3D-printed Chip Research Articles

Super-Resolution-Chip: an in-vitro platform that enables super-resolution microscopy of co-cultures and 3D systems.

The development of organs-on-a-chip platforms has revolutionized in-vitro cellular culture by allowing cells to be grown in an environment that better mimics human physiology. However, there is still a challenge in integrating those platforms with advanced imaging technology. This is extremely important when we want to study molecular changes and subcellular processes on the level of a single molecule using super-resolution microscopy (SRM), which has a resolution beyond the diffraction limit of light. Currently, existing platforms that include SRM have certain limitations, either as they only support 2D monocultures, without flow or as they demand a lot of production and handling. In this study, we developed a Super-Res-Chip platform, consisting of a 3D-printed chip and a porous membrane, that could be used to co-culture cells in close proximity either in 2D or in 3D while allowing SRM on both sides of the membrane. To demonstrate the functionality of the device, we co-cultured in endothelial and epithelial cells and used direct stochastic optical reconstruction microscopy (dSTORM) to investigate how glioblastoma cells affect the expression of the gap-junction protein Connexin43 in endothelial cells grown in 2D and in 3D. Cluster analysis of Connexin43 distribution revealed no difference in the number of clusters, their size, or radii, but did identify differences in their density. Furthermore, the spatial resolution was high also when the cells were imaged through the membrane (20-30 nm for x-y) and 10-20 nm when imaged directly both for 2D and 3D conditions. Overall, this chip allows to characterize of complex cellular processes on a molecular scale in an easy manner and improved the capacity for imaging in a single molecule resolution complex cellular organization.

3D-Printed Tumor-on-Chip for the Culture of Colorectal Cancer Microspheres: Mass Transport Characterization and Anti-Cancer Drug Assays.

Tumor-on-chips have become an effective resource in cancer research. However, their widespread use remains limited due to issues related to their practicality in fabrication and use. To address some of these limitations, we introduce a 3D-printed chip, which is large enough to host ~1 cm3 of tissue and fosters well-mixed conditions in the liquid niche, while still enabling the formation of the concentration profiles that occur in real tissues due to diffusive transport. We compared the mass transport performance in its rhomboidal culture chamber when empty, when filled with GelMA/alginate hydrogel microbeads, or when occupied with a monolithic piece of hydrogel with a central channel, allowing communication between the inlet and outlet. We show that our chip filled with hydrogel microspheres in the culture chamber promotes adequate mixing and enhanced distribution of culture media. In proof-of-concept pharmacological assays, we biofabricated hydrogel microspheres containing embedded Caco2 cells, which developed into microtumors. Microtumors cultured in the device developed throughout the 10-day culture showing >75% of viability. Microtumors subjected to 5-fluorouracil treatment displayed <20% cell survival and lower VEGF-A and E-cadherin expression than untreated controls. Overall, our tumor-on-chip device proved suitable for studying cancer biology and performing drug response assays.

Quantitative study for control of air–liquid segmented flow in a 3D-printed chip using a vacuum-driven system

The formation of droplets or bubbles in a microfluidic system is a significant topic requiring device miniaturization and a small volume of samples. Especially, a two-phase segmented flow can be applied to micro-mixing for chemical reactions and the treatment of heat and mass transfer. In this study, a flow of liquid slugs and bubbles was generated in a 3D-printed chip and controlled by a single pump creating a vacuum at the outlet. The pump and chip device were integrated to form a simple and portable system. The size and flow rate of liquid slugs, obtained through image processing techniques, were analyzed considering several parameters related to hydraulic resistance and pressure drop. In addition, the effect of segmentation on mixing was observed by measuring the intensity change using two different colored inks. The hydraulic resistance of air and liquid flows can be controlled by changing the tube length of air flow and the viscosity of liquid flow. Because the total pressure drop along the channel was produced using a single pump at the outlet of the channel, the size and flow rate of the liquid slugs showed a near linear relation depending on the hydraulic resistances. In contrast, as the total pressure varied with the flow rate of the pump, the size of the liquid slugs showed a nonlinear trend. This indicates that the frequency of the liquid slug formation induced by the squeezed bubble may be affected by several forces during the development of the liquid slugs and bubbles. In addition, each volume of liquid slug segmented by the air is within the range of 10–1 to 2 µL for this microfluidic system. The segmentation contributes to mixing efficiency based on the increased homogeneity factor of liquid. This study provides a new insight to better understand the liquid slug or droplet formation and predict the segmented flow based on the relationship between the resistance, flow rate, and pressure drop.

Portable all-in-one automated microfluidic system (PAMICON) with 3D-printed chip using novel fluid control mechanism

State-of-the-art microfluidic systems rely on relatively expensive and bulky off-chip infrastructures. The core of a system—the microfluidic chip—requires a clean room and dedicated skills to be fabricated. Thus, state-of-the-art microfluidic systems are barely accessible, especially for the do-it-yourself (DIY) community or enthusiasts. Recent emerging technology—3D-printing—has shown promise to fabricate microfluidic chips more simply, but the resulting chip is mainly hardened and single-layered and can hardly replace the state-of-the-art Polydimethylsiloxane (PDMS) chip. There exists no convenient fluidic control mechanism yet suitable for the hardened single-layered chip, and particularly, the hardened single-layered chip cannot replicate the pneumatic valve—an essential actuator for automatically controlled microfluidics. Instead, 3D-printable non-pneumatic or manually actuated valve designs are reported, but their application is limited. Here, we present a low-cost accessible all-in-one portable microfluidic system, which uses an easy-to-print single-layered 3D-printed microfluidic chip along with a novel active control mechanism for fluids to enable more applications. This active control mechanism is based on air or gas interception and can, e.g., block, direct, and transport fluid. As a demonstration, we show the system can automatically control the fluid in microfluidic chips, which we designed and printed with a consumer-grade 3D-printer. The system is comparably compact and can automatically perform user-programmed experiments. All operations can be done directly on the system with no additional host device required. This work could support the spread of low budget accessible microfluidic systems as portable, usable on-the-go devices and increase the application field of 3D-printed microfluidic devices.

Heater Integrated Lab-on-a-Chip Device for Rapid HLA Alleles Amplification towards Prevention of Drug Hypersensitivity.

HLA-B*15:02 screening before administering carbamazepine is recommended to prevent life-threatening hypersensitivity. However, the unavailability of a point-of-care device impedes this screening process. Our research group previously developed a two-step HLA-B*15:02 detection technique utilizing loop-mediated isothermal amplification (LAMP) on the tube, which requires two-stage device development to translate into a portable platform. Here, we report a heater-integrated lab-on-a-chip device for the LAMP amplification, which can rapidly detect HLA-B alleles colorimetrically. A gold-patterned micro-sized heater was integrated into a 3D-printed chip, allowing microfluidic pumping, valving, and incubation. The performance of the chip was tested with color dye. Then LAMP assay was conducted with human genomic DNA samples of known HLA-B genotypes in the LAMP-chip parallel with the tube assay. The LAMP-on-chip results showed a complete match with the LAMP-on-tube assay, demonstrating the detection system’s concurrence.

Additive manufacturing of a 3D vascular chip based on cytocompatible hydrogel

Development of a three-dimensional (3D) vascular microfluidic chip is one of the important challenges for the study vascular-related diseases and new drugs. To date, however, realization of in vivo-like blood vessel chips based on soft- or photo-lithographic fabrication methods have limitations such as complex fabrication processes, tedious manual labor, and frequent assembly failure. In this study, we describe the development of a 3D cell culture chip by utilizing a stereolithographic printer. The microfluidic chip was fabricated using a resin made of 30% (v/v in water) poly(ethylene glycol) diacrylate (MW = 700) (30% PEG-DA-700), a UV initiator (Irgacure-819 (IRG)) and a photosensitizer (2-isopropylthioxanthone (ITX)) for constructing a cytocompatible hydrogel structure. The 3D-printed cell culture chip is transparent, enabling cell culture and observation of cell proliferation. Endothelial cells were cultured to mimic in vivo-like blood vessel architecture within the 3D-printed chip. After EAhy926 cell cultivation, cell viability was confirmed by a live/dead cell assay and cultured cells were stained with Calcein-AM (Green) and Ethidium homodim-1 (Red). In addition, endothelial cells were successfully cultured in a 3D-printed vascular tube-like architecture in PEG-DA-700. Creating a vascular microfluidic chip with 30% PEG-DA-700 hydrogel using automated 3D-printing processes reduces fabrication steps and time, avoids any assembly and bonding, and economizes manufacturing cost. We believe our stereolithographic 3D-printing method for fabricating a 3D-printed vascular chip provides a cost-effective, convenient, simple, and reproducible route to vascular-related disease modelling and drug screening analyses.

Characterization and Electrochemical Analysis of Microelectrodes and the Interface with a Fabricated 3D-Printed Microfluidic Chip in an Upper-Division Analytical Course

This work explores the interface between 3D-printing, material sciences, and microfluidics with electrochemical detection in an undergraduate laboratory. This work includes a module for the characterization and electrochemical analysis of microelectrodes that spans 4 weeks (3 h per week). Laboratory exercises include the fabrication of a 3D-printed device, examination of 3D-printing techniques, scanning electron microscopy (SEM), and electrochemistry. This interdisciplinary curricula exposed students to the process of designing a functioning microfluidic device. Students began by designing the microfluidic chip with AutoDesk Inventor. To gain a better understanding of the microelectrodes utilized in a microfluidic system, students explored the surfaces of various microelectrodes with SEM. On the basis of the visualization of the microelectrodes with SEM, students formed a hypothesis on the impact of electrode surface area on the sensitivity and limit of detection. Cyclic voltammetry in a classical three-electrode system was used to experimentally examine the relationship between electrode surface area and sensitivity. The module concluded with the use of the fabricated 3D-printed chip and amperometry to develop a calibration curve and determination of an unknown concentration of analyte. This work highlights the integration of 3D-printing, SEM, microfluidics, and electrochemistry into the upper-level undergraduate curriculum.

Portable Pumpless 3D-Printed Chip for On-Site Colorimetric Screening of Hg2+ in Lake Water

The early detection of mercury ions in water sources should be emphasized to prevent human exposure to mercury through drinking water or food consumption. Here, we used a three-dimensional (3D)-printed multiarray chip to achieve colorimetric detection of inorganic mercury ions in environmental matrices such as lake water. The chip designed for the multidetection of inorganic mercury did not require an electrical syringe pump or a complex analytical system, thereby making it suitable for on-site detection. The chip could detect inorganic mercury ions via rolling circle amplification, by selectively harnessing thymine-Hg2+-thymine coordination chemistry. Additionally, the activities of rolling circle amplification in simple and environmental matrices were compared. The limits of detection for inorganic mercury ions were 3.4 and 4.1 ??g/L in the simple and environmental matrices, respectively. Further, the chip was successfully applied for the detection of mercury in actual lake water samples collected from locations nearby inland fish farms. The results were quantitatively comparable to those obtained using a conventional mercury analyzer.

Simple and low-cost production of hybrid 3D-printed microfluidic devices.

The use of three-dimensional (3D) printing for the fabrication of microfluidic chips has attracted considerable attention among researchers. This low-cost fabrication method allows for rapid prototyping and the creation of complex structures; however, these devices lack optical transparency, which greatly hinders the characterization and quantification of experiment results. To address this problem, integrating a transparent substrate with a 3D-printed chip is an effective approach. In this study, we present a solvent bonding method of poly(methyl methacrylate) (PMMA) and acrylonitrile butadiene styrene (ABS) thermoplastic materials for the creation of optically detectable 3D-printed microfluidic devices. To achieve an excellent bonding between PMMA and ABS substrates, we used spray coating as a method for the distribution of ethanol solution followed by UV exposure and post-annealing step to improve the bonding strength. We fabricated a microfluidic chip with S-microchannel to characterize the bonding protocol, and other two application-oriented microfluidic chips, including a 3D split-and-recombine-based passive micromixer, and an integrated microchip for the mixing of two streams of liquid prior to the formation of double-emulsion droplets, to evaluate the efficacy of the proposed scheme. As a result, at least eight bars of the bonding strength between PMMA/ABS substrates was achieved, and the ability of producing optically detectable 3D-printed microfluidic devices based on this bonding method was confirmed.

Partitioning of hydrogels in 3D-printed microchannels.

Hydrogels allow for controlling the diffusion rate and amount of solute according to the hydrogel network and thus have found many applications in drug delivery, biomaterials, toxicology, and tissue engineering. This paper describes a 3D-printed microfluidic chip for the straightforward partitioning of hydrogel barriers between microchannels. We use a previously-reported 3-channel architecture whereby the middle channel is filled with a hydrogel - acting like a porous barrier for diffusive transport - and the two side channels act as sink and source; the middle channel communicates with the side channels via orthogonal, small capillary channels that are also responsible for partitioning the hydrogel during filling. Our 3D-printed microfluidic chip is simple to fabricate by stereolithography (SL), inexpensive, reproducible, and convenient, so it is more adequate for transport studies than a microchip fabricated by photolithographic procedures. The chip was fabricated in a resin made of poly(ethylene glycol) diacrylate (PEG-DA) (MW = 258) (PEG-DA-258). The SL process allowed us to print high aspect ratio (37 : 1) capillary channels (27 μm-width and 1 mm-height) and enable the trapping of liquid-phase hydrogels in the hydrogel barrier middle channel. We studied the permeability of hydrogel barriers made of PEG-DA (MW = 700) (PEG-DA-700, 10% polymer content by wt. in water) - as a model of photopolymerizable barriers - and agarose (MW = 120 000, 2% polymer content by wt. in water) - as a model of thermally-gelled barriers. We measured the diffusion of fluorescein, 10k-dextran-Alexa 680 and BSA-Texas Red through these barriers. Fluorescein diffusion was observed through both 10% PEG-DA-700 and 2% agarose barriers while 10k-dextran-Alexa 680 and BSA-Texas Red diffused appreciably only through the 2% agarose hydrogel barrier. Our microfluidic chip facilitates the tuning of such barriers simply by altering the hydrogel materials. The straightforward trapping of selective barriers in 3D-printed microchannels should find wide applicability in drug delivery, tissue engineering, cell separation, and organ-on-a-chip platforms.

A Portable Smartphone-Based Sensing System Using a 3D-Printed Chip for On-Site Biochemical Assays.

Recently, smartphone-based chromogenic sensing with paper-based microfluidic technology has played an increasingly important role in biochemical assays. However, generally there were three defects: (i) the paper-based chips still required complicated fabrication, and the hydrophobic boundaries on the chips were not clear enough; (ii) the chromogenic signals could not be steadily captured; (iii) the smartphone apps were restricted to the detection of specific target analytes and could not be extended for different assays unless reprogrammed. To solve these problems, in this study, a portable smartphone-based sensing system with a 3D-printed chip was developed. A 3D-printed imaging platform was designed to significantly reduce sensing errors generated during signal capture, and a brand-new strategy for signal processing in downloadable apps was established. As a proof-of-concept, the system was applied for detection of organophosphorus pesticides and multi-assay of fruit juice, showing excellent sensing performance. For different target analytes, the most efficient color channel could be selected for signal analysis, and the calibration equation could be directly set in user interface rather than programming environment, thus the developed system could be flexibly extended for other biochemical assays. Consequently, this study provides a novel methodology for smartphone-based biochemical sensing.

Assessing the Reusability of 3D-Printed Photopolymer Microfluidic Chips for Urine Processing.

Three-dimensional (3D) printing is emerging as a method for microfluidic device fabrication boasting facile and low-cost fabrication, as compared to conventional fabrication approaches, such as photolithography, for poly(dimethylsiloxane) (PDMS) counterparts. Additionally, there is an increasing trend in the development and implementation of miniaturized and automatized devices for health monitoring. While nonspecific protein adsorption by PDMS has been studied as a limitation for reusability, the protein adsorption characteristics of 3D-printed materials have not been well-studied or characterized. With these rationales in mind, we study the reusability of 3D-printed microfluidics chips. Herein, a 3D-printed cleaning chip, consisting of inlets for the sample, cleaning solution, and air, and a universal outlet, is presented to assess the reusability of a 3D-printed microfluidic device. Bovine serum albumin (BSA) was used a representative urinary protein and phosphate-buffered solution (PBS) was chosen as the cleaning agent. Using the 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) fluorescence detection method, the protein cross-contamination between samples and the protein uptake of the cleaning chip were assessed, demonstrating a feasible 3D-printed chip design and cleaning procedure to enable reusable microfluidic devices. The performance of the 3D-printed cleaning chip for real urine sample handling was then validated using a commercial dipstick assay.

3D Printing Solutions for Microfluidic Chip-To-World Connections.

The connection of microfluidic devices to the outer world by tubes and wires is an underestimated issue. We present methods based on 3D printing to realize microfluidic chip holders with reliable fluidic and electric connections. The chip holders are constructed by microstereolithography, an additive manufacturing technique with sub-millimeter resolution. The fluidic sealing between the chip and holder is achieved by placing O-rings, partly integrated into the 3D-printed structure. The electric connection of bonding pads located on microfluidic chips is realized by spring-probes fitted within the printed holder. Because there is no gluing or wire bonding necessary, it is easy to change the chip in the measurement setup. The spring probes and O-rings are aligned automatically because of their fixed position within the holder. In the case of bioanalysis applications such as cells, a limitation of 3D-printed objects is the leakage of cytotoxic residues from the printing material, cured resin. This was solved by coating the 3D-printed structures with parylene-C. The combination of silicon/glass microfluidic chips fabricated with highly-reliable clean-room technology and 3D-printed chip holders for the chip-to-world connection is a promising solution for applications where biocompatibility, optical transparency and accurate sample handling must be assured. 3D printing technology for such applications will eventually arise, enabling the fabrication of complete microfluidic devices.

Rapid and Low-Cost CRP Measurement by Integrating a Paper-Based Microfluidic Immunoassay with Smartphone (CRP-Chip).

Traditional diagnostic tests for chronic diseases are expensive and require a specialized laboratory, therefore limiting their use for point-of-care (PoC) testing. To address this gap, we developed a method for rapid and low-cost C-reactive protein (CRP) detection from blood by integrating a paper-based microfluidic immunoassay with a smartphone (CRP-Chip). We chose CRP for this initial development because it is a strong biomarker of prognosis in chronic heart and kidney disease. The microfluidic immunoassay is realized by lateral flow and gold nanoparticle-based colorimetric detection of the target protein. The test image signal is acquired and analyzed using a commercial smartphone with an attached microlens and a 3D-printed chip–phone interface. The CRP-Chip was validated for detecting CRP in blood samples from chronic kidney disease patients and healthy subjects. The linear detection range of the CRP-Chip is up to 2 μg/mL and the detection limit is 54 ng/mL. The CRP-Chip test result yields high reproducibility and is consistent with the standard ELISA kit. A single CRP-Chip can perform the test in triplicate on a single chip within 15 min for less than 50 US cents of material cost. This CRP-Chip with attractive features of low-cost, fast test speed, and integrated easy operation with smartphones has the potential to enable future clinical PoC chronic disease diagnosis and risk stratification by parallel measurements of a panel of protein biomarkers.

3D-printed chip for detection of methicillin-resistant Staphylococcus aureus labeled with gold nanoparticles.

Methicillin-resistant Staphylococcus aureus (MRSA) is a dangerous pathogen occurring not only in hospitals but also in foodstuff. Currently, discussions on the issue of the increasing resistance, and timely and rapid diagnostic of resistance strains have become more frequent and sought. Therefore, the aim of this study was to design an effective platform for DNA isolation from different species of microorganisms as well as the amplification of mecA gene that encodes the resistance to β-lactam antibiotic formation and is contained in MRSA. For this purpose, we fabricated 3D-printed chip that was suitable for bacterial cultivation, DNA isolation, PCR, and detection of amplified gene using gold nanoparticle (AuNP) probes as an indicator of MRSA. Confirmation of the MRSA presence in the samples was based on a specific interaction between mecA gene with the AuNP probes and a colorimetric detection, which utilized the noncross-linking aggregation phenomenon of DNA-functionalized AuNPs. To test the whole system, we analyzed several real refractive indexes, in which two of them were positively scanned to find the presence of mecA gene. The aggregation of AuNP probes were reflected by 75% decrease of absorbance (λ = 530 nm) and change in AuNPs size from 3 ± 0.05 to 4 ± 0.05 nm (n = 5). We provide the one-step identification of mecA gene using the unique platform that employs the rapid, low-cost, and easy-to-use colorimetric method for MRSA detection in various samples.