Dimitris E. Nikitopoulos

Highly Efficient Circulating Tumor Cell Isolation from Whole Blood and Label-Free Enumeration Using Polymer-Based Microfluidics with an Integrated Conductivity Sensor

A novel microfluidic device that can selectively and specifically isolate exceedingly small numbers of circulating tumor cells (CTCs) through a monoclonal antibody (mAB) mediated process by sampling large input volumes (>/=1 mL) of whole blood directly in short time periods (<37 min) was demonstrated. The CTCs were concentrated into small volumes (190 nL), and the number of cells captured was read without labeling using an integrated conductivity sensor following release from the capture surface. The microfluidic device contained a series (51) of high-aspect ratio microchannels (35 mum width x 150 mum depth) that were replicated in poly(methyl methacrylate), PMMA, from a metal mold master. The microchannel walls were covalently decorated with mABs directed against breast cancer cells overexpressing the epithelial cell adhesion molecule (EpCAM). This microfluidic device could accept inputs of whole blood, and its CTC capture efficiency was made highly quantitative (>97%) by designing capture channels with the appropriate widths and heights. The isolated CTCs were readily released from the mAB capturing surface using trypsin. The released CTCs were then enumerated on-device using a novel, label-free solution conductivity route capable of detecting single tumor cells traveling through the detection electrodes. The conductivity readout provided near 100% detection efficiency and exquisite specificity for CTCs due to scaling factors and the nonoptimal electrical properties of potential interferences (erythrocytes or leukocytes). The simplicity in manufacturing the device and its ease of operation make it attractive for clinical applications requiring one-time use operation.

Titer plate formatted continuous flow thermal reactors for high throughput applications: fabrication and testing

A high throughput, multi-well (96) polymerase chain reaction (PCR) platform, based on a continuous flow (CF) mode of operation, was developed. Each CFPCR device was confined to a footprint of 8 × 8 mm2, matching the footprint of a well on a standard micro-titer plate. While several CFPCR devices have been demonstrated, this is the first example of a high-throughput multi-well continuous flow thermal reactor configuration. Verification of the feasibility of the multi-well CFPCR device was carried out at each stage of development from manufacturing to demonstrating sample amplification. The multi-well CFPCR devices were fabricated by micro-replication in polymers, polycarbonate to accommodate the peak temperatures during thermal cycling in this case, using double-sided hot embossing. One side of the substrate contained the thermal reactors and the opposite side was patterned with structures to enhance thermal isolation of the closely packed constant temperature zones. A 99 bp target from a λ-DNA template was successfully amplified in a prototype multi-well CFPCR device with a total reaction time as low as ~5 min at a flow velocity of 3 mm s−1 (15.3 s cycle−1) and a relatively low amplification efficiency compared to a bench-top thermal cycler for a 20-cycle device; reducing the flow velocity to 1 mm s−1 (46.2 s cycle−1) gave a seven-fold improvement in amplification efficiency. Amplification efficiencies increased at all flow velocities for 25-cycle devices with the same configuration.

Non-intrusive measurements of bubble size and velocity

A non-intrusive measuring technique based on video-imaging has been developed for the measurement of bubble size, velocity and frequency. Measurements carried out with this method have been compared to those obtained by an optimized phase-Doppler system in standard configuration, for a wide range of bubble sizes produced from single injectors in a quiescent environment. The two measuring techniques have yielded velocities and frequencies that are in very good agreement while the size of spherical bubbles was consistently measured by both methods. The phase-Doppler system was also used to size oblate-spheroidal bubbles moving with their equatorial plane parallel to the scattering plane, yielding measurements reasonably close to the average radius of curvature of the bubbles in the neighborhood of the equatorial plane, as calculated from the video-imaging data. Both methods were used for detailed velocity measurements of the bubble-stream in the neighborhood of the injector tip. The observed bubble-velocity variation with the distance from the injector tip does not always display the usual increasing trend leading into the terminal velocity. When injection conditions are near the transition from discrete to jet injection mode and the bubbles are small, the latter decelerate into a terminal velocity due to direct interaction of successive bubbles at the injector tip. The measured terminal velocities of bubble-chains for a variety of bubble sizes and injection frequencies, are successfully predicted by using a far-field wake approximation to account for the drafting effect which is responsible for bubble-chain velocities higher than those of single bubbles.

Surface Modification of Droplet Polymeric Microfluidic Devices for the Stable and Continuous Generation of Aqueous Droplets

Droplet microfluidics performed in poly(methyl methacrylate) (PMMA) microfluidic devices resulted in significant wall wetting by water droplets formed in a liquid-liquid segmented flow when using a hydrophobic carrier fluid such as perfluorotripropylamine (FC-3283). This wall wetting led to water droplets with nonuniform sizes that were often trapped on the wall surfaces, leading to unstable and poorly controlled liquid-liquid segmented flow. To circumvent this problem, we developed a two-step procedure to hydrophobically modify the surfaces of PMMA and other thermoplastic materials commonly used to make microfluidic devices. The surface-modification route involved the introduction of hydroxyl groups by oxygen plasma treatment of the polymer surface followed by a solution-phase reaction with heptadecafluoro-1,1,2,2-tetrahydrodecyl trichlorosilane dissolved in fluorocarbon solvent FC-3283. This procedure was found to be useful for the modification of PMMA and other thermoplastic surfaces, including polycyclic olefin copolymer (COC) and polycarbonate (PC). Angle-resolved X-ray photoelectron spectroscopy indicated that the fluorination of these polymers took place with high surface selectivity. This procedure was used to modify the surface of a PMMA droplet microfluidic device (DMFD) and was shown to be useful in reducing the wetting problem during the generation of aqueous droplets in a perfluorotripropylamine (FC-3283) carrier fluid and could generate stable segmented flows for hours of operation. In the case of PMMA DMFD, oxygen plasma treatment was carried out after the PMMA cover plate was thermally fusion bonded to the PMMA microfluidic chip. Because the appended chemistry to the channel wall created a hydrophobic surface, it will accommodate the use of other carrier fluids that are hydrophobic as well, such as hexadecane or mineral oils.

Polymer-based microfluidic devices for biomedical applications

Two types of Microfluidic bioanalytical systems were designed and fabricated in polymer substrates using the LIGA process. A continuous flow polymerase chain reaction (CFPCR) Microfluidic device was fabricated in polycarbonate (PC), which utilized isothermal zone and shuttling the sample through each zone to achieve amplification. A 20-cycle PCR amplification of a fragment of a plasmid DNA template was achieved in 5.3 min. The results were comparable to those obtained in commercial laboratory-scale PCR system. The second system consisted of a microchip contating a low-density array assembled into the Microfluidic channel, which was hot-embossed in poly(methyl methacrylate) (PMMA). The detection of low-abundant mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancer was successfully performed. The array accessed microfluidics in order to enhance the kinetic associated with hybridization.

Detailed mass transfer distribution in a ribbed coolant passage with a 180° bend

Abstract Detailed, three-dimensional mass (heat) transfer distributions along four active walls of a square duct containing a sharp 180° bend are presented. The duct simulates two passes of an internal coolant channel in a gas turbine engine with inactive (insulated) ribs mounted on two opposite walls. Mass (heat) transfer measurements, taken using the naphthalene sublimation technique, are presented for Reynolds numbers between 5000 and 40000, for a rib-height-to-hydraulic-diameter ratio ( e / D h ) of 0.1 and rib-pitch-to-rib-height ratios ( P / e ) of 10.5 and 21. Ribbed wall measurements show periodically developed mass transfer after three hydraulic diameters from the entrance, which agrees well with previous studies. Mass transfer distribution on ribbed walls has more span-wise uniformity than the smooth side-walls that experience high mass transfer rates close to the rib ends and near the corners downstream of each rib. The effect of the bend is clearly visible in the ribbed duct following the bend. The observed local Sherwood number distributions are explained on the basis of the secondary flow developed within the bend and possible separation from the inner wall after the bend.

Heat Transfer in 1:4 Rectangular Passages With Rotation

The paper presents an experimental study of heat/mass transfer coefficient in 1:4 rectangular channel with smooth or ribbed walls for Reynolds number in the range of 5000-40,000 and rotation numbers in the range of 0-0.12. Such passages are encountered close to the mid-chord sections of the turbine blade. Normal ribs (e/D h =0.3125 and P/e =8) are placed on the leading and the trailing sides only. The experiments are conducted in a rotating two-pass coolant channel facility using the naphthalene sublimation technique. For purposes of comparison, selected measurements are also performed in a 1:1 cross section. The local mass-transfer data in the fully developed region is averaged to study the effect of the Reynolds and the rotation numbers. The spanwise mass transfer distributions in the smooth and the ribbed cases are also examined.

Passive micro-assembly of modular, hot embossed, polymer microfluidic devices using exact constraint design

Low-cost microfluidic platforms have the potential to change accepted practices in many fields, including biology and medicine, in the near future. Micro-assembly of molded polymer microfluidic devices is one approach to cost-effective mass production of modular, microfluidic instruments. Polymer, passive alignment structures were used to precisely assemble molded polymer components to prevent infinitesimal motions and minimize the misalignment between assembled components and devices. The motion and constraint of the assemblies were analyzed using screw theory to identify combinations of passive alignment structures that would provide exact constraint of all degrees of freedom of the two mating parts without over-constraint. One option identified by kinematic analysis was a set of three v-groove and hemisphere-tipped pin joints, which are well known from precision engineering and suitable for microfabrication. To validate the passive alignment scheme, brass mold inserts containing alignment structures were micro-milled and used to hot emboss components in polycarbonate (PC). Dimensional and location variations of prototype alignment structures were measured to quantify the difference between the as-designed and actual dimensions and the locations of the alignment structures. The dimensional variation was 0.2–3% less than the designed dimensions and the location variation was 0.7% less. The alignment accuracy of an assembly was characterized by measuring the mismatch and vertical variation between molded alignment standards embossed on each pair of mating plates. With molded, polymer alignment structures the mean mismatch and mean vertical variation were as low as 13 ± 3 µm in the lateral plane along the x- and y-axes and −6 ± 15 µm with respect to the nominal value of 107 µm. This micro-assembly technology is applicable to the integration of all microsystems including the interconnection of microfluidic devices, the assembly of hybrid microsystems and the parallel assembly of microdevices.

Modeling and validation of a molded polycarbonate continuous-flow polymerase chain reaction devic

A continuous flow polymerase chain reaction (CFPCR) system was designed, fabricated from molded polycarbonate, and tested. Finite element modeling was used to simulate the thermal and Microfluidic response of the system. The mold insert for the initial prototypes was fabricated using the X-ray LIGA microfabrication process and device components produced by hot embossing polycarbonate. Commercial thin film heaters under PID control were used to supply the necessary heat flux to maintain the steady-state temperatures in the PCR. The simulated transient temperature response at start up was compared to the experimental response. The simulated steady state temperature profile along the channel generated by the finite element analysis was compared to the experimental temperature profile displayed by liquid crystals. Experimental and simulated results were within 5% of each other, validating the thermal design of the CFPCR device.

Heat Transfer in a Two-Pass Internally Ribbed Turbine Blade Coolant Channel With Cylindrical Vortex Generators

The effect of vortex generators on the mass (heat) transferfrom the ribbed passage of a two-pass turbine blade coolant channel is investigated with the intent of optimizing the vortex generator geometry so that significant enhancements in mass/heat transfer can be achieved. In the experimental configuration considered, ribs are mounted on two opposite walls; all four walls along each pass are active and have mass transfer from their surfaces but the ribs are nonparticipating. Mass transfer measurements, in the form of Sherwood number ratios, are made along the centerline and in selected interrib modules. Results are presented for Reynolds number in the range of 5000 to 40, 000, pitch to rib height ratios of 10.5 and 21, and vortex generator-rib spacing to rib height ratios of 0.55 and 1.5. Centerline and spanwise-averaged Sherwood number ratios are presented along with contours of the Sherwood number ratios. Results indicate that the vortex generators lead to substantial increases in the local mass transfer rates, particularly along the side walls, and modest increases in the average mass transfer rates. The vortex generators have the effect of making the interrib profiles along the ribbed walls more uniform. Along the side walls, vortices that characterize the vortex generator wake are associated with significant mass transfer enhancements. The wake effects and the levels of enhancement decrease somewhat with increasing Reynolds number and decreasing pitch.