features. The 3D print template could not be used directly as a mold for hot embossing PMMA because the general-purpose resin has a relatively low heat deflection temperature (73°C at 0.45 MPa). Hence, the 3D printed part was a template for the intermediate molds.

[0064] Utilizing cost-effective resins for constructing the 3D printed template to achieve multi-height microscale features necessitated the identification of an additional replication method suitable as a base for hot embossing thermoplastics. Consequently, PDMS was chosen to cast high-temperature epoxy, creating a secondary positive mold for hot embossing. Prior to PDMS molding of the 3D printed template, it was crucial to thoroughly cure the 3D printed object because a common challenge arises in the leeching of residual uncured photoinitiators from the print. This leaching prevents complete curing of the PDMS in the proximity of the print surface, hindering effective feature transfer. For the specific resin used, the same issue persisted, even with the manufacturer recommended UV exposure and bake treatment (30 minutes at 60°C). Alternative methods, such as coating the print surface, were either cumbersome or time-consuming. A simple solution was adopted by implementing an additional 1 hour device bake at high temperatures (120°C), which was previously optimized for the specific resin formulation. Additionally, a release spray was used to cleanly separate the PDMS from the 3D printed template without altering the features.

[0065] Epoxy Cast'. The cleanly delaminated PDMS replica of the 3D printed template was then used to cast high-temperature epoxy as the base for hot embossing thermoplastics. Drawing inspiration from the use of metal -filled epoxies for robust hot embossing molds which, due to their longer curing times and higher viscosity, are still less than ideal for rapid prototyping, a commercial epoxy exhibiting excellent temperature resistance and relatively low viscosity (6000 cP) was used. Nevertheless, bubbles with similar size ranges to the features formed in the epoxy remained an issue, potentially leading to unintended features in the final product. To address the inevitable bubble formation during the mixing of the two-part epoxy, the epoxy was subjected to a gentle centrifugation (1000 RPM for 5 minutes) before casting.18SUBSTITUTE SHEET (RULE 26)Additionally, when pouring, larger bubbles could be trapped due to the viscous nature of this epoxy, which did not readily fdl PDMS cavities. Employing the common degassing process used in soft lithography, which involves casting the epoxy in a vacuum, proved ineffective in removing bubbles from a mixture more viscous than PDMS. Instead, careful agitation of bubbles with a 20G needle tip was sufficient to dislodge the air bubbles from the features. A small amount of epoxy (<15 m ), enough to submerge the features, was first poured onto the PDMS mold, and bubbles were agitated. After the surface was free of bubbles, additional epoxy was poured to fill the mold. If the epoxy was slightly underfilled, the edges of the epoxy base formed a raised lip against the PDMS mold after curing and was manually sanded down using coarse sandpaper to ensure a flat surface. The flattened epoxy mold exhibited an improved lifespan since premature mold cracking, caused by uneven mold height during hot embossing, was prevented.

[0066] Hot Embossed PMMA'. Rigorous testing and validation was conducted to determine that the high-temperature epoxy mold could withstand the high temperatures and pressures of an embossing cycle on the inexpensive hot embossing setup (<$500) without cracking or significant deformation. Various works have suggested different temperatures, pressures, and time combinations to achieve proper PMMA molding. It was determined that applying 500 lb of force for 5 minutes at 140°C was sufficient for pattern transfer. PMMA devices were separated from the epoxy mold at 90°C, below the glass transition temperature of PMMA (TG=105°C). The placement of a deformable spacer (20: 1 PDMS) surrounding the epoxy mold helped to evenly apply compressing force to emboss. Hot embossing without the spacer led to uncontrollable cracking of the epoxy mold.Time / Cost Assessment

[0067] The described rapid prototyping pipeline offers significant time and cost benefits compared to conventional PDMS prototyping methods. The equipment required for this process is more readily available and / or inexpensive compared to sophisticated microfabrication tools. SLA 3D printers with excellent print resolution are now commercially available in the price range of $100s-$ 1000s. Other requisite capital equipment, such as a vacuum system, centrifuge, and oven, are commonly found in research settings. The per-device material cost for all combined steps maintains at least a 10-fold reduction compared to standard soft lithography and other proposed methods. It is anticipated that multilayer photolithography would incur even higher manufacturing costs due to its requirement for multiple photomasks, and the complexity of the process often leads to additional expenses, resulting from extended usage fees for microfabrication facility, besides failed iterations.

[0068] The described protocol not only offers minimal time constraints but also demonstrates efficiency. Additionally, the time constraints are relatively minimal with this described19SUBSTITUTE SHEET (RULE 26)protocol. When executed sequentially, it takes less than 48 hours to convert initial geometry ideation to the final product, with only 2.5 hours of labor time (Table 2). The majority of the 48-hour manufacturing duration is attributed to idle time, involving automated 3D printing and epoxy curing. Even the brief active steps required are not technically challenging. In contrast, a significant time constraint of soft lithography involves waiting for the delivery of photomasks . Additionally, photolithography is a lengthy and technically complex process conducted by skilled personnel in cleanroom facilities. In comparison, all steps of this protocol may be completed in-house. The significant time reduction compared to standard soft lithography is a result of the optimization of each step, which could otherwise take several days.Table 2: A breakdown of the time for each step of the rapid fabrication protocol compared to single layer photolithography. Active time refers to processing time involving manual labor (i.e. mixing / degassing materials, post-processing 3D printed templates). The remaining idle time refers to passive steps without user intervention (i.e. epoxy curing, 3D printing). Most of the photomask processing time is incurred from shipping from third party vendors compared to in-house printing of 3D printed templates.Validation of Capillaric Circuits

[0069] FIGS. 4A and 4B represent a demonstration of pre-programmed flow manipulation within the designed CC. FIG. 4A is a schematic 400 of a single device fully loaded with diluted food dye at the instance when flow out of Branch 1 was initiated. CC components and features of interest are labeled. The neck of the RBV and TV are denoted by the top and bottom arrows, respectively. FIG. 4B is a plot of branch depletion time (n=18) after all the solutions were loaded, prior to depletion of Branch 1.

[0070] CCs were selected to demonstrate the feasibility of the described rapid prototyping workflow. The plasma treatment of the PMMA features was necessary to create hydrophilic surfaces for achieving designed fluid flow. Plasma-treated PMMA maintains its hydrophilicity over weeks, and if an alternative method for treating PMMA or preserving PMMA hydrophilicity for longer durations is desired, additional polymer coatings or UV treatment can20SUBSTITUTE SHEET (RULE 26)be implemented. The contact angle of water on the tape and plasma-treated PMMA were experimentally determined to be 85° and 41°, respectively. The tape was precisely cut into the pre -determined size (23 mm x 40 mm), such that the inlet wells and capillary pump outlet via holes were accessible for loading solutions using pipettes. An exposed capillary pump outlet for wicking (FIG. 4A) was critical in triggering the assay. Overhanging tape made it more difficult to have the filter paper contact the solution for wicking. To further simplify the operation of the device, cutting the wicking paper to have a pointed edge allows easy placement, ensuring contact with the square, 200 pm capillary pump outlet.

[0071] A CC was designed with 4 sequentially flowing branches (Branches 1 through 4) alongside the wetting channel (Branch W) (FIG. 4A). After completely loading Branches 1-4 (RBV, Reservoir, and TV), fluid did not dispense into the main channel, validating the successful implementation of the TV. After adding the wetting solution to the wetting branch, the resistor and capillary pump outlet were filled with the solution. Placing the filter paper at the pump wicking outlet initiated sequential branch depletion, dependent on the sequence of RBV cross section. Branches depleted in descending order of cross-sectional area of its corresponding RBV. The average depletion time for Branches 1-4 to transport the volume of the solution from the reservoirs ranged between 40 and 50 seconds. (FIG. 4B). The depletion time for these branches is determined by tracking the movement of the tailing edge of the flow from the neck of RBV to the neck of TV (Fig. 4A). The wetting branch, which did not have a TV, had a slower flow time. The flow of the wetting branch was defined as the trailing edge of the solution moving from its neck of RBV and through the connecting channel, extending up to the exit of the resistor.

[0072] FIGS. 5 A and 5B demonstrate a proof-of-concept, automated, quantitative immunofluorescence protein quantification using antibody-coated beads in the designed CC. FIG. 5 A is a schematic representation of different reagents loaded into the CC 500, in accordance with the present disclosure. FIG. 5B is a plot comparing the mean fluorescence intensity of pre -determined regions of interest across different devices testing various BSA- FITC concentrations, in accordance with the present disclosure. An inset 510A shows a brightfield image of the channel 510 with the compacted bead column. Regions of interest for fluorescence analysis are boxed and annotated as 512, 514, 516. Note: to match the orientation of the image with the schematic, the image was inverted horizontally. Scale bar = 500 pm. (FIG. 5B) Plot comparing the mean fluorescence intensity of pre-determined regions of interest (N=3, dotted boxes in (A)) across different devices testing various BSA-FITC concentrations. The calculated LOD (500 ng / mL) was plotted with black lines. Representative fluorescence images for each protein concentration are depicted above the plot. Scale bar = 200 pm. Immunofluorescence Protein Quantification Assay21SUBSTITUTE SHEET (RULE 26)

[0073] As a proof of concept, a simple, immunofluorescence assay was performed on-chip to quantify protein concentrations in solution. Biotinylated anti-BSA antibodies were conjugated with streptavidin-coated agarose beads before being loaded into the CC to form a reaction pack bed, providing ample surface area and excellent binding capacity for protein-specific immobilization. Then, Branch 1 was loaded with a fluorescent BSA-FITC solution 504 spanning a multiple order of magnitudes in concentration (100 ng / mL - 1 mg / mL) while the remaining branches were loaded with 25 pL total wash buffer 506. This represents sequential triple washes of ~8 pL each to remove excess unbound protein (FIG. 5A). Loading the resuspended bead solution 508 into the wetting branch (W) induced the start of the on-chip flow. The 40-165 pm agarose beads in solution aggregated and compacted into a dense column in the straight channel, feeding into the 150 pm resistor channel (FIG. 5A). The bead column did not impede predicted and tested preprogrammed flow operation duration or sequence. The concentration of beads was tuned (-250 beads / pL) to ensure that the bead column, forming effective reaction packed bed, did not extend past the TV of Branch 1 (<5 mm from the exit of the connecting channel, leading to the resistor). Experimentally, an excessively long column disrupted the flow and caused the branches to flow out of order.

[0074] After triggering CC fluid flow, the beads acted as the assay substrate for affinity -based protein quantification through immunofluorescence intensity. Following fluid flow, the bead column was imaged using the appropriate filter set. Serial protein dilutions were quantified via fluorescence of antibody-coated beads accumulated on the device to assess the limit of detection. Fluorescence intensity analysis of the beads post-assay completion is shown in FIG. 5B. A four-parameter logistic equation was calculated to fit the serial dilution fluorescence intensities to a standard S curve. The LOD was estimated to be 500 ng / mL using the negative control and minimum tested concentration fluorescence intensities. If desired, additional branches and reagents may be added to further increase the sensitivity of detection or complexity of the assay.

[0075] The present disclosure provides a rapid and cost-effective pipeline for prototyping thermoplastic microfluidic devices. An SLA 3D printer was utilized to mold devices with complex geometries, which are then transferred to PMMA through intermediate steps involving PDMS and epoxy moldings. The efficacy of this pipeline was demonstrated by the successful fabrication and iteration of CC designs, leading to the development of a proof-of-concept bioassay. The process is compatible with various 3D printers, allowing for tradeoffs in price, print size, and resolution. Similarly, the method is transferrable to other types of thermoplastics with a simple and inexpensive hot embossing process optimization. With this process, ideas may transition from CAD files to a plastic product in less than 48 hours, most of which is idle time. The total material costs for the entire manufacturing process are low, processes may be22SUBSTITUTE SHEET (RULE 26)completed outside of a well -maintained microfabrication cleanroom, and requisite equipment are already largely available in research labs.

[0076] The fabrication method can enable iterating on channel design using appropriate materials capable of scaled-up production. This prototyping method enables testing required chemical or other biological assay conditions directly with the material to be utilized for massive production, eliminating the need for additional re -optimization of designed assays. After iterating through designs with this method, the finalized epoxy mold may be converted into a metal mold for robust, long-term thermoplastic forming. Depending on the application and final device requirements, PMMA devices can be sealed to other thermoplastics using thermal or solvent bonding instead of tape Future works will focus on exploring other biologically relevant assays that could not be realized through conventional lateral flow assays but would benefit from inexpensive, decentralized platforms. In all, this pipeline reveals new possibilities for rapid prototyping of microfluidic devices and provides a foundation for further advancement in the field.

[0077] While the present teachings have been illustrated with respect to one or more implementations, alterations and / or modifications may be made to the illustrated examples without departing from the spirit and scope of the appended claims. For example, it may be appreciated that while the process is described as a series of acts or events, the present teachings are not limited by the ordering of such acts or events. Some acts may occur in different orders and / or concurrently with other acts or events apart from those described herein. Also, not all process stages may be required to implement a methodology in accordance with one or more aspects or embodiments of the present teachings. It may be appreciated that structural objects and / or processing stages may be added, or existing structural objects and / or processing stages may be removed or modified. Further, one or more of the acts depicted herein may be carried out in one or more separate acts and / or phases. Furthermore, to the extent that the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof are used in either the detailed description and the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.” The term “at least one of’ is used to mean one or more of the listed items may be selected. Further, in the discussion and claims herein, the term “on” used with respect to two materials, one “on” the other, means at least some contact between the materials, while “over” means the materials are in proximity, but possibly with one or more additional intervening materials such that contact is possible but not required. Neither “on” nor “over” implies any directionality as used herein. The term “conformal” describes a coating material in which angles of the underlying material are preserved by the conformal material. The term “about” indicates that the value listed may be somewhat altered, as long as the alteration does not result in nonconformance of the23SUBSTITUTE SHEET (RULE 26)process or structure to the illustrated embodiment. The terms “couple,” “coupled,” “connect,” “connection,” “connected,” “in connection with,” and “connecting” refer to “in direct connection with” or “in connection with via one or more intermediate elements or members.” Finally, the terms “exemplary” or “illustrative” indicate the description is used as an example, rather than implying that it is an ideal. Other embodiments of the present teachings may be apparent to those skilled in the art from consideration of the specification and practice of the disclosure herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the present teachings being indicated by the following claims.24SUBSTITUTE SHEET (RULE 26)

Claims

WHAT IS CLAIMED IS:

1. A method of fabricating a microfluidic device, comprising: creating a device template comprising one or more features on a surface of the device template; casting an inverse replica cavity mold of the device template; casting an embossing stamp by pouring a stamp material into the inverse replica cavity mold of the device template; forming the embossing stamp; and pressing the embossing stamp into a thermoplastic material to form a microfluidic device.

2. The method of fabricating a microfluidic device of claim 1, wherein the device template is created using a three-dimensional printing method.

3. The method of fabricating a microfluidic device of claim 2, wherein the three-dimensional printing method further comprises SLA.

4. The method of fabricating a microfluidic device of claim 1, wherein the microfluidic device comprises one or more channels comprising a cross-sectional dimension of from about 30 pm to about 1000 pm.

5. The method of fabricating a microfluidic device of claim 4, wherein the one or more channels comprises a cross-sectional dimension of from about 150 pm to about 600 pm.

6. The method of fabricating a microfluidic device of claim 1, wherein the thermoplastic material comprises a polyacrylate, an acrylonitrile butadiene styrene, a polyamide, a poly(amide- imide), a polylactic acid, a polycarbonate, or a combination thereof.

7. The method of fabricating a microfluidic device of claim 1, the thermoplastic material comprises poly (methyl methacrylate).

8. The method of fabricating a microfluidic device of claim 1, wherein a composition of the inverse replica cavity mold comprises a silicone or polydimethylsiloxane (PDMS).

9. The method of fabricating a microfluidic device of claim 1, further comprising designing the device template with the use of computer aided design (CAD).25SUBSTITUTE SHEET (RULE 26)10. The method of fabricating a microfluidic device of claim 1, wherein forming the embossing stamp further comprises heating the stamp material.

11. The method of fabricating a microfluidic device of claim 1, further comprising heating the embossing stamp subsequent to casting the embossing stamp.

12. The method of fabricating a microfluidic device of claim 11, wherein the embossing stamp is heated to a temperature from about 120°C to about 160°C.

13. The method of fabricating a microfluidic device of claim 1, further comprising heating the thermoplastic material prior to pressing the embossing stamp into a thermoplastic material.

14. The method of fabricating a microfluidic device of claim 1, wherein the method of fabricating the microfluidic device is conducted in an uncontrolled environment.

15. The method of fabricating a microfluidic device of claim 1, wherein an embossing stamp material comprises a thermosetting resin.

16. The method of fabricating a microfluidic device of claim 15, wherein the embossing stamp material comprises a non-metal.

17. A method of fabricating a microfluidic device, comprising: creating a device template using a three-dimensional printing method, the device template comprising one or more features on a surface of the device template; casting an inverse replica cavity mold of the device template by immersing the surface of the device template in a molding composition; casting an embossing stamp by pouring a thermosetting resin into inverse replica cavity mold of the device template; curing the thermosetting resin to fully form the embossing stamp; and pressing the embossing stamp into a thermoplastic material to form a microfluidic device.

18. The method of fabricating a microfluidic device of claim 17, wherein the microfluidic device comprises one or more channels comprising a cross-sectional dimension of from about 50 pm to about 1000 pm.

19. The method of fabricating a microfluidic device of claim 17, wherein the method of fabricating the microfluidic device is conducted in an uncontrolled environment.

20. The method of fabricating a microfluidic device of claim 17, further comprising heating the embossing stamp subsequent to casting the embossing stamp to a temperature from about 120°C to about 160°C.26SUBSTITUTE SHEET (RULE 26)

Images