Mesofluidic Device Research Articles

Biomimetic models of fish gill rakers as lateral displacement arrays for particle separation.

Ram suspension-feeding fish, such as herring, use gill rakers to separate small food particles from large water volumes while swimming forward with an open mouth. The fish gill raker function was tested using 3D-printed conical models and computational fluid dynamics simulations over a range of slot aspect ratios. Our hypothesis predicting the exit of particles based on mass flow rates, dividing streamlines (i.e. stagnation streamlines) at the slots between gill rakers, and particle size was supported by the results of experiments with physical models in a recirculating flume. Particle movement in suspension-feeding fish gill raker models was consistent with the physical principles of lateral displacement arrays ('bump arrays') for microfluidic and mesofluidic separation of particles by size. Although the particles were smaller than the slots between the rakers, the particles skipped over the vortical region that was generated downstream from each raker. The particles 'bumped' on anterior raker surfaces during posterior transport. Experiments in a recirculating flume demonstrate that the shortest distance between the dividing streamline and the raker surface preceding the slot predicts the maximum radius of a particle that will exit the model by passing through the slot. This theoretical maximum radius is analogous to the critical separation radius identified with reference to the stagnation streamlines in microfluidic and mesofluidic devices that use deterministic lateral displacement and sieve-based lateral displacement. These conclusions provide new perspectives and metrics for analyzing cross-flow and cross-step filtration in fish with applications to filtration engineering.

Isolation of enriched small RNA from cell-lysate using on-chip isotachophoresis.

In spite of the growing interest in the roles and applications of small RNAs (sRNAs), sRNA isolation methods are inconsistent, tedious, and dependent on the starting number of cells. In this work, we employ ITP to isolate sRNAs from the cell-lysate of K562 (chronic myelogenous leukemia) cells in a polydimethylsiloxane (PDMS) mesofluidic device. Our method specifically purifies sRNA of <60 nucleotides from lysate of a wide range of cell number spanning from 100 to 1000000 cells. We measured the amount of sRNA using the Agilent Bioanalyzer and further verified the extraction efficiency by reverse transcription quantitative PCR. Our method was shown to be more efficient in sRNA extraction than commercial sRNA isolation kits, especially when using smaller numbers of starting cells. Our assay presents a simple and rapid sRNA extraction method with 20min assay time and no intermediate transfer steps.

Fully Automated Electric-Field-Driven Liquid Phase Microextraction System with Renewable Organic Membrane As a Front End to High Performance Liquid Chromatography.

This article reports for the first time a programmable-flow-based mesofluidic platform that accommodates electric-field-driven liquid phase microextraction (μ-EME) in a fully automated mode. The miniaturized system is composed of a computer-controlled microsyringe pump and a multiposition rotary valve for handling aqueous and organic solutions at a low microliter volume and acts as a front-end to online liquid chromatographic separation. The organic membrane is automatically renewed and disposed of in every analytical cycle, thus minimizing analyte carry-over effects while avoiding analyst intervention. The proof-of-concept applicability of the automated mesofluidic device is demonstrated by the liquid chromatographic determination of nonsteriodal anti-inflammatory drugs in μ-EME processed complex samples (such as urine and influent wastewater) using online heart-cut approaches. Using 5 μL of 1-octanol, 7.5 μL of untreated sample and 7.5 μL of acceptor solution (25 mM NaOH), and 250 V for only 10 min in a stopped-flow mode, the extraction recoveries for the μ-EME of ibuprofen, ketoprofen, naproxen, and diclofenac exceed 40% in real samples. The flow-through system features moderately selective extraction regardless of the sample matrix constituents with repeatability values better than 13%.

High-throughput mechanotransduction in Drosophila embryos with mesofluidics.

Developing embryos create complexity by expressing genes to coordinate movement which generates mechanical force. An emerging theory is that mechanical force can also serve as an input signal to regulate developmental gene expression. Experimental methods to apply mechanical stimulation to whole embryos have been limited, mainly to aspiration, indentation, or moving a coverslip; these approaches stimulate only a few embryos at a time and require manual alignment. A powerful approach for automation is microfluidic devices, which can precisely manipulate hundreds of samples. However, using microfluidics to apply mechanical stimulation has been limited to small cellular systems, with fewer applications for larger scale whole embryos. We developed a mesofluidic device that applies the precision and automation of microfluidics to the Drosophila embryo: high-throughput automatic alignment, immobilization, compression, real-time imaging, and recovery of hundreds of live embryos. We then use twist:eGFP embryos to show that the mechanical induction of twist depends on the dose and duration of compression. This device allows us to quantify responses to compression, map the distribution of ectopic twist, and measure embryo stiffness. For building mesofluidic devices, we describe modifications on ultra-thick photolithography, derive an analytical model that predicts the deflection of sidewalls, and discuss parametric calibration. This "mesomechanics" approach combines the high-throughput automation and precision of microfluidics with the biological relevance of live embryos to examine mechanotransduction. These analytical models facilitate the design of future devices to process multicellular organisms such as larvae, organoids, and mesoscale tissue samples.

Development of 3D printed mesofluidic devices to elute and concentrate DNA.

To understand structural variation for personal genomics, an extensive ensemble of large DNA molecules will be required to span large structural variations. Nanocoding, a whole-genome analysis platform, can analyze large DNA molecules for the construction of physical restriction maps of entire genomes. However, handling of large DNA is difficult and a system is needed to concentrate large DNA molecules, while keeping the molecules intact. Insert technology was developed to protect large DNA molecules during routine cell lysis and molecular biology techniques. However, eluting and concentrating DNA molecules has been difficult in the past. Utilizing 3D printed mesofluidic device, a proof of principle system was developed to elute and concentrate lambda DNA molecules at the interface between a solution and a poly-acrylamide roadblock. The matrix allowed buffer solution to move through the pores in the matrix; however, it slowed down the progression of DNA in the matrix, since the molecules were so large and the pore size was small. Using fluorescence intensity of the insert, 84% of DNA was eluted from the insert and 45% of DNA was recovered in solution from the eluted DNA. DNA recovered was digested with a restriction enzyme to determine that the DNA molecules remained full length during the elution and concentration of DNA.

Rapid Fabrication of Chip-Based Physiometers for Neurobehavioral Toxicity Assays Using Rotifers Brachionus calyciflorus.

An increased interest in implementations of Lab-on-a-Chip (LOC) technologies for in-situ analysis of multicellular metazoan model organisms and their embryonic stages demands development of new prototyping techniques. Due to size of multicellular organisms the fabrication of soft-lithography molds requires features with high aspect ratios as well as deposition of layers with significant thicknesses. This makes them time consuming and difficult to fabricate using conventional photolithography techniques. In this work we describe development of a rapid technique capable of generating thick films achieved with high viscosity SU-8 and used in fabricating master templates for high aspect ratio micro- and mesofluidic devices. The cost effective and rapid method eliminated the need for multiple spin coating cycles as well as edge bead artifacts while preserving low surface roughness and superior surface uniformity. Due to elimination of spin coating steps, typically constrained to clean room facilities, the new method allows to significantly reduce microfabrication costs. We have utilized the prototyping technique to develop proof-of-concept chip-based devices capable of effectively caging freshwater rotifers Brachionus calyciflorus for high-definition video-microscopy analysis. The combination of time-resolved video-microscopy and chip-based physiometers enabled us to demonstrate new applications for neurobehavioral assays utilizing non-invasive sub-lethal end-points.

3D-Printed Microflow Injection Analysis Platform for Online Magnetic Nanoparticle Sorptive Extraction of Antimicrobials in Biological Specimens as a Front End to Liquid Chromatographic Assays.

In this work, the concept of 3D-printed microflow injection (3D-μFI) embodying a dedicated multifunctional 3D-printed stator onto a rotary microvalve along with a mesofluidic sample preparation platform is proposed for the first time. A transparent 3D-printed stereolithographic mesofluidic chip device accommodating polyaniline (PANI) decorated magnetic nanoparticles (32.5 ± 3.8 mg) is harnessed to in-line sorptive microextraction as a front end to liquid chromatography with peak focusing. As a proof-of-concept application, the 3D-μFI assembly was resorted to matrix cleanup and automatic programmable-flow determination of organic emerging contaminants (4-hydroxybenzoate analogues and triclosan as antimicrobial model analytes) in human saliva and urine samples. By using a sample volume of 1.0 mL with a loading flow rate of 200 μL min-1, an eluent volume of 120 μL at 80 μL min-1, and online HPLC injection of 300 μL of the mixture of eluate and Milli-Q water (in a 1:2 ratio) to prevent band broadening effects of the most polar analytes, the limits of detection (3σ criterion) ranged from 1.1 to 4.5 ng mL-1 for methylparaben (MP), ethylparaben (EP), propylparaben (PrP), phenylparaben (PhP), butylparaben (BP), and triclosan (TCS). Enhancement factors of 16-25 were obtained for the target analytes. Spike recoveries ranged from 84 to 117% for both saliva and urine samples. The online 3D-μFI hyphenated method is synchronized with the chromatographic separation and features a chip lifetime of more than 20 injections with minimal losses of moderately nonpolar compounds on the walls of the mesofluidic device.

Prototyping of Meso- and Microfluidic Devices with Embedded TiO2 Photocatalyst for Photodegradation of an Organic Dye

We present prototyping of meso- and microfluidic photocatalytic devices, functionalized through incorporation of TiO2 nanoparticles in polydimethylsiloxane (PDMS), and comparison of their efficiencies for the degradation of rhodamine B (10−5 mol/L). The prototyping of the photocatalytic devices involves simple and low-cost procedures, which includes microchannels fabrication on PDMS, deposition and impregnation of TiO2 on PDMS, and, finally, plugging on the individual parts. For the microfluidic device with 13 μL internal volume, photocatalytic TiO2–PDMS composite was sealed by another PDMS component activated by O2 plasma (PDMS–TiO2–PDMS). For the mesofluidic device, a homemade polyetheretherketone (PEEK) flow cell with 800 μL internal volume was screwed on a steel support with a glass slide and the photocatalytic composite. The photocatalytic activities of the devices were evaluated using two different pumping flow systems: a peristaltic pump and a syringe pump, both at 0.05 mL/min under the action of 3...

Development of 3D Printed Devices to Extract DNA Molecules for Genome Analysis

The high-throughput analysis of individual DNA is altering the pace and scope of biomedical investigation. Personal genomics will require large ensembles of individual genomes to develop a database of genomic variations. Within the human genome, more base pairs are altered as a result of structural variation than point mutations (Eichler et al., Nature Rev. Gen., 2011, 12, 363). In order to ascertain larger variations or large complex variations in human cancer genome, longer molecules (average molecule size > 700 kb) are necessary. In this regard, the Optical Mapping System (Teague et al., PNAS, 2010, 107, 10848) and Nanocoding (Jo et al., PNAS, 2007, 104, 2673; Kounovsky-Shafer, et al., Macromolecules, 2013, 46, 8356) were developed as a single molecule platform for construction of physical maps that span entire genomes. However, even these platforms find it difficult to span across large (>400 kb) complex genomic regions. The main goal of our research is to develop devices to elute DNA molecules from a gel matrix—prevents shearing of DNA molecules during cell lysis—and concentrate extremely long DNA molecules (>700 kb). Therefore, we are leveraging 3D printing to fabricate meso-fluidic devices to elute lambda DNA from gel using an electric field. For these devices, dimensions of the devices are varied in channel width; whereas, height and length of the device remains constant. In order to ascertain the device that elutes the highest concentration of lambda DNA, we are using a multi-prong approach: measuring fluorescence intensity of YOYO-1 stained DNA that are eluted into solution, as well as the fluorescence intensity of YOYO-1 stained DNA that are within gel matrix during the experiment, and measuring the concentration of DNA that remains in the gel matrix after the conclusion of the experiment using gel electrophoresis.

Early in-situ cellularization of a supramolecular vascular graft is modified by synthetic stromal cell-derived factor-1α derived peptides

In an in-situ approach towards tissue engineered cardiovascular replacement grafts, cell-free scaffolds are implanted that engage in endogenous tissue formation. Bioactive molecules can be incorporated into such grafts to facilitate cellular recruitment. Stromal cell derived factor 1α (SDF1α) is a powerful chemoattractant of lymphocytes, monocytes and progenitor cells and plays an important role in cellular signaling and tissue repair. Short SDF1α-peptides derived from its receptor-activating domain are capable of activating the SDF1α–specific receptor CXCR4. Here, we show that SDF1α-derived peptides can be chemically modified with a supramolecular four-fold hydrogen bonding ureido-pyrimidinone (UPy) moiety, that allows for the convenient incorporation of the UPy-SDF1α-derived peptides into a UPy-modified polymer scaffold. We hypothesized that a UPy-modified material bioactivated with these UPy-SDF1α-derived peptides can retain and stimulate circulating cells in an anti-inflammatory, pro-tissue formation signaling environment. First, the early recruitment of human peripheral blood mononuclear cells to the scaffolds was analyzed in vitro in a custom-made mesofluidic device applying physiological pulsatile fluid flow. Preferential adhesion of lymphocytes with reduced expression of inflammatory factors TNFα, MCP1 and lymphocyte activation marker CD25 was found in the bioactivated scaffolds, indicating a reduction in inflammatory signaling. As a proof of concept, in-vivo implantation of the bioactivated scaffolds as rat abdominal aorta interposition grafts showed increased cellularity by CD68+ cells after 7 days. These results indicate that a completely synthetic, cell-free biomaterial can attract and stimulate specific leukocyte populations through supramolecular incorporation of short bioactive SDF1α derived peptides.

Nanomanufacturing of Tobacco Mosaic Virus-Based Spherical Biomaterials Using a Continuous Flow Method.

Nanomanufacturing of nanoparticles is critical for potential translation and commercialization. Continuous flow devices can alleviate this need through unceasing production of nanoparticles. Here we demonstrate the scaled-up production of spherical nanoparticles functionalized with biomedical cargos from the rod-shaped plant virus tobacco mosaic virus (TMV) using a mesofluidic, continued flow method. Production yields were increased 30-fold comparing the mesofluidic device versus batch methods. Finally, we produced MRI contrast agents of select sizes, with per particle relaxivity reaching 979,218 mM–1 s–1 at 60 MHz. These TMV-based spherical nanoparticle MRI contrast agents are in the top echelon of relaxivity per nanoparticle.

OpenSource Lab‐on‐a‐Chip Physiometer for Accelerated Zebrafish Embryo Biotests

Zebrafish (Danio rerio) embryo assays have recently come into the spotlight as convenient experimental models in both biomedicine and ecotoxicology. As a small aquatic model organism, zebrafish embryo assays allow for rapid physiological, embryo-, and genotoxic tests of drugs and environmental toxins that can be simply dissolved in water. This protocol describes prototyping and application of an innovative, miniaturized, and polymeric chip-based device capable of immobilizing a large number of living fish embryos for real-time and/or time-lapse microscopic examination. The device provides a physical address designation to each embryo during analysis, continuous perfusion of medium, and post-analysis specimen recovery. Miniaturized embryo array is a new concept of immobilization and real-time drug perfusion of multiple individual and developing zebrafish embryos inside the mesofluidic device. The OpenSource device presented in this protocol is particularly suitable to perform accelerated fish embryo biotests in ecotoxicology and phenotype-based pharmaceutical screening.

Pressure-driven mesofluidic platform integrating automated on-chip renewable micro-solid-phase extraction for ultrasensitive determination of waterborne inorganic mercury

A dedicated pressure-driven mesofluidic platform incorporating on-chip sample clean-up and analyte preconcentration is herein reported for expedient determination of trace level concentrations of waterborne inorganic mercury. Capitalizing upon the Lab-on-a-Valve (LOV) concept, the mesofluidic device integrates on-chip micro-solid phase extraction (μSPE) in automatic disposable mode followed by chemical vapor generation and gas–liquid separation prior to in-line atomic fluorescence spectrometric detection. In contrast to prevailing chelating sorbents for Hg(II), bare poly(divinylbenzene-N-vinylpyrrolidone) copolymer sorptive beads were resorted to efficient uptake of Hg(II) in hydrochloric acid milieu (pH=2.3) without the need for metal derivatization nor pH adjustment of prior acidified water samples for preservation to near-neutral conditions. Experimental variables influencing the sorptive uptake and retrieval of target species and the evolvement of elemental mercury within the miniaturized integrated reaction chamber/gas–liquid separator were investigated in detail.Using merely <10mg of sorbent, the limits of detection and quantification at the 3sblank and 10sblank levels, respectively, for a sample volume of 3mL were 12 and 42ngL−1 Hg(II) with a dynamic range extending up to 5.0μgL−1. The proposed mesofluidic platform copes with the requirements of regulatory bodies (US-EPA, WHO, EU-Commission) for drinking water quality and surface waters that endorse maximum allowed concentrations of mercury spanning from 0.07 to 6.0μgL−1. Demonstrated with the analysis of aqueous samples of varying matrix complexity, the LOV approach afforded reliable results with relative recoveries of 86–107% and intermediate precision down to 9% in the renewable μSPE format.

Simplified Mesofluidic Systems for the Formation of Micron to Millimeter Droplets and the Synthesis of Materials

We present and validate simple mesofluidic devices for producing monodisperse droplets and materials. The significance of this work is a demonstration that simple and complex droplet formulations can be prepared uniformly using off-the-shelf small-diameter tubing, barbed tubing adapters, and needles. With these simple tools, multiple droplet-forming devices and a new particle concentrator were produced and validated. We demonstrate that the droplet-forming devices could produce low-dispersity particles from 25 to 1200 Km and that these results are similar to results from more complicated devices. Through a study of the fluid dynamics and a dimensional analysis of the data, we have correlated droplet size with two dimensionless groups, capillary number and viscosity ratio. The flowfocusing device is more sensitive to both parameters than the T-junction geometry. The modular character of our mesofluidic devices allowed us to rapidly assemble compound devices that use flow-focusing and T-junction devices in series to create complex droplet-in-microcapsule materials. This work demonstrates that flow chemistry does not require complicated tools, and an inexpensive tool-kit can allow anyone with interest to enter the field.

Highly Parallel Translation of DNA Sequences into Small Molecules

A large body of in vitro evolution work establishes the utility of biopolymer libraries comprising 1010 to 1015 distinct molecules for the discovery of nanomolar-affinity ligands to proteins.[1], [2], [3], [4], [5] Small-molecule libraries of comparable complexity will likely provide nanomolar-affinity small-molecule ligands.[6], [7] Unlike biopolymers, small molecules can offer the advantages of cell permeability, low immunogenicity, metabolic stability, rapid diffusion and inexpensive mass production. It is thought that such desirable in vivo behavior is correlated with the physical properties of small molecules, specifically a limited number of hydrogen bond donors and acceptors, a defined range of hydrophobicity, and most importantly, molecular weights less than 500 Daltons.[8] Creating a collection of 1010 to 1015 small molecules that meet these criteria requires the use of hundreds to thousands of diversity elements per step in a combinatorial synthesis of three to five steps. With this goal in mind, we have reported a set of mesofluidic devices that enable DNA-programmed combinatorial chemistry in a highly parallel 384-well plate format. Here, we demonstrate that these devices can translate DNA genes encoding 384 diversity elements per coding position into corresponding small-molecule gene products. This robust and efficient procedure yields small molecule-DNA conjugates suitable for in vitro evolution experiments.

A Spectroscopic Monitoring Module Based on a Ceramic Microfluidic Platform

A 3-dimensional mesofluidic biological monitoring module has been successfully designed and fabricated using a low-temperature co-fired-ceramic (LTCC) technology. This mesofluidic device consists of a network of micro-channels and a spherical mixing cavity. The selection of appropriate commercially available ceramic tapes has been done with regard to their biocompatibility performance. Specific processing procedures required for the realization of such complex structure are demonstrated. Three dimensional numerical flow simulations have been conducted to characterize the concentration profiles of liquids at a specific measuring port and verified by experiment. The module was successfully applied to study complex chemical reaction kinetics complemented by mathematical modelling.Fig. 1Scheme of the monitoring moduleView Large Image | View Hi-Res Image | Download PowerPoint Slide