3D Microfluidic Device Research Articles

On-Chip Droplet Splitting with High Volume Ratios Using a 3D Conical Microstructure-Based Microfluidic Device.

This work reports a simple microfluidic method for splitting a mother droplet into two daughter droplets with high and precise volume ratios. To achieve this, a droplet-splitting microfluidic device embedded with a three-dimensional (3D) conical microstructure is fabricated, in which the high splitting ratios of monodisperse mother droplets are achieved. The volume ratio of the split daughter droplets can reach up to 265. In addition, we examined factors that affect the splitting ratio of the daughter droplets and found that the ratio is affected by the flow rates of the two individual outlet channels, the injection length of the conical microstructure, and the diameter of the original mother droplets. Numerical simulations of these parameters were conducted to gain a clearer understanding of the splitting behavior. The proposed droplet splitting device with a conical microstructure enables on-chip sample extraction and droplet volume control, which can be a powerful tool for various droplet-based applications in microfluidic devices such as viral infectivity assays and sequencing heterogeneous populations.

Gene expression-phenotype association study reveals the dual role of TNF-α/TNFR1 signaling axis in confined breast cancer cell migration

AimsWhile enhanced tumor cell migration is a key process in the tumor dissemination, mechanistic insights into causal relationships between tumor cells and mechanical confinement are still limited. Here we combine the use of microfluidic platforms to characterize confined cell migration with genomic tools to systematically unravel the global signaling landscape associated with the migratory phenotype of breast cancer (BC) cells. Meterials and methodsThe spontaneous migration capacity of seven BC cell lines was evaluated in 3D microfluidic devices and their migration capacity was correlated with publicly available molecular signatures. The role of identified signaling pathways on regulating BC migration capacity was determined by receptor stimulation through ligand binding or inhibition through siRNA silencing. Downstream effects on cell migration were evaluated in microfluidic devices, while the molecular changes were monitored by RT-qPCR. Key findingsExpression of 715 genes was correlated with BC cells migratory phenotype, revealing TNF-α as one of the top upstream regulators. Signal transduction experiments revealed that TNF-α stimulates the confined migration of triple negative, mesenchymal-like BC cells that are also characterized by high TNFR1 expression, but inhibits the migration of epithelial-like cells with low TNFR1 expression. TNFR1 was strongly associated with the migration capacity and triple-negative, mesenchymal phenotype. Downstream of TNF/TNFR1 signaling, transcriptional regulation of NFKB seems to be important in driving cell migration in confined spaces. SignificanceTNF-α/TNFR1 signaling axis reveals as a key player in driving BC cells confined migration, emerging as a promising therapeutic strategy in targeting dissemination and metastasis of triple negative, mesenchymal BC cells.

Functional Integration of Synthetic Cells into 3D Microfluidic Devices for Artificial Organ-On-Chip Technologies.

Microfluidics plays a pivotal role in organ-on-chip technologies and in the study of synthetic cells, especially in the development and analysis of artificial cell models. However, approaches that use synthetic cells as integral functional components for microfluidic systems to shape the microenvironment of natural living cells cultured on-chip are not explored. Here, colloidosome-based synthetic cells are integrated into 3D microfluidic devices, pioneering the concept of synthetic cell-based microenvironments for organs-on-chip. Methods are devised to create dense and stable networks of silica colloidosomes, enveloped by supported lipid bilayers, within microfluidic channels. These networks promote receptor-ligand interactions with on-chip cultured cells. Furthermore, a technique is introduced for the controlled release of growth factors from the synthetic cells into the channels, using a calcium alginate-based hydrogel formation within the colloidosomes. To demonstrate the potential of the technology, a modular plug-and-play lymph-node-on-a-chip prototype that guides the expansion of primary human T cells by stimulating receptor ligands on the T cells and modulating their cytokine environment is presented. This integration of synthetic cells into microfluidic systems offers a new direction for organ-on-chip technologies and suggests further avenues for exploration in potential therapeutic applications.

Nanoporous, Gas Permeable PEGDA Ink for 3D Printing Organ‐on‐a‐Chip Devices

AbstractPolydimethylsiloxane (PDMS), commonly used in organ‐on‐a‐chip (OoC) systems, faces limitations in replicating complex geometries, hindering its effectiveness in creating 3D OoC models. In contrast, poly(ethylene glycol)diacrylate (PEGDA‐250), favored for its fabrication ease and resistance to small molecule absorption, is increasingly used for 3D printing microfluidic devices. However, applications in cell culture have been limited due to poor cell adhesion. Here, a nanoporous PEGDA ink (P‐PEGDA) is introduced to enhance cell adhesion. P‐PEGDA is formulated with a porogen, photopolymerized, followed by the porogen removal. Utilizing P‐PEGDA, complex microstructures, and membranes as thin as 27 µm are 3D‐printed. Porogen concentrations from 10 to 30% are tested yielding constructs with increasing porosity and oxygen permeability surpassing PDMS, without compromising printing resolution. Tests across four cell lines show >80% cell viability, with a notable 77‐fold increase in MDA‐MB‐231 cell coverage on the porous scaffolds. Finally, an OoC model comprising a gyroid scaffold with a central opening filled with a cancer spheroid is introduced. This setup, after a 14 days co‐culture, demonstrates significant endothelial sprouting and integration within the spheroid. The P‐PEGDA formulation is suitable for high‐resolution 3D printing of constructs for 3D cell culture and OoC owing to its printability, gas permeability, biocompatibility, and cell adhesion.

Biomimetic gradient scaffolds for the tissue engineering and regeneration of rotator cuff enthesis.

Rotator cuff tear is one of the most common musculoskeletal disorders, which often results in recurrent shoulder pain and limited movement. Enthesis is a structurally complex and functionally critical interface connecting tendon and bone that plays an essential role in maintaining integrity of the shoulder joint. Despite the availability of advanced surgical procedures for rotator cuff repair, there is a high rate of failure following surgery due to suboptimal enthesis healing and regeneration. Novel strategies based on tissue engineering are gaining popularity in improving tendon-bone interface (TBI) regeneration. Through incorporating physical and biochemical cues into scaffold design which mimics the structure and composition of native enthesis is advantageous to guide specific differentiation of seeding cells and facilitate the formation of functional tissues. In this review, we summarize the current state of research in enthesis tissue engineering highlighting the development and application of biomimetic scaffolds that replicate the gradient TBI. We also discuss the latest techniques for fabricating potential translatable scaffolds such as 3D bioprinting and microfluidic device. While preclinical studies have demonstrated encouraging results of biomimetic gradient scaffolds, the translation of these findings into clinical applications necessitates a comprehensive understanding of their safety and long-term efficacy.

Numerical and experimental investigation of chaotic advection and diffusion mixing effects in 3D multihelical microfluidics for liposome synthesis

The recent advances in nanomaterials’ production by microfluidics with high throughput became critical for industry-scale production. Numerical simulation has been part of this development, allowing faster and more detailed study of the process that would be costly only experimentally. Here, we investigated the diffusion and chaotic advection effects on cationic and stealth liposome production in a 3D multihelical chaotic-advection microfluidic device. Suitable conditions for chaotic advection were primarily investigated with computational fluid dynamics (CFD) assessing diverse flow rate ratio (FRR) and total flow rate (TFR). The results showed that Reynolds ≈ 100 and Dean number > 50 can trigger chaotic advection for this device, corresponding to a TFR of 5 mL/min. We produced the liposomes in the same conditions as the simulations to evaluate the impact of these parameters on the colloidal physicochemical properties. A clear difference was shown between TFR ≤ 1 and TFR ≥ 5 mL/min for size and polydispersity (PDI). An increase in PDI was observed for FRR 10, in which the mixing index (M.I.) played an important role. The effect of composition proved an important factor, as stealth liposomes were not deeply affected by TFR change. Cryo-Transmission Electron Microscopy (Cryo-TEM) analysis showed that both cationic and stealth liposomes resulted in unilamellar liposomes. Coupling numerical simulation with experimental characterization proved an efficient process evaluation in microfluidics. Finally, the high mass throughput (2.8 g/h) with the chaotic advection microfluidic device shows a potential microchip for industrial scale-up and parallelization.

Abstract 935: Co-clinical endometrial cancer (EC) patient-derived organoids (PDO) to assess functional effects of replication stress (RS)-targeting agents

Abstract Background: EC is the most commonly diagnosed gynecologic cancer in the US and has both rising incidence and mortality. Uterine serous cancer (USC) and uterine carcinosarcoma (UCS) are high grade ECs characterized by TP53 mutation and molecular alterations suggesting high replication stress (RS) and cell cycle dysregulation. These molecular alterations could engender sensitivity to RS-targeting strategies such as WEE1 inhibition. In a single arm phase 2 of 35 patients, the WEE1 inhibitor adavosertib demonstrated clinical activity in recurrent USC, with an overall response rate of 29.4%. To support the development of predictive biomarkers of sensitivity to RS targeting agents, we report on the establishment of co-clinical EC PDOs from pre- and on-treatment biopsies from patients with USC or UCS enrolled to a translational trial of adavosertib. Methods: Patients with USC or UCS consented for trial participation underwent pre- and on-treatment biopsies while receiving adavosertib monotherapy. Biopsies were dissociated and passaged as 3D organoid cultures in Matrigel in an optimized media. Established PDOs were seeded in 3D microfluidic devices, treated with adavosertib in a dose-dependent manner and assessed for viability after 6 days of treatment by dual labeling live/dead imaging. IC50 values were calculated using log(inhibitor) vs response - variable slope (four parameters) model in GraphPad Prism. To assess the effect of adavosertib on replication fork speed, DNA fiber assays were conducted with PDOs treated with adavosertib for 72 hours. Finally, FFPE blocks were created from PDOs for assessment of DNA damage repair biomarkers by IHC. Results: 19 biopsies were obtained from 11 participating patients. 6 biopsies did not yield sufficient tumor cells for PDO generation. From the remaining 13 samples, 5 PDOs were successfully established (3 USC and 2 UCS), with a higher success rate when 3 biopsy cores were received (3 of 4; 75%) compared to when 2 biopsy cores were received (2 of 9; 22 %). Dual labeling live/dead imaging of PDOs treated with adavosertib resulted in IC50 values ranging from 113nM to 856nM. DNA fiber assays revealed consistent decrease in fork speed with adavosertib exposure, consistent with the hypothesized effect of adavosertib in increasing RS in these cells. A dose dependent effect on replication fork speed was observed, with greater decreases in fork speed with increasing concentrations of adavosertib. Due to the limited number of samples and the number of PDOs generated from on-treatment as opposed to pre-treatment biopsies, clinical correlation between PDO adavosertib sensitivity and clinical activity was not formally assessed. IHC analysis of potential DNA damage biomarkers is ongoing. Conclusions: We describe the successful establishment of co-clinical EC PDOs for functional evaluation of sensitivity to RS targeting agents. Citation Format: Elena Ivanova, Aisha L. Saldanha, Shrabasti Roychoudhury, Bose Kochupurakkal, Ari P. Zlota, Clare E. Padrick, Ha V. Vo, Eli K. Greenberg, Hannah Sawyer, Carina Feeney, Courtney H. Qi, Swati Narayan, Jennifer D. Curtis, Nabihah Tayob, Marisa R. Nucci, Panagiotis Konstantinopoulos, Dipanjan Chowdhury, Geoffrey Shapiro, Cloud P. Paweletz, Ursula A. Matulonis, Joyce F. Liu. Co-clinical endometrial cancer (EC) patient-derived organoids (PDO) to assess functional effects of replication stress (RS)-targeting agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 935.

YAP/TEAD involvement in resistance to paclitaxel chemotherapy in lung cancer.

The Yes-associated protein (YAP) oncoprotein has been linked to both metastases and resistance to targeted therapy of lung cancer cells. We aimed to investigate the effect of YAP pharmacological inhibition, using YAP/TEA domain (TEAD) transcription factor interaction inhibitors in chemo-resistant lung cancer cells. YAP subcellular localization, as a readout for YAP activation, cell migration, and TEAD transcription factor functional transcriptional activity were investigated in cancer cell lines with up-regulated YAP, with and without YAP/TEAD interaction inhibitors. Parental (A549) and paclitaxel-resistant (A549R) cell transcriptomes were analyzed. The half-maximal inhibitory concentration (IC50) of paclitaxel or trametinib, which are Mitogen-Activated protein kinase and Erk Kinase (MEK) inhibitors, combined with a YAP/TEAD inhibitor (IV#6), was determined. A three-dimensional (3D) microfluidic culture device enabled us to study the effect of IV#6/paclitaxel combination on cancer cells isolated from fresh resected lung cancer samples. YAP activity was significantly higher in paclitaxel-resistant cell lines. The YAP/TEAD inhibitor induced a decreased YAP activity in A549, PC9, and H2052 cells, with reduced YAP nuclear staining. Wound healing assays upon YAP inhibition revealed impaired cell motility of lung cancer A549 and mesothelioma H2052 cells. Combining YAP pharmacological inhibition with trametinib in K-Ras mutated A549 cells recapitulated synthetic lethality, thereby sensitizing these cells to MEK inhibition. The YAP/TEAD inhibitor lowered the IC50 of paclitaxel in A549R cells. Differential transcriptomic analysis of parental and A549R cells revealed an increased YAP/TEAD transcriptomic signature in resistant cells, downregulated upon YAP inhibition. The YAP/TEAD inhibitor restored paclitaxel sensitivity of A549R cells cultured in a 3D microfluidic system, with lung cancer cells from a fresh tumor efficiently killed by YAP/TEAD inhibitor/paclitaxel doublet. Evidence of the YAP/TEAD transcriptional program's role in chemotherapy resistance paves the way for YAP therapeutic targeting.

Twisted fiber microfluidics: a cutting-edge approach to 3D spiral devices.

The development of 3D spiral microfluidics has opened new avenues for leveraging inertial focusing to analyze small fluid volumes, thereby advancing research across chemical, physical, and biological disciplines. While traditional straight microchannels rely solely on inertial lift forces, the novel spiral geometry generates Dean drag forces, eliminating the necessity for external fields in fluid manipulation. Nevertheless, fabricating 3D spiral microfluidics remains a labor-intensive and costly endeavor, hindering its widespread adoption. Moreover, conventional lithographic methods primarily yield 2D planar devices, thereby limiting the selection of materials and geometrical configurations. To address these challenges, this work introduces a streamlined fabrication method for 3D spiral microfluidic devices, employing rotational force within a miniaturized thermal drawing process, termed as mini-rTDP. This innovation allows for rapid prototyping of twisted fiber-based microfluidics featuring versatility in material selection and heightened geometric intricacy. To validate the performance of these devices, we combined computational modeling with microtomographic particle image velocimetry (μTPIV) to comprehensively characterize the 3D flow dynamics. Our results corroborate the presence of a steady secondary flow, underscoring the effectiveness of our approach. Our 3D spiral microfluidics platform paves the way for exploring intricate microflow dynamics, with promising applications in areas such as drug delivery, diagnostics, and lab-on-a-chip systems.

Hybrid Electrospinning Printing for Nanofiber Self-Supporting 3D Microfluidic Devices

Hybrid Electrospinning Printing for Nanofiber Self-Supporting 3D Microfluidic Devices

Synergistic effects of fibrin-enriched adipose decellularized extracellular matrix (AdECM) and microfluidic model on vascularization.

Vasculature is essential for maintaining the cellular function and balance of organs and tumors. As a key component of the tumor microenvironment (TME), it significantly influences tumor characteristics. Angiogenesis, heavily influenced by the extracellular matrix (ECM), which acts as a structural scaffold and growth factor reservoir, is regulated by various factors. Notably, adipose tissues and adipose-derived stromal cells contribute angiogenic and anti-apoptotic factors that promote angiogenesis. Sustained vasculature is essential for tissue engineering and ex vivo disease modeling. Lack of shear stress from fluid flow leads to vascular instability and regression. Microfluidic models replicate three-dimensional (3D) cultures from original tissues, encapsulate microenvironmental factors, and maintain consistent fluid flow. In our study, we established decellularized adipose ECM (AdECM) derived from bovine sources and engineered a 3D-printed microfluidic device. We observed significant increases in both the length and diameter of vascular networks after coculturing HUVECs and HDFs in a fibrin gel containing 0.5% AdECM. Additionally, gene expression related to ECM remodeling and angiogenesis was significantly enhanced in vasculature cultivated in fibrin gel containing 0.5% AdECM compared to that in fibrin gel alone. The enhanced vasculogenesis was further amplified and sustained by the 3D microfluidic device placed on a rocker during extended cultivation, primarily through the activation of the PI3K and JAK-mediated pathways. Our ex vivo model with vascularized colon tumoroids revealed that integrating AdECM within a microfluidic device correlates with increased tumoroid growth. Therefore, our study underscores the synergistic impact of AdECM and microfluidic device in promoting and sustaining vasculature. This synergy may have significant implications for tissue regeneration and ex vivo disease modeling, facilitating drug testing and efficacy evaluation.

Construction of fluorescent sensor array and three-dimensional microfluidic paper based analytical device for specific identification and visual determination of antibiotics in food.

For antibiotics misuse since the global outbreak of COVID 19, a novel strategy for discriminating and detecting antibiotics is proposed based on the graphene quantum dots with multi-doped heteroatoms including F, N and P (M-GQDs), which exhibit blue emission (419.0nm) under the excitation of 336.0nm. Specifically, the fluorescence of M-GQDs is quenched by tetracyclines (TCs) owing to inner filter effect (IFE) and enhanced by alkane-modified fluoroquinolones (AFQs), which is attributed to restricted conformational rotation based on π-π stacking, hydrogen-bonding and electrostatic interactions. Meanwhile, the electron-accepting property of oxazine ring in oxazine-modified fluoroquinolones (OFQs) increases emission peak at 498.0nm and decreases emission peak at 419.0nm as the color changes from blue to cyan. Moreover, a cascade system integrated with 3D microfluidic paper-based analytical device (3D-μPAD) is applied successfully for visually distinguishing three antibiotics, which shows great potential and versatility of M-GQDs for food safety monitoring.

Controlled tumor heterogeneity in a co-culture system by 3D bio-printed tumor-on-chip model

Cancer treatment resistance is a caused by presence of various types of cells and heterogeneity within the tumor. Tumor cell–cell and cell-microenvironment interactions play a significant role in the tumor progression and invasion, which have important implications for diagnosis, and resistance to chemotherapy. In this study, we develop 3D bioprinted in vitro models of the breast cancer tumor microenvironment made of co-cultured cells distributed in a hydrogel matrix with controlled architecture to model tumor heterogeneity. We hypothesize that the tumor could be represented by a cancer cell-laden co-culture hydrogel construct, whereas its microenvironment can be modeled in a microfluidic chip capable of producing a chemical gradient. Breast cancer cells (MCF7 and MDA-MB-231) and non-tumorigenic mammary epithelial cells (MCF10A) were embedded in the alginate-gelatine hydrogels and printed using a multi-cartridge extrusion bioprinter. Our approach allows for precise control over position and arrangements of cells in a co-culture system, enabling the design of various tumor architectures. We created samples with two different types of cells at specific initial locations, where the density of each cell type was carefully controlled. The cells were either randomly mixed or positioned in sequential layers to create cellular heterogeneity. To study cell migration toward chemoattractant, we developed a chemical microenvironment in a chamber with a gradual chemical gradient. As a proof of concept, we studied different migration patterns of MDA-MB-231 cells toward the epithelial growth factor gradient in presence of MCF10A cells in different ratios using this device. Our approach involves the integration of 3D bioprinting and microfluidic devices to create diverse tumor architectures that are representative of those found in various patients. This provides an excellent tool for studying the behavior of cancer cells with high spatial and temporal resolution.

Biocompatible High-Resolution 3D-Printed Microfluidic Devices: Integrated Cell Chemotaxis Demonstration.

We demonstrate a method to effectively 3D print microfluidic devices with high-resolution features using a biocompatible resin based on avobenzone as the UV absorber. Our method relies on spectrally shaping the 3D printer source spectrum so that it is fully overlapped by avobenzone's absorption spectrum. Complete overlap is essential to effectively limit the optical penetration depth, which is required to achieve high out-of-plane resolution. We demonstrate the high resolution in practice by 3D printing 15 μm square pillars in a microfluidic chamber, where the pillars are separated by 7.7 μm and are printed with 5 μm layers. Furthermore, we show reliable membrane valves and pumps using the biocompatible resin. Valves are tested to 1,000,000 actuations with no observable degradation in performance. Finally, we create a concentration gradient generation (CG) component and utilize it in two device designs for cell chemotaxis studies. The first design relies on an external dual syringe pump to generate source and sink flows to supply the CG channel, while the second is a complete integrated device incorporating on-chip pumps, valves, and reservoirs. Both device types are seeded with adherent cells that are subjected to a chemoattractant CG, and both show clear evidence of chemotactic cellular migration. Moreover, the integrated device demonstrates cellular migration comparable to the external syringe pump device. This demonstration illustrates the effectiveness of our integrated chemotactic assay approach and high-resolution biocompatible resin 3D printing fabrication process. In addition, our 3D printing process has been tuned for rapid fabrication, as printing times for the two device designs are, respectively, 8 and 15 min.

Hinge-like paper-based dual-channel enzyme-free ratiometric fluorescent microfluidic platform for simultaneous visual detection of carbaryl and glyphosate

On-site multi-pesticide residues detection is particularly urgent and challenging. Here, we fabricated an enzyme-free ratiometric fluorescent detection system in combination with a hinge-like dual-channel 3D microfluidic paper analytical device (3D μPAD) for simultaneous visual detection of carbaryl and glyphosate. Blue-emission 1-naphthol (Em. 470 nm) was hydrolyzed from carbaryl, while yellow-emission 2,3-diaminophenazine (Em. 570 nm) was produced with the aid of Cu2+ for glyphosate sensing. Inner-filter effect between 1-naphthol or 2,3-diaminophenazine and green-emission carbon dots (Em. 510 nm) realized two ratiometric fluorescent detection systems. Remarkable color variation of green–blue for carbaryl (50.00–1100 μΜ) and yellow–green for glyphosate (5.00–600 μΜ) were observed on a dual-channel 3D μPAD without crosstalk. Their detection limits were 1.11 and 0.63 μΜ, respectively. The strategy realized simultaneous visual detection of carbaryl and glyphosate in food/herbal with excellent accuracy (spiked recoveries, 91.00–107.2%), high precision (RSD ≤ 8.43%), and superior selectivity.

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.

A 3D Microfluidic Device with Vertical Channels toward In Vitro Reconstruction of Blood-Brain Barrier.

This paper reports a three-dimensional microfluidic device with an array of vertical channels to enable regulated, continuous, vertical flows to emulate the environment for in vitro culturing of brain and blood vessel organoids. This is expected to ultimately lead to in vitro reconstruction of the blood-brain barrier that is of high interest to studies on mental illness mechanisms and drug delivery to the brain. Twelve vertical microfluidic channels, each with 300 µm diameter and 5 mm height, were formed in the high-permeability agar gel surrounding the organoid to realize the vertical circulation flows and to allow lateral diffusion flows. The combined vertical flow rate of all channels ranges from 2.1 to 6.8 mL/min under different control parameters. A 30-day-old human blood vessel organoid was planted into the device for initial culturing and flow function tests. The result indicates that the organoid was properly activated with effective flow generation in the culturing site of the device.

Hemagglutination Assay via Optical Density Characterization in 3D Microtrap Chips.

Hemagglutination assay has been used for blood typing and detecting viruses, thus applicable for the diagnosis of infectious diseases, including COVID-19. Therefore, the development of microfluidic devices for fast detection of hemagglutination is on-demand for point-of-care diagnosis. Here, we present a way to detect hemagglutination in 3D microfluidic devices via optical absorbance (optical density, OD) characterization. 3D printing is a powerful way to build microfluidic structures for diagnostic devices. However, mixing liquid in microfluidic chips is difficult due to laminar flow, which hampers practical applications such as antigen-antibody mixing. To overcome the issue, we fabricated 3D microfluidic chips with embedded microchannel and microwell structures to induce hemagglutination between red blood cells (RBCs) and antibodies. We named it a 3D microtrap chip. We also established an automated measurement system which is an integral part of diagnostic devices. To do this, we developed a novel way to identify RBC agglutination and non-agglutination via the OD difference. By adapting a 3D-printed aperture to the microtrap chip, we obtained a pure absorbance signal from the microchannels by eliminating the background brightness of the microtrap chip. By investigating the underlying optical physics, we provide a 3D device platform for detecting hemagglutination.

A 3D sliding-strip microfluidic device for the simultaneous determination of multiple target analytes

A simple paper-based microfluidic device was fabricated to simultaneously detect multiple targets. Microfluidic paper-based analytical devices (μPAD) comprise a single-layer moving sliding PAD (SPAD) to control the flow channel switch together with a folding origami PAD (OPAD) to test the target analytes. The facile assembly without any splicing materials avoids cross-contamination and non-specific adsorption of joining materials that may be caused by multi-target detection. The concentration of Fe(III), Ni(II), Cr(VI), and nitrite in standard solutions and actual aqueous solutions was successfully determined using the designed μPAD. The μPAD was able to achieve LOD of 3.3 mg/L, 1.3 mg/L, 0.35 mg/L, 0.28 mg/L for Fe (III), Ni (II), Cr (VI), and nitrite, respectively. The designed SOPAD exhibits improved stability, with a deviation of less than 7% compared to conventional analytical methods (ICP-OES and UV). Our work demonstrates that this 3D PAD holds great promise and a wide scope in environmental monitoring, biochemical analysis, food testing and other testing industries.

A 3D paper microfluidic device for enzyme-linked assays: Application to DNA analysis.

A paper microfluidic device capable of conducting enzyme-linked assays is presented: a microfluidic enzyme-linked paper analytical device (μEL-PAD). The system exploits a wash-free sandwich coupling to form beads/analyte/enzyme complexes, which are subsequently added to the vertical flow device composed of wax-printed paper, waxed nitrocellulose membrane and absorbent/barrier layers. The nitrocellulose retains the bead complexes without disrupting the flow, enabling for an efficient washing step. The entrapped complexes then interact with the chromogenic substrate stored on the detection paper, generating a color change on it, quantified with an open-source smartphone software. This is a universal paper-based technology suitable for high-sensitivity quantification of many analytes, such as proteins or nucleic acids, with different enzyme-linked formats. Here, the potential of the μEL-PAD is demonstrated to detect DNA from Staphylococcus epidermidis. After generation of isothermally amplified genomic DNA from bacteria, Biotin/FITC-labeled products were analyzed with the μEL-PAD, exploiting streptavidin-coated beads and antiFITC-horseradish peroxidase. The μEL-PAD achieved a limit of detection (LOD) and quantification<10 genome copies/μL, these being at least 70- and 1000-fold lower, respectively, than a traditional lateral flow assay (LFA) exploiting immobilized streptavidin and antiFITC-gold nanoparticles. It is envisaged that the device will be a good option for low-cost, simple, quantitative, and sensitive paper-based point-of-care testing.