3D Microfluidic Device Research Articles

3D paper-based microfluidic device: a novel dual-detection platform of bisphenol A.

A novel platform of microfluidic paper-based analytical devices (μPADs) for dual detection of bisphenol A (BPA), a model analyte, was fabricated using an electronic digital plotter to create the stacked layer of μPADs and generate the lateral-flow channel without using an external pump. Two detection techniques, including electrochemical detection and laser desorption ionization mass spectrometric detection (LDI-MS), were used complementarily to improve the precision in the detection of BPA. The fluid sample was delivered to both detection zones by the capillary action, automatically generated from the fabricated microfluidic device. For an electrochemical sensor, two ballpoint pens filled with silver nanoparticles (AgNPs) and multiwall carbon nanotube (MWCNT) ink were used to print onto the paper with a contact printing method using a digital plotter. To further improve the sensitivity, zinc oxide (ZnO) was used to modify both electrochemical and LDI-MS detection zones. For BPA detection, high electrocatalytic properties and strong UV absorption of ZnO promote the electron transfer in the electrochemical sensor and ionization efficiency in LDI-MS with low interferences compared with a conventional organic matrix. Under optimal conditions, this platform showed a dual detection capability for BPA with a detection limit of 0.35 μM for electrochemical detection and with an ultralow detection limit of 0.01 pM for LDI-MS. This novel platform might be very useful for trace analyses requiring high precision detection of various analytes.

Human Induced Pluripotent Stem-Cardiac-Endothelial-Tumor-on-a-Chip to Assess Anticancer Efficacy and Cardiotoxicity

Cancer remains a leading health threat in the United States, and cardiovascular drug toxicity is a primary cause to eliminate a drug from FDA approval. As a result, the demand to develop new anticancer drugs without cardiovascular toxicity is high. Human induced pluripotent stem (iPS) cell-derived tissue chips provide potentially a cost-effective preclinical drug testing platform, including potential avenues for personalized medicine. We have developed a three-dimensional microfluidic device that simultaneously cultures tumor cell spheroids with iPS-derived cardiomyocytes (iPS-CMs) and iPS-derived endothelial cells (iPS-EC). The iPS-derived cells include a GCaMP6 fluorescence reporter to allow real-time imaging to monitor intracellular calcium transients. The multiple-chambered tissue chip features electrodes for pacing of the cardiac tissue to assess cardiomyocyte function such as the maximum capture rate and conduction velocity. We measured the inhibition concentration (IC50) of the anticancer drugs, Doxorubicin (0.1 μM) and Oxaliplatin (4.2 μM), on the tissue chip loaded with colon cancer cells (SW620). We simultaneously evaluated the cardiotoxicity of these anticancer drugs by assessing the drug effect on the spontaneous beat frequency and conduction velocity of iPS-derived cardiac tissue. Consistent with in vivo observations, Doxorubicin reduced the spontaneous beating rate and maximum capture rate at or near the IC50 (0.04 and 0.22 μM, respectively), whereas the toxicity of Oxaliplatin was only observed at concentrations beyond the IC50 (33 and 9.9 μM, respectively). Our platform demonstrates the feasibility to simultaneously assess cardiac toxicity and antitumor effects of drugs and could be used to enhance personalized drug testing safety and efficacy. Impact statement Drug development using murine models for preclinical testing is no longer adequate nor acceptable both financially for the pharmaceutical industry as well as for generalized or personalized assessment of safety and efficacy. Innovative solutions using human cells and tissues provide exciting new opportunities. In this study, we report on the creation of a 3D microfluidic device that simultaneously cultures human tumor cell spheroids with cardiomyocytes and endothelial cells derived from the same induced pluripotent stem cell line. The platform provides the opportunity to assess efficacy of anticancer agents while simultaneously screening for potential cardiovascular toxicity in a format conducive for personalized medicine.

Abstract B065: TANK-Binding Kinase 1 (TBK1) as a novel cancer immunotherapy target

Abstract Despite the unprecedented success of immune checkpoint blockade (ICB) in melanoma and other cancers, tackling innate (primary) resistance remains a major challenge and robust biomarkers to guide treatment are lacking. Clinical trials are already underway evaluating novel immune modulatory agents in combination with anti-PD-1/PD-L1 therapies in an effort to overcome innate resistance. Despite increasing reports of ‘rational’ combination strategies, these therapies remain “one size fits all”, due the lack of robust biomarkers to guide clinical decision-making. There is an unmet need for novel approaches, tools, techniques, and methods for pre-clinical and clinical use to better understand mechanisms of response and resistance to immune checkpoint inhibitors and next-generation anti-tumor immune modulatory drugs. We have adapted a 3D microfluidic device to the short-term culture of murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS)for functional ex vivo profiling of PD-1 blockade in a model tumor immune microenvironment (TIME) to facilitate identification of mediators of response and resistance to ICB and to guide development of next-generation immunotherapy combinations. The MDOTS/PDOTS platform provides a window into the complex and dynamic events of the TIME during response (and resistance) to ICB and is an ideal model system for pre-clinical evaluation of novel cancer immunotherapy targets. Focused evaluation of novel treatment combinations using MDOTS identified TANK-binding kinase 1 (TBK1) as a novel cancer immunotherapy target, mirroring in vivoefficacy of this treatment approach. TBK1 is a Ser/Thr kinase involved in innate immune signaling and is an emerging target for anti-cancer therapy. Importantly, independent orthogonal data from two different laboratories has also identified TBK1 as a cancer immunotherapy target.We have confirmed that Tbk1 deletion and/or TBK1 pharmacologic inhibition enhances response to in vivo anti-PD-1 therapy, and have characterized the impact of deletion of Tbk1 (CRISPR) or pharmacologic inhibition of TBK1 (Cmpd1) on the tumor-immune microenvironment.Lastly, we confirmed that resistance to PD-1 blockade can be overcome with pharmacologic TBK1 inhibition using murine- and patient-derived organotypic tumor spheroids in 3D microfluidic culture. These findings confirm TBK1 as a target to overcome resistance to PD-1 blockade, further supporting the pre-clinical and clinical development of this novel combination strategy. REFERENCES 1. Jenkins RW, Barbie DA, Flaherty KT. Mechanisms of resistance to immune checkpoint inhibitors. Br J Cancer. 2018;118(1):9-16. doi: 10.1038/bjc.2017.434. PubMed PMID: 29319049; PMCID: PMC5765236. 2. Manguso RT, Pope HW, Zimmer MD, Brown FD, Yates KB, Miller BC, Collins NB, Bi K, LaFleur MW, Juneja VR, Weiss SA, Lo J, Fisher DE, Miao D, Van Allen E, Root DE, Sharpe AH, Doench JG, Haining WN. In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target. Nature. 2017. doi: 10.1038/nature23270. PubMed PMID: 28723893. 3. Pan D, Kobayashi A, Jiang P, Ferrari de Andrade L, Tay RE, Luoma AM, Tsoucas D, Qiu X, Lim K, Rao P, Long HW, Yuan GC, Doench J, Brown M, Liu XS, Wucherpfennig KW. A major chromatin regulator determines resistance of tumor cells to T cell-mediated killing. Science. 2018;359(6377):770-5. doi: 10.1126/science.aao1710. PubMed PMID: 29301958. Citation Format: Ajaykumar Vishwakarma, Yi Sun, Amina Fu, Emily Robitschek, Arvin Iracheta-Vellve, Susanna Stinson, Aliasger Salem, Robert Manguso, Russell Jenkins. TANK-Binding Kinase 1 (TBK1) as a novel cancer immunotherapy target [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B065. doi:10.1158/1535-7163.TARG-19-B065

Mixing Performance of a Cost-effective Split-and-Recombine 3D Micromixer Fabricated by Xurographic Method.

This paper presents experimental and numerical investigations of a novel passive micromixer based on the lamination of fluid layers. Lamination-based mixers benefit from increasing the contact surface between two fluid phases by enhancing molecular diffusion to achieve a faster mixing. Novel three-dimensional split and recombine (SAR) structures are proposed to generate fluid laminations. Numerical simulations were conducted to model the mixer performance. Furthermore, experiments were conducted using dyes to observe fluid laminations and evaluate the proposed mixer’s characteristics. Mixing quality was experimentally obtained by means of image-based mixing index (MI) measurement. The multi-layer device was fabricated utilizing the Xurography method, which is a simple and low-cost method to fabricate 3D microfluidic devices. Mixing indexes of 96% and 90% were obtained at Reynolds numbers of 0.1 and 1, respectively. Moreover, the device had an MI value of 67% at a Reynolds number of 10 (flow rate of 116 µL/min for each inlet). The proposed micromixer, with its novel design and fabrication method, is expected to benefit a wide range of lab-on-a-chip applications, due to its high efficiency, low cost, high throughput and ease of fabrication.

Multiple self-cleaning paper-based electrochemical ratiometric biosensor based on the inner reference probe and exonuclease III-assisted signal amplification strategy

Rapid and accurate detection of nucleic acids plays a major role in biological research and clinical diagnostics. Here, a 3D multiple self-cleaning electrochemical ratiometric microfluidic paper-based analytical device (SER-μPAD) has been constructed for manganese super oxide dismutase (MnSOD) gene detection on the basis of the inner reference probe and exonuclease Ш (Exo Ш)-assisted analytes recycling amplification method. To simplify manual operations, a multipath self-cleaning tab that could manipulate fluid transport was introduced into the paper-based device, realizing time-programmable self-cleaning of the electrode. For achieving sensitive detection of MnSOD gene, the methylene blue (MB)-modified capture probe (CP) as the inner reference element was first self-assembled on triangular Au nanosheets modified paper working electrode to provide a built-in correction and improve the detection accuracy. When MnSOD gene existed, it hybridized with the hairpin-structured signal probe, triggering the cyclic amplification with the assistance of Exo Ш selective digestion to engender numerous residual DNA labeled with ferrocene (Fc) that could be captured on electrode surface by CPs. Hence, the Fc tags were close to the electrode surface, resulting in the oxidation peak current of Fc (IFc) increase, while that of MB (IMB) was constant on account of the unchanged distance between the MB tags and the electrode. The value of IFc/IMB was linear with MnSOD gene concentration from 10 nM to 1200 nM, and the detection limit was 3.91 nM. This strategy provides an accurate, robust, and sensitive method for nucleic acids detection and shows great potential in the construction of portable devices.

Multiplexed chemiluminescence determination of three acute myocardial infarction biomarkers based on microfluidic paper-based immunodevice dual amplified by multifunctionalized gold nanoparticles

Acute myocardial infarction (AMI) causes significant mortality and morbidity. The determination of multiple AMI biomarkers is very important for the timely diagnosis of AMI. In this work, simultaneous determination of three AMI biomarkers were achieved by virtue of a three-dimensional (3D) microfluidic paper-analytical device (μPAD) with temporally resolved chemiluminescence (CL) emissions for the first time. A dual-signal amplification strategy was introduced including by employing primary antibody functionalized gold nanoparticles (Ab1-GNPs) immobilized on the detection zone as amplified capture probes, and Co(II) catalyst, secondary antibody, luminol multifunctionalized gold nanoparticles (Co(II)-Ab2-luminol-GNPs) with excellent CL activity as amplified signal probes. CL immunoreactions were performed at three detection zone of the fabricated 3D μPAD by assembling Ab1-GNPs, antigen, and Co(II)-Ab2-luminol-GNPs to form sandwich-type immunocomplexes. Auto separated CL signals with temporal resolution were obtained by time delayed transport of H2O2 to different detection zones for multiplexed analysis. The CL signal obtained by using Co(II)-Ab2-luminol-GNPs as signal probe (10576 a.u.) were about 20-fold higher than that by using conventional horseradish peroxidase labeled antibody modified luminol-GNPs as signal probe (531 a.u.). Finally, three AMI biomarkers including heart-type fatty acid-binding protein (H-FABP), cardiac troponin I (cTnI) and copeptin were quantitatively analyzed in one CL detection run by reading the CL intensity of the obtained three CL emission peaks. The detection range were ultra-wide ranged from 0.1 pg/mL to 1 μg/mL, 0.5 pg/mL to 1 μg/mL and 1 pg/mL to 1 mg/mL with the detection limits down to 0.06 pg/mL, 0.3 pg/mL and 0.4 pg/mL for H-FABP, cTnI and copeptin detection, respectively. The developed μPAD based immunoassay performing multiplexed analysis ability, high sensitivity, ultra-wide dynamic range, favorable selectivity, accessible accuracy and reproducibility, have great application potential for the early diagnosis of AMI.

Three-Dimensional Modeling of Avascular Tumor Growth in Both Static and Dynamic Culture Platforms.

Microfluidic cell culture platforms are ideal candidates for modeling the native tumor microenvironment because they can precisely reconstruct in vivo cellular behavior. Moreover, mathematical modeling of tumor growth can pave the way toward description and prediction of growth pattern as well as improving cancer treatment. In this study, a modified mathematical model based on concentration distribution is applied to tumor growth in both conventional static culture and dynamic microfluidic cell culture systems. Apoptosis and necrosis mechanisms are considered as the main inhibitory factors in the model, while tumor growth rate and nutrient consumption rate are modified in both quiescent and proliferative zones. We show that such modification can better predict the experimental results of tumor growth reported in the literature. Using numerical simulations, the effects of the concentrations of the nutrients as well as the initial tumor radius on the tumor growth are investigated and discussed. Furthermore, tumor growth is simulated by taking into account the dynamic perfusion into the proposed model. Subsequently, tumor growth kinetics in a three-dimensional (3D) microfluidic device containing a U-shaped barrier is numerically studied. For this case, the effect of the flow rate of culture medium on tumor growth is investigated as well. Finally, to evaluate the impact of the trap geometry on the tumor growth, a comparison is made between the tumor growth kinetics in two frequently used traps in microfluidic cell culture systems, i.e., the U-shaped barrier and microwell structures. The proposed model can provide insight into better predicting the growth and development of avascular tumor in both static and dynamic cell culture platforms.

Three-dimensional microfluidic paper-based device for multiplexed colorimetric detection of six metal ions combined with use of a smartphone.

A simple double-layered three-dimensional (3D) microfluidic paper-based analytical device (μPAD) was designed for the simultaneous determination of six metal ions-Fe(III), Ni(II), Cr(VI), Cu(II), Al(III), and Zn(II)-for the first time. The 3D μPAD was composed of two paper layers: a top pretreatment layer and a bottom colorimetric detection layer. The sample solution added to the central sample reservoir of the 3D μPAD could be automatically divided into eight flow pathways and be automatically pretreated while flowing through the pretreatment zones located in the microfluidic channels, and automatically carried out the chromogenic reactions after reaching the detection zones. Random diffusion of the chromogenic reagents was effectively prevented by transport of the pretreated sample solution to the detection zones through 3D microfluidic channels with an L-type circuitous flow route design, resulting in highly increased color uniformity and reproducibility. Combined with use of a flat LED lamp as an upward lighting source and a smartphone as a convenient detector, improved color perception, highly enhanced sensitivity, and an extended detection range were obtained. Finally, the double-layered 3D μPAD was applied to the multiplexed determination of the six metal ions in mixtures and environmental samples with satisfactory results. Detection limits as low as 0.2, 0.3, 0.1, 0.03, 0.08, and 0.04 mg/L for Fe(III), Ni(II), Cr(VI), Cu(II), Al(III), and Zn(II) detection, respectively, were achieved, which are about one order of magnitude lower than obtained with previously reported μPADs for the detection of metal ions. The present 3D μPAD is simple, fast, selective, sensitive, and user-friendly, and holds great application potential for multiplexed on-site analysis.

3D Micromachined Polyimide Mixing Devices for in Situ X-ray Imaging of Solution-Based Block Copolymer Phase Transitions.

Advances in modern interface- and material sciences often rely on the understanding of a system's structure-function relationship. Designing reproducible experiments that yield in situ time-resolved structural information at fast time scales is therefore of great interest, e.g., for better understanding the early stages of self-assembly or other phase transitions. However, it can be challenging to accurately control experimental conditions, especially when samples are only available in small amounts, prone to agglomeration, or if X-ray compatibility is required. We address these challenges by presenting a microfluidic chip for triggering dynamics via rapid diffusive mixing for in situ time-resolved X-ray investigations. This polyimide/Kapton-only-based device can be used to study the structural dynamics and phase transitions of a wide range of colloidal and soft matter samples down to millisecond time scales. The novel multiangle laser ablation three-dimensional (3D) microstructuring approach combines, for the first time, the highly desirable characteristics of Kapton (high X-ray stability with low background, organic solvent compatibility) with a 3D flow-focusing geometry that minimizes mixing dispersion and wall agglomeration. As a model system, to demonstrate the performance of these 3D Kapton microfluidic devices, we selected the non-solvent-induced self-assembly of biocompatible and amphiphilic diblock copolymers. We then followed their structural evolution in situ at millisecond time scales using on-the-chip time-resolved small-angle X-ray scattering under continuous-flow conditions. Combined with complementary results from 3D finite-element method computational fluid dynamics simulations, we find that the nonsolvent mixing is mostly complete within a few tens of milliseconds, which triggers initial spherical micelle formation, while structural transitions into micelle lattices and their deswelling only occur on the hundreds of milliseconds to second time scale. These results could have an important implication for the design and formulation of amphiphilic polymer nanoparticles for industrial applications and their use as drug-delivery systems in medicine.

T-shirt ink for one-step screen-printing of hydrophobic barriers for 2D- and 3D-microfluidic paper-based analytical devices

This work presents the use of polyvinyl chloride (PVC) fabric ink, commonly employed for screening t-shirts, as new and versatile material for printing hydrophobic barrier on paper substrate for microfluidic paper-based analytical devices (μPADs). Low-cost, screen-printing apparatus (e.g., screen mesh, squeegee, and printing table) and materials (e.g. PVC ink and solvent) were employed to print the PVC ink solution onto Whatman filter paper No. 4. This provides a one-step strategy to print flow barriers without the need of further processing except evaporation for 3–5 min in a fume hood to remove the solvent. The production of the single layer μPADs is reasonably high with up to 77 devices per screening with 100% success rate. This method produces very narrow fluidic channel 486 ± 14 μm in width and hydrophobic barrier of 642 ± 25 μm thickness. Reproducibility of the production of fluidic channels and zones is satisfactory with RSDs of 2.9% (for 486-μm channel, n = 10), 3.7% (for 2-mm channel, n = 50) and 1.5% (for 6-mm diameter circular zone, n = 80). A design of a 2D-μPAD produced by this method was employed for the colorimetric dual-measurements of thiocyanate and nitrite in saliva. A 3D-μPADs with multiple layers of ink-screened paper was designed and constructed to demonstrate the method's versatility. These 3D-μPADs were designed for gas-liquid separation with in-situ colorimetric detection of ethanol vapor on the μPADs. The 3D-μPADs were applied for direct quantification of ethanol in beverages and highly colored pharmaceutical products. The printed barrier was resistant up to 8% (v/v) ethanol without liquid creeping out of the barrier.

High-resolution temporally resolved chemiluminescence based on double-layered 3D microfluidic paper-based device for multiplexed analysis

In this work, a double-layered three-dimensional (3D) microfluidic paper-based analytical device (μPAD) with high resolution temporally resolved chemiluminescence (CL) emissions were designed for multiplexed CL analysis. The temporally resolved CL emissions were obtained by virtue of the 3D branched microfluidic channel design, which create time delays for luminol transported from one detection zone to another. The peak intensity and peak shape of the temporally resolved CL emissions were quite stable and base-line separated with resolution as high as 21.2–24.4. Then, the fabricated μPAD was applied to multiplexed determination of glucose, lactate, cholesterol, and choline as model analytes. The sample was added to four detection zone modified with CL catalyst cobalt ion and different oxidase by virtue of chitosan. When luminol flowed to μPAD, four temporally resolved CL peaks were successively generated from the cobalt ion catalyzed CL reactions between luminol and generated H2O2 from the specific enzymatic reactions between the oxidase and the analytes. The generated four CL emission peaks in the CL kinetic curve increased in proportion to the concentrations of glucose, lactate, cholesterol, and choline, respectively. Finally, four linear calibration curves were obtained for the detection of glucose (0.01–1.0 mmol/L), lactate (0.02–5.0 mmol/L), cholesterol (0.01–0.4 mmol/L), and choline (0.001–1.0 mmol/L). The detection limits were as low as 8 μmol/L, 15 μmol/L, 6 μmol/L, and 0.07 μmol/L for glucose, lactate, choline, and cholesterol detection, respectively. The present work provides a new strategy for the fabrication of simple and sensitive 3D μPAD with high resolution temporally resolved CL emissions for multiplexed CL analysis, which holds great application potential for point-of-care diagnosis.

Development of a micro-distillation microfluidic paper-based analytical device as a screening tool for total ammonia monitoring in freshwaters

An easy-to-use, portable 3D microfluidic paper-based analytical device (μPAD) for the determination of total ammonia (i.e., ammonia + ammonium) in freshwaters is described. It consists of two layers of paper patterned with hydrophilic circular zones, one impregnated with sodium hydroxide (sample zone) and another (detection zone) with an acid-base indicator (nitrazine yellow (NY) or bromothymol blue (BTB)), separated by a μ-distillation chamber. Ammonium ions present in the water sample are converted into ammonia gas by reaction with sodium hydroxide in the sample zone. Ammonia then diffuses through a headspace and reacts with an acid-base indicator in the detection zone, the reflectance of which can be related to the total ammonia concentration. The analytical signal at 7.8 mg L−1 offered by the μ-distillation chamber-based μPAD is more than double of that obtained using a gas-permeable membrane.The proposed μPAD is characterised by a limit of detection of 0.32 or 0.47 mg N L-1 and working concentration ranges of 0.5–3.0 mg N L-1 or 2.0–10 mg N L-1 when using NY or BTB indicators, respectively. This is the first μPAD whose working range covers almost the entire trigger value range (0.32–2.3 mg N L-1) for ammonia nitrogen in freshwater systems which makes it suitable as a field screening tool for ammonia in freshwaters. An inter- and intra-device repeatability of 7.6% and 9.0%, respectively, has been achieved for the NY-based μPAD (3 mg N L-1) and 13 and 2.5% (8 mg N L-1) for the BTB-based μPAD. The NY-based μPAD is stable under vacuum for at least 12 days at room temperature, and 56 days if stored in a freezer (≤−20 °C).

Enabling cell recovery from 3D cell culture microfluidic devices for tumour microenvironment biomarker profiling

The tumour microenvironment (TME) has recently drawn much attention due to its profound impact on tumour development, drug resistance and patient outcome. There is an increasing interest in new therapies that target the TME. Nonetheless, most established in vitro models fail to include essential cues of the TME. Microfluidics can be used to reproduce the TME in vitro and hence provide valuable insight on tumour evolution and drug sensitivity. However, microfluidics remains far from well-established mainstream molecular and cell biology methods. Therefore, we have developed a quick and straightforward collagenase-based enzymatic method to recover cells embedded in a 3D hydrogel in a microfluidic device with no impact on cell viability. We demonstrate the validity of this method on two different cell lines in a TME microfluidic model. Cells were successfully retrieved with high viability, and we characterised the different cell death mechanisms via AMNIS image cytometry in our model.

Rapid and Inexpensive Fabrication of Multi-Depth Microfluidic Device using High-Resolution LCD Stereolithographic 3D Printing

With the dramatic increment of complexity, more microfluidic devices require 3D structures, such as multi-depth and -layer channels. The traditional multi-step photolithography is time-consuming and labor-intensive and also requires precise alignment during the fabrication of microfluidic devices. Here, we present an inexpensive, single-step, and rapid fabrication method for multi-depth microfluidic devices using a high-resolution liquid crystal display (LCD) stereolithographic (SLA) three-dimensional (3D) printing system. With the pixel size down to 47.25 μm, the feature resolutions in the horizontal and vertical directions are 150 μm and 50 μm, respectively. The multi-depth molds were successfully printed at the same time and the multi-depth features were transferred properly to the polydimethylsiloxane (PDMS) having multi-depth channels via soft lithography. A flow-focusing droplet generator with a multi-depth channel was fabricated using the presented 3D printing method. Experimental results show that the multi-depth channel could manipulate the morphology and size of droplets, which is desired for many engineering applications. Taken together, LCD SLA 3D printing is an excellent alternative method to the multi-step photolithography for the fabrication of multi-depth microfluidic devices. Taking the advantages of its controllability, cost-effectiveness, and acceptable resolution, LCD SLA 3D printing can have a great potential to fabricate 3D microfluidic devices.

3D Printed 3D-Microfluidics: Recent Developments and Design Challenges

Multi-layer structures with functionalities, varying height channels, complex microfluidic structures like valves, mixers and high aspect ratio channels (hereby referred as 3D-Microfluidics) are very difficult to fabricate using conventional microfluidics fabrication methods. On the other hand, hig h geometric complexity, less manufacturing time, low cost, multiple material options, improved mechanical properties and dimensional accuracy have made 3D printing a very popular technology for macroscale manufacturing. The resolution and accuracy of state-of-the-art 3D printers are well established on micro-scale with a potential for nanoscale. The 3D printing process is additive in nature and can produce intricate 3-dimensional (3D) shapes and structures in micrometer range, with a few specialized printers claiming to have accuracy down to 500 nm. Due to its ability to print intricate 3D shapes in very small dimensions with high accuracy, 3D printing is considered as a strong alternative technique to fabricate complex and difficult to manufacture 3D Microelectromechanical Systems (MEMS) and 3D-Microfluidic devices. This paper discusses the brief history as well as current challenges and limitations of conventional 3D-Microfluidics fabrication techniques and elaborates the research progress reported in the last years related to 3D printing of 3D-Microfluidics. The discussion includes a critical analysis of the state-of-the-art and presents challenges that are yet to be addressed to successfully broaden the use of this novel technique for the aforementioned devices. A case study is also presented to analyze the potential use of desktop 3D printers for fabrication of 3D-Microfluidics for lab-on-a-chip and other applications. Finally, future trends of 3D printed 3D-Microfluidic devices are discussed.

Melamine promotes calcium crystal formation in three-dimensional microfluidic device

Melamine, which induces proximal tubular (PT) cell damage has a greater nephrotoxic effect when combined with cyanuric and uric acids; however, it is unknown whether such effect can stimulate calcium phosphate (CaP)/calcium oxalate (CaOx) stone formation. Here, we show that melamine acts as an inducer of CaP, CaOx and CaP + CaOx (mixed) crystal formations in a time and concentration-dependent manner by stabilizing those crystals and further co-aggregating with melamine. To explore the physiological relevance of such melamine-augmented calcium crystal formation, we used 2-dimensional (2D) and 3D microfluidic (MF) device, embedded with PT cells, which also resembled the effect of melamine-stimulated CaP, CaOx and mixed crystal formation. Significantly, addition of preformed CaP and/or CaOx crystal in the presence of melamine, further potentiated those crystal formations in 3D MFs, which helped the growth and aggregation of mixed crystals. Our data show that the mechanism of such predisposition of stone formation could be largely due to co-crystallization between melamine and CaP/CaOx and pronounced effect on induction of stone-forming pathway activation in 3D MF. Taken together, melamine-induced CaP and/or CaOx crystal formation ex-vivo will help us in understanding the larger role of melamine as an environmental toxicant in producing the pathology in similar cellular microenvironments.

3D Droplet-Based Microfluidic Device Easily Assembled from Commercially Available Modules Online Coupled with ICPMS for Determination of Silver in Single Cell.

More recently, single-cell analysis based on ICPMS has made considerable headway while a challenge remains to differentiate single cell from doublets during the analysis. One burgeoning solution is to encapsulate single cell into droplets on the platform of the microfluidic chip. However, the manufacture of the droplet-based microfluidic chip requires sophisticated fabrication and limits its potential application. In this paper, we presented an off-the-shelf three-dimensional (3D) microfluidic device by assembling commercially available parts without any proficient manufacturing process. Uniform monodisperse microdroplet was generated from the 3D microfluidic device with a size variation of 1.5%, and the innner diameter of the 3D microfluidic device was the same as the nebulizer (150 μm). The proposed 3D microfluidic device-time-resolved ICPMS system was applied to detect silver in single AgNPs (51 nm), and the result is in good agreement with conventional acid digestion method, demonstrating the accuracy of the method. Silver uptake behaviors in HepG2 cells were then studied by incubating with Ag+ or AgNPs under biocompatible conditions. The results revealed that the cell-to-cell variability in terms of the diversity of cells incubated with AgNPs was wider than those cells incubated with Ag+ from the aspect of the content distribution of silver at the single-cell level.

High-Precision Stereolithography of Biomicrofluidic Devices.

Stereolithography (SL) is emerging as an attractive alternative to soft lithography for fabricating microfluidic devices due to its low cost and high design efficiency. Low molecular weight poly(ethylene glycol)diacrylate (MW = 258) (PEG-DA-258) has been used for SL 3D-printing of biocompatible microdevices at submillimeter resolution. However, 3D-printing resins that simultaneously feature high transparency, high biocompatibility, and high resolution are still lacking. It is found that photosensitizer isopropyl thioxanthone can, in a concentration-dependent manner, increase the absorbance of the resin (containing PEG-DA-258 and photoinitator Irgacure-819) by over an order of magnitude. This increase in absorbance allows for SL printing of microdevices at sub pixel resolution with commercially available desktop printers and without compromising transparency or biocompatibility. The assembly-free, rapid (<15 h) 3D-printing of a variety of complex 3D microfluidic devices such as a 3D-fluid router, a passive chaotic micro-mixer, an active micro-mixer with pneumatic microvalves, and high-aspect ratio (37:1) microchannels of single pixel width is demonstrated. These manufacturing capabilities are unavailable in conventional microfluidic rapid prototyping techniques. The low absorption of small hydrophobic molecules and microfluidic labeling of cultured mammalian cells in 3D-printed PEG-DA-258 microdevices is demonstrated, indicating the potential of PEG-DA-based fabrication of cell-based assays, drug discovery, and organ-on-chip platforms.

A Review on the Recent Advancement in “Tumour Spheroids-on-a-Chip”

Introduction: Three-dimensional (3D) cell cultures are identified as more accurate and representative models of human cancers than conventional two-dimensional monolayer cell cultures. However, currently established 3D culturing techniques are technically challenging, time- and resource-consuming, and performed using traditional laboratory tissue culturing equipment. In recent years, microfluidics has been introduced into biomedical research, allowing cells and tissues to be cultured in microfabricated devices. The current challenge is to adapt existing 3D cell culturing techniques to microfluidic devices, allowing for the fabrication of low-cost, rapid evaluation devices to facilitate biomedical research and clinical application. The aim of this review was to evaluate microfluidics and 3D cell culture research with particular relevance to oncological research. Methods: Journal articles were acquired from different scientific databases and were identified using specific keywords. Three-Dimensional Cell Culturing Microfluidic Concepts: Various 3D cell culturing microfluidic devices have been designed, based on existing 3D cell culturing methods. This includes non-cell adherent-based devices, matrix-embedding, hanging drop, and droplet-based culturing methods. These platforms facilitate the culturing, treatment, and analysis of 3D spheroids, while simultaneously scaling down traditional experimental requirements. Limitations and Future Perspectives: Beyond superficial analysis, a major drawback in the current scope of 3D cell culturing microfluidic devices is the inability to extract spheroids for examining histology. Polydimethylsiloxane is the preferred material to fabricate devices but may need revision for commercializing microfluidic platforms in the future. Integrating 3D bioprinting and organoid cultures could potentially improve the quality of 3D models in microfluidic devices. Conclusion: 3D spheroids are an effective representation of in vivo cancers and microfluidics has streamlined the culture, treatment, and analysis of 3D models. Considerable improvements have been made in combining the two entities, but further work is required to manufacture 3D cell culturing microfluidic devices on a commercial scale.

On-the-fly exchangeable microfluidic nozzles for facile production of various monodisperse micromaterials.

Microfluidic manufacturing platforms have advanced the production of monodisperse, shape-controlled, and chemically defined micromaterials. However, conventional microfabrication platforms are typically designed and fabricated as single-purpose and single-use tools, which limits their efficiency, versatility, and overall potential. We here present an on-the-fly exchangeable nozzle concept that operates in a transparent, 3D, and reusable microfluidic device produced without cleanroom technology. The facile exchange and repositioning of the nozzles readily enables the production of monodisperse water-in-oil and oil-in-water emulsions, solid and core-shell microspheres, microfibers, and even Janus type micromaterials with controlled diameters ranging from 10 to 1000 μm using a single microfluidic device.