DNA genetics, Fluorescence, Genetic Testing, Glass, Humans, Microfluidic Analytical Techniques methods, Physical Phenomena, Polymerase Chain Reaction methods, Temperature, DNA analysis, Microfluidic Analytical Techniques instrumentation, and Polymerase Chain Reaction instrumentation
In this article, low cost microfluidic devices have been used for simultaneous amplification and analysis of DNA. Temperature gradient flow PCR was performed, during which the unique fluorescence signature of the amplifying product was determined. The devices were fabricated using xurography, a fast and highly flexible prototype manufacturing method. Each complete iterative design cycle, from concept to prototype, was completed in less than 1 h. The resulting devices were of a 96% glass composition, thereby possessing a high thermal stability during continuous-flow PCR. Volumetric flow rates up to 4 microl/min induced no measurable change in the temperature distribution within the microchannel. By incorporating a preliminary channel passivation protocol, even the first microliters through the system exhibited a high amplification efficiency, thereby demonstrating the biocompatibility of this fabrication technique for DNA amplification microfluidics. The serpentine microchannel induced 23 temperature gradient cycles in 15 min at a 2 microl/min flow rate. Fluorescent images of the device were acquired while and/or after the PCR mixture filled the microchannel. Because of the relatively high initial concentration of the phage DNA template (PhiX174), images taken after 10 min (less than 15 PCR cycles) could be used to positively identify the PCR product. A single fluorescent image of a full device provided the amplification curve for the entire reaction as well as multiple high resolution melting curves of the amplifying sample. In addition, the signal-to-noise ratio associated with the spatial fluorescence was characterized as a function of spatial redundancy and acquisition time.
Presently, cardiovascular interventions such as stent deployment and balloon angioplasty are performed under x-ray guidance. However, x-ray fluoroscopy has poor soft tissue contrast and is limited by imaging in a single plane, resulting in imprecise navigation of endovascular instruments. Moreover, x-ray fluoroscopy exposes patients to ionizing radiation and iodinated contrast agents. Magnetic resonance imaging (MRI) is a safe and enabling modality for cardiovascular interventions. Interventional cardiovascular MR (iCMR) is a promising approach that is in stark contrast with x-ray fluoroscopy, offering high-resolution anatomic and physiologic information and imaging in multiple planes for enhanced navigational accuracy of catheter-based devices, all in an environment free of radiation and its deleterious effects. While iCMR has immense potential, its translation into the clinical arena is hindered by the limited availability of MRI-visible catheters, wire guides, angioplasty balloons, and stents. Herein, we aimed to create application-specific, devices suitable for iCMR, and demonstrate the potential of iCMR by performing cardiovascular catheterization procedures using these devices. Tools, including catheters, wire guides, stents, and angioplasty balloons, for endovascular interventions were functionalized with a polymer coating consisting of poly(lactide-co-glycolide) (PLGA) and superparamagnetic iron oxide (SPIO) nanoparticles, followed by endovascular deployment in the pig. Findings from this study highlight the ability to image and properly navigate SPIO-functionalized devices, enabling interventions such as successful stent deployment under MRI guidance. This study demonstrates proof-of-concept for rapid prototyping of iCMR-specific endovascular interventional devices that can take advantage of the capabilities of iCMR.
Efficient intracellular cargo delivery is a key hurdle for the translation of many emerging stem cell and cellular reprogramming therapies. Recently, a microfluidic-based device constructed from silicon was shown to transduce macromolecules into cells via shear-induced formation of plasma membrane pores. However, the scalability and widespread application of the current platform is limited since physical deformation-mediated delivery must be optimized for each therapeutic application. Therefore, we sought to create a low-cost, versatile device that could facilitate rapid prototyping and application-specific optimization in most academic research labs. Here we describe the design and implementation of a microfluidic device constructed from Polydimethylsiloxane (PDMS) that we call Cyto-PDMS (Cytoplasmic PDMS-based Delivery and Modification System). Using a systematic Cyto-PDMS workflow, we demonstrate intracellular cargo delivery with minimal effects on cellular viability. We identify specific flow rates at which a wide range of cargo sizes (1-70 kDa) can be delivered to the cell interior. As a proof-of-principle for the biological utility of Cyto-PDMS, we show (i) F-actin labeling in live human fibroblasts and (ii) intracellular delivery of recombinant Cre protein with appropriate genomic recombination in recipient fibroblasts. Taken together, our results demonstrate that Cyto-PDMS can deliver small-molecules to the cytoplasm and biologically active cargo to the nucleus without major effects on viability. We anticipate that the cost and versatility of PDMS can be leveraged to optimize delivery to a broad array of possible cell types and thus expand the potential impact of cellular therapies.
Tian P, Chen C, Hu J, Qi J, Wang Q, Chen JC, Cavanaugh J, Peng Y, and Cheng MM
Biomedical microdevices [Biomed Microdevices] 2017 Nov 23; Vol. 20 (1), pp. 4. Date of Electronic Publication: 2017 Nov 23.
Animals, Carbon chemistry, Dielectric Spectroscopy, Electric Conductivity, Electric Stimulation instrumentation, Electrodes, Implanted, Electromyography instrumentation, Equipment Design, Male, Rats, Sprague-Dawley, Signal-To-Noise Ratio, Thermogravimetry, Electrodes, and Printing, Three-Dimensional
Three-dimensional (3D) printing is an emerging technique in the field of biomedical engineering and electronics. This paper presents a novel biofabrication method of implantable carbon electrodes with several advantages including fast prototyping, patient-specific and miniaturization without expensive cleanroom. The method combines stereolithography in additive manufacturing and chemical modification processes to fabricate electrically conductive carbon electrodes. The stereolithography allows the structures to be 3D printed with very fine resolution and desired shapes. The resin is then chemically modified to carbon using pyrolysis to enhance electrochemical performance. The electrochemical characteristics of 3D printing carbon electrodes are assessed by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The specific capacitance of 3D printing carbon electrodes is much higher than the same sized platinum (Pt) electrode. In-vivo electromyography (EMG) recording, 3D printing carbon electrodes exhibit much higher signal-to-noise ratio (40.63 ± 7.73) than Pt electrodes (14.26 ± 6.83). The proposed biofabrication method is envisioned to enable 3D printing in many emerging applications in biomedical engineering and electronics.
Integration of microelectronics with microfluidics enables sophisticated lab-on-a-chip devices for sensing and actuation. In this paper, we investigate a novel method for in-situ microfluidics fabrication and packaging on wafer level. Two novel photo-patternable adhesive polymers were tested and compared, PA-S500H and DXL-009. The microfluidics fabrication method employs photo lithographical patterning of spin coated polymer films of PA or DXL and direct bonding of formed microfluidics to a top glass cover using die-to-wafer level bonding. These new adhesive materials remove the need for additional gluing layers. With this approach, we fabricated disposable microfluidic flow cytometers and evaluated the performance of those materials in the context of this application. DXL-009 exhibits lower autofluorescence compared to PA-S500H which improves detection sensitivity of fluorescently stained cells. Results obtained from the cytotoxicity test reveals that both materials are biocompatible. The functionality of these materials was demonstrated by detection of immunostained monocytes in microfluidic flow cytometers. The flexible, fully CMOS compatible fabrication process of these photo-patternable adhesive materials will simplify prototyping and mass manufacturing of sophisticated microfluidic devices with integrated microelectronics.
Microfluidic perfusion systems (MPS) are well suited to perform multiparametric measurements with small amounts of tissue to function as an Organ on Chip device (OOC). Such microphysiolgical characterization is particularly valuable in research on the stimulus-secretion-coupling of pancreatic islets. Pancreatic islets are fully functional competent mini-organs, which serve as fuel sensors and transduce metabolic activity into rates of hormone secretion. To enable the simultaneous measurement of fluorescence and oxygen consumption we designed a microfluidic perfusion system from borosilicate glass by 3D femtosecond laser ablation. Retention of islets was accomplished by a plain well design. The characteristics of flow and shear force in the microchannels and wells were simulated and compared with the measured exchange of the perfusion media. Distribution of latex beads, MIN6 cell pseudo islets and isolated mouse islets in the MPS was characterized in dependence of flow rate and well depth. Overall, the observations suggested that a sufficient retention of the islets at low shear stress, together with sufficient exchange of test medium, was achieved at a well depth of 300 μm and perfusion rates between 40 and 240 μl/min. This enabled multiparametric measurement of oxygen consumption, NAD(P)H autofluorescence, cytosolic Ca 2+ concentration, and insulin secretion by isolated mouse islets. After appropriate correction for different lag times, kinetics of these processes could be compared. Such measurements permit a more precise insight into metabolic changes underlying the regulation of insulin secretion. Thus, rapid prototyping using laser ablation enables flexible adaption of borosilicate MPS designs to different demands of biomedical research.
Equipment Design, Equipment Failure, Time Factors, Costs and Cost Analysis, Disposable Equipment economics, Lab-On-A-Chip Devices economics, and Polymers
This work presents the fabrication of a microfluidic autoregulatory valve which is composed of several layers of thin polymer films (i.e., polyvinyl chloride (PVC), polyethylene terephthalate (PET) double-sided tape, and polydimethylsiloxane (PDMS)). Briefly, pulsed UV laser is employed to cut the microstructures of through grooves or holes in the thermoplastic polymer films, and then the polymer-film valves are precisely assembled through laminating the PDMS membranes to the thermoplastic polymer films through the roll-lamination method. The effective bonding between the PVC film and the PDMS membrane is realized using the planar seal method, and the valve is sandwiched and compressed by a home-made housing to achieve the good seal effect. Then, the flow performances of the prototype valve are examined, and constant flow autoregulation is realized under the static or dynamic test pressures. The long-term response of the valve is also studied and minimum flow-rate decrements are found over a long actuation time. The fabrication method proposed in this work is successful for the low-cost and fast prototyping of the polymer-film valve. We believe our method will also be broadly applicable for fabrication of other low-cost and disposable polymer-film microfluidic devices.
In this work, we report a simple fabrication method for microelectrodes on a polymethylmethacrylate substrate, using a low-cost laser platform based on a CD-DVD unit for direct rapid-prototyping. We used this laser microfabrication technique to etch any desired design on polymethylmethacrylate substrates to produce microchannels with controlled geometry, with a highly repeatable micron-scale resolution. Those shallow microchannels were then filled with a conductive paste of material of our choice that was converted into microelectrodes of desired shapes and geometries after drying. To validate our process, different geometries, sizes and materials were used as electrodes, and then tested for amperometry and impedance measurements. Development of these microelectrodes is motivated by their potential application in sensors and biosensors, such as glucose and cell counting, as demonstrated in this paper.
Light, Viscosity, Microtechnology methods, and Phacoemulsification instrumentation
Microstereolithography (microSL) technology can fabricate complex, three-dimensional (3D) microstructures, although microSL has difficulty producing macrostructures with micro-scale features. There are potentially many applications where 3D micro-features can benefit the overall function of the macrostructure. One such application involves a medical device called a coaxial phacoemulsifier where the tip of the phacoemulsifier is inserted into the eye through a relatively small incision and used to break the lens apart while removing the lens pieces and associated fluid from the eye through a small tube. In order to maintain the eye at a constant pressure, the phacoemulsifier also includes an irrigation solution that is injected into the eye during the procedure through a coaxial sleeve. It has been reported, however, that the impinging flow from the irrigation solution on the corneal endothelial cells in the inner eye can damage these cells during the procedure. As a result, a method for reducing the impinging flow velocities and the resulting shear stresses on the endothelial cells during this procedure was explored, including the design and development of a complex, 3D micro-vane within the sleeve. The micro-vane introduces swirl into the irrigation solution, producing a flow with rapidly dissipating flow velocities. Fabrication of the sleeve and fitting could not be accomplished using microSL alone, and thus, a two-part design was accomplished where a sleeve with the micro-vane was fabricated with microSL and a threaded fitting used to attach the sleeve to the phacoemulsifier was fabricated using an Objet Eden 333 rapid prototyping machine. The new combined device was tested within a water container using particle image velocimetry, and the results showed successful swirling flow with an ejection of the irrigation fluid through the micro-vane in three different radial directions corresponding to the three micro-vanes. As expected, the sleeve produced a swirling flow with rapidly dissipating streamwise flow velocities where the maximum measured streamwise flow velocities using the micro-vane were lower than those without the micro-vane by 2 mm from the tip where they remained at approximately 70% of those produced by the conventional sleeve as the flow continued to develop. It is believed that this new device will reduce damage to endothelial cells during cataract surgery and significantly improve patient outcomes from this procedure. This unique application demonstrates the utility of combining microSL with a macro rapid prototyping technology for fabricating a real macro-scale device with functional, 3D micro-scale features that would be difficult and costly to fabricate using alternative manufacturing methods.