Fabrication Protocol for Thermoplastic Microfluidic Devices: Nanoliter Volume Bioreactors for Cell Culturing.

Microfluidic devices consist of microchannels etched or embossed into substrates made of polymer, glass or silicon. Intricate connections of the microchannels to reactors with some smart mechanical structures such as traps or curvatures fulfil the desired functionalities such as mixing, separation, flow control or setting the environment for biochemical reactions. Here, we describe the fabrication methods of a thermoplastic microbioreactor step by step. First, material selection is made, then, production methods are determined with the equipment that can be easily procured in a laboratory. COP with its outstanding characteristics among many polymers was chosen. Two types of microbioreactors, with and without electrodes, are designed with AutoCAD and L-edit softwares. Photolithography and electrochemical wet etching are used for master mold preparation. Thermal evaporator is employed for pure chromium and gold deposition on COP substrate and etchants are used to form the interdigitated electrodes. Once the master mold produced, hot embossing is used to obtain the designed shape on drilled and planarized COP. Cover COP, with or without electrodes, is bonded to the hot embossed COP via thermo-compression and thermoplastic microfluidic device is realized. Tubings are connected to the device and a bridge between the macro and micro world is established. Yeast or mammalian cells labeled or tagged with GFP/RFP on specific gene products are loaded into the microfluidic device, and real time data on cell dimensions and fluorescence intensity are collected using inverted fluorescence microscope, and finally image processing is used to analyze the acquired data.

Magnetic Resonance Imaging-Compatible Optically Powered Miniature Wireless Modular Lorentz Force Actuators.

Minimally invasive medical procedures under magnetic resonance imaging (MRI) guidance have significant clinical promise. However, this potential has not been fully realized yet due to challenges regarding MRI compatibility and miniaturization of active and precise positioning systems inside MRI scanners, i.e., restrictions on ferromagnetic materials and long conductive cables and limited space around the patient for additional instrumentation. Lorentz force-based electromagnetic actuators can overcome these challenges with the help of very high, axial, and uniform magnetic fields (3-7 Tesla) of the scanners. Here, a miniature, MRI-compatible, and optically powered wireless Lorentz force actuator module consisting of a solar cell and a coil with a small volume of 2.5 × 2.5 × 3.0 mm3 is proposed. Many of such actuator modules can be used to create various wireless active structures for future interventional MRI applications, such as positioning needles, markers, or other medical tools on the skin of a patient. As proof-of-concept prototypes toward such applications, a single actuator module that bends a flexible beam, four modules that rotate around an axis, and six modules that roll as a sphere are demonstrated inside a 7 Tesla preclinical MRI scanner.

Thermoplastic microfluidic bioreactors with integrated electrodes to study tumor treating fields on yeast cells.

Tumor-treating fields (TTFields) are alternating electrical fields of intermediate frequency and low intensity that can slow or inhibit tumor growth by disrupting mitosis division of cancerous cells through cell cycle proteins. In this work, for the first time, an in-house fabricated cyclo-olefin polymer made microfluidic bioreactors are integrated with Cr/Au interdigitated electrodes to test TTFields on yeast cells with fluorescent protein:Nop56 gene. A small gap between electrodes (50 µm) allows small voltages (<150 mV) to be applied on the cells; hence, uninsulated gold electrodes are used in the non-faradaic region without causing any electrochemical reaction at the electrode-medium interface. Electrochemical modeling as well as impedance characterization and analysis of the electrodes are done using four different cell nutrient media. The experiments with yeast cells are done with 150 mV, 150 kHz and 30 mV, 200 kHz sinusoidal signals to generate electrical field magnitudes of 6.58 V/cm and 1.33 V/cm, respectively. In the high electrical field experiment, the cells go through electroporation. In the experiment with the low electrical field magnitude for TTFields, the cells have prolonged mitosis from typical 80-90 min to 200-300 min. Our results confirm the validity of the electrochemical model and the importance of applying a correct magnitude of the electrical field. Compared to the so far reported alternatives with insulated electrodes, the here developed thermoplastic microfluidic bioreactors with uninsulated electrodes provide a new, versatile, and durable platform for in vitro cell studies toward the improvement of anti-cancer therapies including personalized treatment.

Cell trapping microfluidic chip made of Cyclo olefin polymer enabling two concurrent cell biology experiments with long term durability.

Cyclo Olefin Polymer (COP) based microbioreactors on a microfluidic chip were produced in house by hot-embossing and thermo-compression bonding methods. The chip allows two different experiments to be performed on trapped cells at the same time. On one side of the chip, red fluorescent protein (RFP) tagged nucleolar Nop56 protein was used to track changes in cell cycle as well as protein synthesis within the yeast cells under the application of the anti-tumor agent hydroxyurea (HU). Simultaneously, on the other side of the chip, the response of yeast cells to the drug metformin, mTOR inhibitor, was investigated to reveal the role of TOR signaling in ribosome biogenesis and cell proliferation. The results of 20 h long experiments are captured by taking brightfield and fluorescent microscopy images of the trapped cells every 9 min. The expression of Nop56 protein of ribosome assembly and synthesis was densely observed during G1 phase of cell cycle, and later towards the end of cell cycle the ribosomal protein expression slowed down. Under HU treatment, the morphology of yeast cells changed, but after cessation of HU, the biomass synthesis rate was sustained as monitored by the cell perimeter. Under metformin treatment, the perimeters of single cells were observed to decrease, implying a decrease in biomass growth; however these cells continued their proliferation during and after the drug application. The relation between ribosome biogenesis and cell cycle was successfully investigated on single cell basis, capturing cell-to-cell variations, which cannot be tracked by regular macroscale bioreactors.

Real-Time Performance of a Tactile Neuroprosthesis on Awake Behaving Rats.

With the advancement of electrode and equipment technology, neuroprosthetics have become a promising alternative to partially compensate for the loss of sensorimotor function in amputees and patients with neurological diseases. Cortical neural interfaces are suitable especially for spinal cord injuries and amyotrophic lateral sclerosis. Although considerable success has been achieved in the literature by spike decoding of motor signals from the human brain, somatosensory feedback is essential for better motor control, interaction with objects, and the embodiment of prosthetic devices. In this paper, we present a tactile neuroprosthesis for rats based on intracortical microstimulation (ICMS). The rats wore mechanically-isolated boots covered with tactile sensors while performing a psychophysical detection task. The vibrotactile stimuli were measured by the artificial sensors and by using a real-time processor, this information was converted to electrical current pulses for ICMS. Some parameters of the real-time processor algorithm were specific to individual rats and were based on psychometric equivalence functions established earlier. Rats could detect the effects of the vibrotactile stimuli better (i.e., higher sensitivity indices) when the tactile neuroprosthesis was switched on compared to the boot only condition during active movement. In other words, the rats could decode the tactile information embedded in ICMS and use that in a behaviorally relevant manner. The presented animal model without peripheral nerve injury or amputation is also a promising tool to test various hardware and software components of neuroprosthetic systems in general.

A low cost PS based microfluidic platform to investigate cell cycle towards developing a therapeutic strategy for cancer.

Inhibition of DNA damage response pathway in combination with DNA alkylating agents may enhance the selective killing of cancer cells leading to better therapeutic effects. MDM2 binding protein (MTBP) in human has a role in G1 phase (interphase of cell cycle) and its overexpression leads to breast and ovarian cancers. Sld7 is an uncharacterized protein in budding yeast and a potential functional homologue of MTBP. To investigate the role of Sld7 as a therapeutic target, the behavior of the wild-type cells and sld7∆ mutants were monitored in 0.5 nL microbioreactors. The brightfield microscopy images were used to analyze the change in the cell size and to determine the durations of G1 and S/G2/M phases of wild type cells and mutants. With the administration of the alkylating agent, the cell size decreased and the duration of cell cycle increased. The replacement of the medium with the fresh one enabled the cells to repair their DNA. The application of calorie restriction together with DNA alkylating agent to mutant cells resulted in smaller cell size and longer G1 phase compared to those in control environment. For therapeutic purposes, the potential of MTBP in humans or Sld7 in yeast as a drug target deserves further exploration. The fabrication simplicity, robustness and low-cost of this microfluidic bioreactor made of polystyrene allowed us to perform yeast culturing experiments and show a potential for further cell culturing studies. The device can successfully be used for therapeutic applications including the discovery of new anti-microbial, anti-inflammatory, anti-cancer drugs.

Advances in microfluidic devices made from thermoplastics used in cell biology and analyses.

Silicon and glass were the main fabrication materials of microfluidic devices, however, plastics are on the rise in the past few years. Thermoplastic materials have recently been used to fabricate microfluidic platforms to perform experiments on cellular studies or environmental monitoring, with low cost disposable devices. This review describes the present state of the development and applications of microfluidic systems used in cell biology and analyses since the year 2000. Cultivation, separation/isolation, detection and analysis, and reaction studies are extensively discussed, considering only microorganisms (bacteria, yeast, fungi, zebra fish, etc.) and mammalian cell related studies in the microfluidic platforms. The advantages/disadvantages, fabrication methods, dimensions, and the purpose of creating the desired system are explained in detail. An important conclusion of this review is that these microfluidic platforms are still open for research and development, and solutions need to be found for each case separately.

Modelling and Realization of a Water-Gated Field Effect Transistor (WG-FET) Using 16-nm-Thick Mono-Si Film.

We introduced a novel water-gated field effect transistor (WG-FET) which uses 16-nm-thick mono-Si film as active layer. WG-FET devices use electrical double layer (EDL) as gate insulator and operate under 1 V without causing any electrochemical reactions. Performance parameters based on voltage distribution on EDL are extracted and current-voltage relations are modelled. Both probe- and planar-gate WG-FETs with insulated and uninsulated source-drain electrodes are simulated, fabricated and tested. Best on/off ratios are measured for probe-gate devices as 23,000 A/A and 85,000 A/A with insulated and uninsulated source-drain electrodes, respectively. Planar-gate devices with source-drain insulation had inferior on/off ratio of 1,100 A/A with 600 µm gate distance and it decreased to 45 A/A when gate distance is increased to 3000 µm. Without source-drain electrode insulation, proper transistor operation is not obtained with planar-gate devices. All measurement results were in agreement with theoretical models. WG-FET is a promising device platform for microfluidic applications where sensors and read-out circuits can be integrated at transistor level.

Fabrication of cyclo olefin polymer microfluidic devices for trapping and culturing of yeast cells.

A microfluidic platform is designed and fabricated to investigate the role of uncharacterized YOR060C (Sld7) protein in aging in yeast cells for the first time. Saccharomyces cerevisiae yeast cells are trapped in the series of C-shaped regions (0.5 nL) of COP (cyclo olefin polymer), PMMA (poly methylmethacrylate), or PS (polystyrene) microbioreactors. The devices are fabricated using hot embossing and thermo-compression bonding methods. Photolithography and electrochemical etching are used to form the steel mold needed for hot embossing. The cell cycle processes are investigated by monitoring green fluorescent protein (GFP) tagged Sld7 expressions under normal as well as calorie restricted conditions. The cells are loaded at 1 µL/min flowrate and trapped successfully within each chamber. The medium is continuously fed at 0.1 µL/min throughout the experiments. Fluorescent signals of the low abundant Sld7 proteins could be distinguished only on COP devices. The background fluorescence of COP is found 1.22 and 7.24 times lower than that of PMMA, and PS, respectively. Hence, experiments are continued with COP, and lasted for more than 40 h without any contamination. The doubling time of the yeast cells are found as 72 min and 150 min, and the growth rates as 9.63 × 10-3 min-1 and 4.62 × 10-3 min-1, in 2% glucose containing YPD and YNB medium, respectively. The product concentration (Sld7p:GFP) increased in accordance with cell growth. The dual role of Sld7 protein in both cell cycle and chronological aging needs to be further investigated following the preliminary experimental results.

Thin film based semi-active resonant marker design for low profile interventional cardiovascular MRI devices.

OBJECTIVES: A new microfabrication method to produce low profile radio frequency (RF) resonant markers on catheter shafts was developed. A semi-active RF resonant marker incorporating a solenoid and a plate capacitor was constructed on the distal shaft of a 5 Fr guiding catheter. The resulting device can be used for interventional cardiovascular MRI procedures. MATERIALS AND METHODS: Unlike current semi-active device visualization techniques that require rigid and bulky analog circuit components (capacitor and solenoid), we fabricated a low profile RF resonant marker directly on guiding the catheter surface by thin film metal deposition and electroplating processes using a modified physical vapor deposition system. RESULTS: The increase of the overall device profile thickness caused by the semi-active RF resonant marker (130 µm thick) was lowered by a factor of 4.6 compared with using the thinnest commercial non-magnetic and rigid circuit components (600 µm thick). Moreover, adequate visibility performance of the RF resonant marker in different orientations and overall RF safety were confirmed through in vitro experiments under MRI successfully. CONCLUSION: The developed RF resonant marker on a clinical grade 5 Fr guiding catheter will enable several interventional congenital heart disease treatment procedures under MRI.

Using a low-amplitude RF pulse at echo time (LARFET) for device localization in MRI.

We describe a new method for frequency down-conversion of MR signals acquired with the radio-frequency projections method for device localization. A low-amplitude, off-center RF pulse applied simultaneously with the echo signal is utilized as the reference for frequency down-conversion. Because of the low-amplitude and large offset from the Larmor frequency, the RF pulse minimally interfered with magnetic resonance of protons. We conducted an experiment with the coil placed at different positions to verify this concept. The down-converted signal was transformed into optical signal and transmitted via fiber-optic cable to a receiver unit placed outside the scanner room. The position of the coil could then be determined by the frequency analysis of this down-converted signal and superimposed on previously acquired MR images for comparison. Because of minimal positional errors (≤ 0.8 mm), this new device localization method may be adequate for most interventional MRI applications.

A solution state diode using semiconductor polymer nanorods with nanogap electrodes.

A solution state polymer diode, which uses regioregular poly(3-hexylthiophene-2,5-diyl) (P3HT):dichlorobenzene solution as the semiconductor between highly doped p-type silicon and aluminum electrodes has been built. Electrodes separated by a 40 nm gap enable intra-chain charge carrier transfer through the lengths of single polymer chains. This prevents chain to chain hopping and chain entanglements, increasing carrier mobility. The degradation with time and hysteresis effects of the diodes are measured. An optimal P3HT solution concentration of 6 mg ml(-1) is found. A current density of at least 300 mA cm(-2) is achieved, indicating at least a six-fold improvement in carrier mobility compared to previously fabricated solid state P3HT diodes.

Monolithic valves for microfluidic chips based on thermoresponsive polymer gels.

The direct preparation of thermoresponsive monolithic copolymers by photopatterning of a liquid phase consisting of an aqueous solution of N-isopropylacrylamide, N-ethylacrylamide, N,N'-methylenebisacrylamide, and 4,4'-azobis(4-cyanovaleric acid) has been studied and the products used as valves within the channels of microfluidic devices. The volume change associated with the polymer phase transition at its lower critical solution temperature (LCST) leads to the rapid swelling and the deswelling of the 2.5% cross-linked monolithic gel thus enabling the polymer to close or open the channel and to function as a nonmechanically actuated valve. The LCST at which the valve switches was easily adjusted within a range of 35 degrees C-74 degrees C by varying the proportions of the monovinyl monomers in the polymerization mixture. The closed valve holds pressures of up to 18 MPa without noticeable dislocation, structural damage, or leakage. In contrast, following deswelling by raising the temperature above LCST the valve offers no appreciable flow resistance since its large, micrometer-size pores are open. Laser-triggered photobleaching of a fluorescent dye contained in the liquid phase enabled monitoring of flow through the device and determination of the times required to open and close the valve. The valves are characterized by very fast actuation times in a range of 1-4 s depending on the type of device. No changes in performance were observed even after repeated open-close cycling of the valves.

Flow control valves for analytical microfluidic chips without mechanical parts based on thermally responsive monolithic polymers.

Monolithic plugs of poly(N-isopropylacrylamide) cross-linked with 5% methylenebisacrylamide have been prepared by photoinitiated polymerization within the channel of a microfluidic device. The volume change associated with the polymer phase transition at its lower critical solution temperature of 32 degrees C allows both the rapid swelling and the deswelling of the monoliths enabling the polymer to close or open the channel as it functions as a nonmechanical valve. Thermoelectric elements capable of changing the temperature of the system between 17 and 57 degrees C were used to actuate the valve. Flow through the device was monitored by fluorescence measurements via the laser-triggered photobleaching of a dye contained in the liquid phase. Photobleaching occurs quickly once the flow is stopped, and the time required to open and close the valve was 3.5 and 5.0 s, respectively. No changes in function were observed even after 120 open-close cycles. Although the 2-mm-long valve was prepared from a polymerization mixture consisting of only a 5% aqueous solution of monomers, it resists pressures of up to 1.38 MPa (200 psi) without observable structural damage.