3D-printed Microfluidic Device Research Articles

Open-source interactive design platform for 3D-printed microfluidic devices

Open-source interactive design platform for 3D-printed microfluidic devices

Development of a 3D Microfluidic Analytical Device for the Detection of Pathogenic Bacteria in Commercial Food Samples with Loop-Mediated Isothermal Amplification

Traditional methods of detecting foodborne pathogens take several days to produce the required results. Furthermore, various molecular techniques (e.g., PCR) that also produce reliable results in the detection of pathogenic bacteria have been introduced, but the cost–time ratio required does not allow them to be considered a substantial solution to this specific problem. Three-dimensional (3D) printing technology provides the ability to design and manufacture microfluidic analytical devices using conventional 3D printers, which, in combination with colorimetric loop-mediated isothermal amplification (LAMP), may further simplify the process. The overall reduction in time and cost may provide the opportunity to upscale this diagnostic modality. Moreover, unlike most microfluidic analytical devices, this technique is simpler and more user-friendly, as it does not require any expertise or additional equipment apart from a conventional oven. A 3D-printed microfluidic analytical device in combination with LAMP was developed and tested for the simultaneous detection of foodborne pathogens in food samples. A total of 150 commercial food specimens (50 milk, 50 chicken, 50 lettuce samples) were analyzed for possible contamination with Salmonella typhimurium, Listeria monocytogenes and Escherichia coli. The 3D-printed microfluidic device was 100% precise for both negative (80 samples) and positive samples (7 samples were positive for S. typhimurium, 28 for L. monocytogenes, and 35 for E. coli) for all pathogens. Overall, the amount of data analyzed led to a high level of confidence in the precision of this device. As such, this new 3D device in combination with LAMP provides a precise detection method for food pathogens with a low detection limit.

Universal Biomaterial-on-Chip: a versatile platform for evaluating cellular responses on diverse biomaterial substrates

Microfluidics has emerged as a promising approach for assessing cellular behavior in vitro, providing more physiologically relevant cell culture environments with dynamic flow and shear stresses. This study introduces the Universal Biomaterial-on-Chip (UBoC) device, which enables the evaluation of cell response on diverse biomaterial substrates in a 3D-printed microfluidic device. The UBoC platform offers mechanical stimulation of the cells and monitoring of their response on diverse biomaterials, enabling qualitative and quantitative in vitro analysis both on- and off-chip. Cell adhesion and proliferation were assessed to evaluate the biocompatibility of materials with different physical properties, while mechanical stimulation was performed to investigate shear-dependent calcium signaling in pre-osteoblasts. Moreover, the applicability of the UBoC platform in creating more complex in vitro models by culturing multiple cell types was demonstrated, establishing a dynamic multicellular environment to investigate cellular interfaces and their significance in biological processes. Overall, the UBoC presents an adaptable tool for in vitro evaluation of cellular behavior, offering opportunities for studying various biomaterials and cell interactions in microfluidic environments.Graphical

3D-printed microfluidic device for high-throughput production of lipid nanoparticles incorporating SARS-CoV-2 spike protein mRNA.

Lipid nanoparticles (LNPs) are drug carriers for protecting nucleic acids for cellular delivery. The first mRNA vaccines authorized by the United States Food and Drug Administration are the mRNA-1273 (Moderna) and BNT162b (BioNTech/Pfizer) vaccines against coronavirus disease 2019 (COVID-19). We designed a 3D printed Omnidirectional Sheath-flow Enabled Microfluidics (OSEM) device for producing mRNA-loaded LNPs that closely resemble the Moderna vaccine: we used the same lipid formulations to encapsulate mRNA encoding SARS-CoV-2 spike protein. The OSEM device is made of durable methacrylate-based materials that can support flow rates in the mL min-1 range and was fabricated by stereolithography (SLA), incorporating readily adaptable interfaces using commercial fluidic connectors. Two key features of the OSEM device are: 1) a 4-way hydrodynamic flow focusing region and 2) a staggered herringbone mixer (SHM). Superior to conventional planar fluid junctions, the 4-way sheath flow channel generates an evenly focused, circular center flow that facilitates the formation of LNPs with low polydispersity. Downstream, fluid mixing in the SHM is intensified by incorporating a zig-zag fluidic pathway to deliver high mRNA encapsulation efficiency. We characterized the mRNA-loaded LNPs produced in the OSEM device and showed that the enhanced 3D microfluidic structures enable a 5-fold higher throughput production rate (60 mL min-1) of LNPs compared to commercial multi-thousand-dollar micromixers. The device produced LNPs of diameter less than 90 nm, with low polydispersity (2-8%) and high mRNA encapsulation efficiency (>90%). The 3D-printed device provides a cost-effective and easily prepared solution for high-throughput LNP production.

Surface-Wetting Characteristics of DLP-Based 3D Printing Outcomes under Various Printing Conditions for Microfluidic Device Fabrication.

In recent times, the utilization of three-dimensional (3D) printing technology, particularly a variant using digital light processing (DLP), has gained increasing fascination in the realm of microfluidic research because it has proven advantageous and expedient for constructing microscale 3D structures. The surface wetting characteristics (e.g., contact angle and contact angle hysteresis) of 3D-printed microstructures are crucial factors influencing the operational effectiveness of 3D-printed microfluidic devices. Therefore, this study systematically examines the surface wetting characteristics of DLP-based 3D printing objects, focusing on various printing conditions such as lamination (or layer) thickness and direction. We preferentially examine the impact of lamination thickness on the surface roughness of 3D-printed structures through a quantitative assessment using a confocal laser scanning microscope. The influence of lamination thicknesses and lamination direction on the contact angle and contact angle hysteresis of both aqueous and oil droplets on the surfaces of 3D-printed outputs is then quantified. Finally, the performance of a DLP 3D-printed microfluidic device under various printing conditions is assessed. Current research indicates a connection between printing parameters, surface roughness, wetting properties, and capillary movement in 3D-printed microchannels. This correlation will greatly aid in the progress of microfluidic devices produced using DLP-based 3D printing technology.

(Invited) All-3D-Printed Devices for Electrochemical Sensing

Three-dimensional (3D) printing has gained significant attention in research laboratories, since it provides the end user with the freedom and ability to produce in-house and on-demand specific electrochemical platforms in a fast and affordable way. The most applied 3D-printing technology in the manufacturing of electrochemical sensors is fused deposition modeling (FDM) which is based on the e-printing of the device by thermoplastic filaments. In particular, the use of FDM for the printing of sensors offers noteworthy advantages compared to conventional fabrication technologies, such as desktop-sized equipment, very low capital, operational, and materials costs, easy fabrication process, design transferability (as the respective file format can be transferred through e-mail and printed on every dual extruder 3D printer), and ecofriendliness (since no waste is produced) [1,2].This presentation will discuss our recent developments of all-3D-printed integrated mini devices and their applications to electrochemical sensing [3-6]. The devices are printed from specific filaments (conductive and non-conductive) at different sizes and shapes, by a dual extruder 3D-printer, in order to fit to the demands of numerous applications. More specifically, all-3D-printed electrochemical microtitration wells for the in-situ and micro-volume quantum dot-based bioassays, as well as a 3D-printed wearable glucose monitoring device in the form of an electrochemical ring will be described. Besides, a 3D-printed wearable device in the form of an electrochemical finger for date rape drugs screening and a 3D-printed microfluidic device modified with metallic film for the trace determination of heavy metals will be presented.Acknowledgements:I thank the Special Account for Research Grants of National and Kapodistrian University of Athens for the financial support.References A. Abdalla and B. A. Patel, Curr. Opin. Electrochem., 20 (2020) 78–81.E. J. Carrasco-Correa, E. F. Simó-Alfonso, J. M. Herrero-Martínez, M. Miró, TrAC - Trends Anal. Chem., 136 (2021) 116177.A Baltima, H. Panagopoulou, A. Economou, C. Kokkinos, Anal. Chim. Acta 1159 (2021) 338426.V. Katseli, M. Angelopoulou, C. Kokkinos, Adv. Funct. Mater., 31 (2021) 2102459.V. Katseli, A. Economou, C. Kokkinos, Anal. Chem. 93 (2021) 3331−3336.G. Poulladofonou, C. Freris, A. Economou, C. Kokkinos, Anal. Chem. 94 (2022) 4087−4094.

Multi-Sample Detection of Soil Nitrate Nitrogen Using a Digital Microfluidic Platform

The rapid quantification of nitrate nitrogen concentration plays a pivotal role in monitoring soil nutrient content. Nevertheless, the low detection efficiency limits the application of traditional methods in rapid testing. For this investigation, we utilized a digital microfluidic platform and 3D-printed microfluidics to accomplish automated detection of soil nitrate nitrogen with high sensitivity across numerous samples. The system combines digital microfluidics (DMF), 3D-printed microfluidics, a peristaltic pump, and a spectrometer. The soil solution, obtained after extraction, was dispensed onto the digital microfluidic platform using a micropipette. The digital microfluidic platform regulated the movement of droplets until they reached the injection area, where they were then aspirated into the 3D-printed microfluidic device for absorbance detection. Implementing this approach allows for the convenient sequential testing of multi-samples, thereby enhancing the efficiency of nitrate nitrogen detection. The results demonstrate that the device exhibits rapid detection (200 s for three samples), low reagent consumption (40 µL per sample), and low detection limit (95 µg/L). In addition, the relative error between the detected concentration and the concentration measured by ultraviolet spectrophotometry is kept within 20%, and the relative standard deviation (RSD) of the measured soil samples is between 0.9% and 4.7%. In the foreseeable future, this device will play a significant role in improving the efficiency of soil nutrient detection and guiding fertilization practices.

Synthesis of tunable gold nanostars via 3D-printed microfluidic device with vibrating sharp-tip acoustic mixing

Synthesis of tunable gold nanostars via 3D-printed microfluidic device with vibrating sharp-tip acoustic mixing

Development of an automated flow chemistry affinity-based purification process for DNA-encoded chemistry

An automated flow chemistry platform for DNA-encoded library (DEL) technologies requires the integration of a purification process for DNA-tagged substrates. It facilitates the development of further DEL reactions, building block rehearsal, and library synthesis. Therefore, a recently developed, manual affinity-based batch purification process for DNA-tagged substrates based on dispersive solid-phase extraction (DSPE) was transferred to automated flow chemistry using tailored 3D-printed microfluidic devices and open-source lab automation equipment. The immobilization and purification steps use Watson–Crick base pairing for a compound-encoding single-stranded DNA, which allows for the thorough removal of impurities and contaminations by washing steps and operationally simple recovery of the purified DNA-encoded compounds. This work optimized the annealing step for flow incubation and DNA purification was accomplished by flow DSPE washing/elution steps. The manually performed batch affinity-based purification process was compared with the microfluidic process by determining qualitative and quantitative DNA recovery parameters. It aimed at comparing batch and flow purification processes with regard to DNA recovery and purity to benefit from the high potential for automation, precise process control, and higher information density of the microfluidic purification process for DNA-tagged substrates. Manual operations were minimized by applying an automation strategy to demonstrate the potential for integrating the microfluidic affinity-based purification process for DNA-tagged substrates into an automated DNA-encoded flow chemistry platform.Graphical

Output Force Density Saturation in COMSOL Simulations of Biomimetic Artificial Muscles

Many modern applications, such as undersea drones, exoskeletal suits, all-terrain walker drones, prosthetics, and medical augments, would greatly benefit from artificial muscles. Such may be built through 3D-printed microfluidic devices that mimic biological muscles and actuate electrostatically. Our preliminary results from COMSOL simulations of individual devices and small arrays (2 × 2 × 1) established the basic feasibility of this approach. Herein, we report on the extension of this work to N × N × 10 arrays where Nmax = 13. For each N, parameter sweeps were performed to determine the maximal output force density, which, when plotted vs. N, exhibited saturation behavior for N ≥ 10. This indicates that COMSOL simulations of a 10 × 10 × 10 array of this type are sufficient to predict the behavior of far larger arrays. Also, the saturation force density was ~9 kPa for the 100 μm scale. Both results are very important for the development of 3D-printable artificial muscles and their applications, as they indicate that computationally accessible simulation sizes would provide sufficiently accurate quantitative predictions of the force density output and overall performance of macro-scale arrays of artificial muscle fibers. Hence, simulations of new geometries can be done rapidly and with quantitative results that are directly extendable to full-scale prototypes, thereby accelerating the pace of research and development in the field of actuators.

Continuous Flow Separation of Live and Dead Cells Using Gravity Sedimentation.

The separation of target cell species is an important step for various biomedical applications ranging from single cell studies to drug testing and cell-based therapies. The purity of cell solutions is critical for therapeutic application. For example, dead cells and debris can negatively affect the efficacy of cell-based therapies. This study presents a cost-effective method for the continuous separation of live and dead cells using a 3D resin-printed microfluidic device. Saccharomyces cerevisiae yeast cells are used for cell separation experiments. Both numerical and experimental studies are presented to show the effectiveness of the presented device for the isolation of dead cells from cell solutions. The experimental results show that the 3D-printed microfluidic device successfully separates live and dead cells based on density differences. Separation efficiencies of over 95% are achieved at optimum flow rates, resulting in purer cell populations in the outlets. This study highlights the simplicity, cost-effectiveness, and potential applications of the 3D-printed microfluidic device for cell separation. The implementation of 3D printing technology in microfluidics holds promise for advancing the field and enabling the production of customized devices for biomedical applications.

Engineering Biosensors and Biomedical Detection Devices from 3D-Printed Technology

Limitation of 3D construction ability, complex preparation processes and developing customer demands have promoted people to find low-cost, rapid prototyping, and simple operation methods to produce novel functional devices in the near future. Among various techniques, 3D-printed technology is a promising candidate for the fabrication of biosensors and biomedical detection devices with a wide variety of potential applications. This review offers four important 3D printing techniques for biosensors and biomedical detection devices and their applications. The principle and printing process of 3D-printed technologies will be generalized, and the printing performance of many 3D printers will be compared. Despite the resolution restrictions of 3D-printed, these technologies have already shown promising applications in many biosensors and biomedical detection devices, such as 3D-printed microfluidic devices, 3D-printed optical devices, 3D-printed electrochemical devices, and 3D-printed integrated devices. Some of the most representative examples will also be discussed here, demonstrating that 3D-printed technology can rationally design biosensors and biomedical detection devices and achieve important applications in microfluidic, optical, electrochemical, and integrated devices.

Leveraging the third dimension in microfluidic devices using 3D printing: no longer just scratching the surface.

3D printers utilize cutting-edge technologies to create three-dimensional objects and are attractive tools for engineering compact microfluidic platforms with complex architectures for chemical and biochemical analyses. 3D printing's popularity is associated with the freedom of creating intricate designs using inexpensive instrumentation, and these tools can produce miniaturized platforms in minutes, facilitating fabrication scaleup. This work discusses key challenges in producing three-dimensional microfluidic structures using currently available 3D printers, addressing considerations about printer capabilities and software limitations encountered in the design and processing of new architectures. This article further communicates the benefits of using three-dimensional structures, including the ability to scalably produce miniaturized analytical systems and the possibility of combining them with multiple processes, such as mixing, pumping, pre-concentration, and detection. Besides increasing analytical applicability, such three-dimensional architectures are important in the eventual design of commercial devices since they can decrease user interferences and reduce the volume of reagents or samples required, making assays more reliable and rapid. Moreover, this manuscript provides insights into research directions involving 3D-printed microfluidic devices. Finally, this work offers an outlook for future developments to provide and take advantage of 3D microfluidic functionality in 3D printing. Graphical abstract Creating three-dimensional microfluidic structures using 3D printing will enable key advances and novel applications in (bio)chemical analysis.

Trends in 3D Printed Microfluidic Devices for Biotechnology Research

Trends in 3D Printed Microfluidic Devices for Biotechnology Research

System for Patterning Polydopamine and VAPG Peptide on Polytetrafluoroethylene and Biodegradable Polyesters for Patterned Growth of Smooth Muscle Cells In Vitro.

Biomaterial's surface functionalization for selective adhesion and patterned cell growth remains essential in developing novel implantable medical devices for regenerative medicine applications. We built and applied a 3D-printed microfluidic device to fabricate polydopamine (PDA) patterns on the surface of polytetrafluoroethylene (PTFE), poly(l-lactic acid-co-D,l-lactic acid) (PLA), and poly(lactic acid-co-glycolic acid) (PLGA). Then, we covalently attached the Val-Ala-Pro-Gly (VAPG) peptide to the created PDA pattern to promote the adhesion of the smooth muscle cells (SMCs). We proved that the fabrication of PDA patterns allows for the selective adhesion of mouse fibroblast and human SMCs to PDA-patterned surfaces after only 30 min of in vitro cultivation. After 7 days of SMC culture, we observed the proliferation of cells only along the patterns on PTFE but over the entire surface of the PLA and PLGA, regardless of patterning. This means that the presented approach is beneficial for application to materials resistant to cell adhesion and proliferation. The additional attachment of the VAPG peptide to the PDA patterns did not bring measurable benefits due to the high increase in adhesion and patterned cell proliferation by PDA itself.

Precise Integration of Polymeric Sensing Functional Materials within 3D Printed Microfluidic Devices

This work presents a new architecture concept for microfluidic devices, which combines the conventional 3D printing fabrication process with the stable and precise integration of polymeric functional materials in small footprints within the microchannels in well-defined locations. The approach solves the assembly errors that normally occur during the integration of functional and/or sensing materials in hybrid microfluidic devices. The method was demonstrated by embedding four pH-sensitive ionogel microstructures along the main microfluidic channel of a complex 3D printed microfluidic device. The results showed that this microfluidic architecture, comprising the internal integration of sensing microstructures of diverse chemical compositions, highly enhanced the adhesion force between the microstructures and the 3D printed microfluidic device that contains them. In addition, the performance of this novel 3D printed pH sensor device was investigated using image analysis of the pH colour variations obtained from photos taken with a conventional camera. The device presented accurate and repetitive pH responses in the 2 to 12 pH range without showing any type of device deterioration or lack of performance over time.

Fabrication of a simple 3D-printed microfluidic device with embedded electrochemiluminescence detection for rapid determination of sibutramine in dietary supplements.

Thedesign and fabrication of a simple 3D-printed platform with embedded electrochemiluminescence (ECL) detection for sibutramine determination is described. The microfluidic platform was fabricated by the fused deposition 3D-printing technique with polylactic acid filament, facilitated by computer-aided design (CAD). A three-electrode system was integrated into the device using graphene carbon paste as a working electrode, Ag/AgCl wire as a reference, and a graphite rod as a counter electrode. A further modification was carried out by applying bimetallic Au-Pt nanoparticle-supported multi-walled carbon nanotubes (MWCNT-Au-Pt) on the working electrode surface to enhance the electrocatalytic performance by exploiting the unique properties of nanomaterials. The analytical feasibility of the CAD-ECL sensor was tested through its application for the determination of sibutramine in dietary supplements. Under the optimized conditions, based on the enhancing effect of luminol emission, the device exhibited a linear calibration curve of the logarithmic sibutramine concentration versus ECL intensityin the range 5 × 10-3 to 1ngmL-1. The limit of detection was 3pgmL-1 with a relative standard deviation of 1.7% (n = 15). The 3D-printed prototype can be successfully applied to a small-scale analysis in a simple and cost-effective approach.

3D printed microfluidic devices using TOPAS filament for THz spectroscopic measurements

We present the 3D printing of microfluidic devices suitable for the THz regime using TOPAS filaments. We assessed the 3D-printed microfluidic devices with THz time-domain spectroscopy and X-ray microscopy. We compared the results of polished and unpolished windows of the 3D printed devices for studying the quality of the 3D printing in the THz and VIS regimes. Spectroscopic measurements were performed of water and lysozyme dissolved in water. We found that the results from the polished and unpolished devices were similar in the THz regime. A detailed calculation of the measurement uncertainty revealed that the dominant component of the measurement uncertainly was the gap thickness. Because of their optical and mechanical properties, we are convinced that 3D printing from TOPAS offers a cost-effective and rapid production of microfluidic devices usable in X-ray, UV, VIS, NIR, and THz regimes.

A multistep immunoassay on a 3D-printed capillarity-driven microfluidic device for point-of-care diagnostics

Although efficient diagnostic testing often relies on centralized laboratories, rapid diagnostics at the point-of-care (POC) are increasingly investigated to provide an affordable and easy-to-use alternative that requires minimal external equipment. Implementing complex assays on such POC devices is challenging, and existing methods such as paper-based analytical devices suffer from laborious and heterogeneous fabrication. 3D printing is a promising technology to enable the single-step fabrication of such diagnostic devices. We previously reported monolithic, 3D-printed microfluidics driven by capillarity, and used the platform to implement a proof-of-concept enzyme-linked immunosorbent assay (ELISA) for immunoglobulin E (IgE) detection. In this study, we aimed to improve the time-to-result and detection limit of this approach by (i) designing a second-generation 3D-printed device and (ii) further optimizing the IgE ELISA. Herein, we selected horseradish peroxidase for faster and more sensitive signal amplification compared to the first-generation device, for which we adapted the microfluidic device design to allow sequential flow while using separated reagent channels for each assay step. To implement the ELISA on this new design, we decreased reagent volumes and incubation times to be compatible with POC, and then we translated this assay stepwise from microtiter plates to the completely 3D-printed, second-generation device. Overall, this work gives additional insight into the biocompatibility of these 3D-printed devices, as well as demonstrates the huge potential of powder bed 3D printing to fabricate POC diagnostic devices.

Simultaneous detection of dopamine and ascorbic acid by using a thread-based microfluidic device and multiple pulse amperometry.

This study presents a novel approach for the simultaneous detection of ascorbic acid (AA) and dopamine (DA) using an affordable and user-friendly microfluidic device. Microfluidic devices, when combined with electrochemical detectors like screen-printed electrodes (SPEs), offer numerous advantages such as portability, high sample throughput, and low reagent consumption. In this study, a 3D-printed microfluidic device called a μTED was developed, utilizing textile threads as microfluidic channels and an unmodified SPE as the amperometric detector. The method employed multiple pulse amperometry (MPA) with carefully selected potential values (+0.65 V and -0.10 V). The reduction current signals generated by dopamine o-quinone were used to calculate a correction factor for the oxidation signals of ascorbic acid, enabling simultaneous quantification. The developed microfluidic device ensured a stable flow rate of the carrier solution at 1.19 μL s-1, minimizing the consumption of samples and reagents (injection volume of 2.0 μL). Under the optimized experimental conditions, a linear range from 50 to 900 μmol L-1 was achieved for both DA and AA. The obtained sensitivities were 2.24 μA L mmol-1 for AA and 5.09 μA L mmol-1 for DA, with corresponding limits of detection (LOD) of 2.60 μmol L-1 and 1.54 μmol L-1, respectively. To confirm the effectiveness of the proposed method, it was successfully applied to analyze AA and DA in a commercial blood serum sample spiked at three different concentration levels, with a medium recovery rate of 70%. Furthermore, the MPA technique demonstrated its simplicity by enabling the simultaneous determination of AA and DA without the need for prior separation steps or the use of chemically modified electrodes.