Michael C. Murphy

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.

Fabrication and Preliminary Results for LiGA Fabricated Nickel Micro Gas Chromatograph Columns

High aspect ratio nickel microfluidic columns were fabricated using the LiGA technique. The 2-m-long 50-mum-wide high aspect ratio columns will be the separation component of a handheld gas chromatograph device for detecting semivolatile and volatile compounds. As a first step, 600-mum-deep electrodeposited nickel columns were fabricated. The serpentine columns were sealed and pressure-flow rate characteristics compared with the theoretical values. The response of the sealed columns was studied by running methane gas plugs through uncoated columns with a flame ionization detector at the exit. Negligible flow-induced dispersion was observed in the sealed metal columns. Unretained peak widths of ~15 ms were measured, and the experimental pressure and flow rate distributions matched those predicted by established analytical models within plusmn2.5%. Columns were coated with OV-1 stationary phase using static coating methods. A mixture of four hydrocarbons C6, C8, C10, and C12 was separated in a coated 50 mum by 600 mum by 0.5 m column in less than 2 s at 70 degC

Fully Integrated Thermoplastic Genosensor for the Highly Sensitive Detection and Identification of Multi‐Drug‐Resistant Tuberculosis

Infectious diseases are a major global health burden accounting for approximately 15 million deaths annually, many from drug resistant pathogenic agents, with a significant number of cases occurring in developing countries.[1–7] In particular, the resurgence of tuberculosis (TB) has been accompanied by the rapid spread of multi-drug resistance TB (MDR-TB) resulting from Mycobacterium tuberculosis (Mtb) strains that fail to respond to the first-line drugs, rifampin and isoniazid. Currently, <5% of ~0.5 million MDR-TB cases estimated globally are appropriately diagnosed and treated due in part to the long assay turnaround time associated with conventional culture-based drug susceptibility testing.[8]

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.

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.

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.

Next-Generation qPCR for the High- Throughput Measurement of Gene Expression in Multiple Leukocyte Subsets

Clinical studies of gene expression are increasingly using the whole blood, peripheral blood mononuclear cells, and leukocyte subsets involved in the innate and adaptive immune responses. However, the small amount of RNA available in the clinical setting is a limitation for commonly used methods such as quantitative polymerase chain reactions (qPCR) and microarrays. Our aim was to design 96 gene assays to simultaneously measure gene expression in the whole blood and seven leukocyte subsets using a new-generation qPCR method—high-throughput nanofluidic reverse transcription qPCR (HT RT-qPCR). The leukocyte subset purity was 94% to 98% for seven subsets and was less for the γδ T-cell receptor subset (80%). The HT RT-qPCR replicate sample measurements were highly reproducible (r = 0.997, p < 2.2 × 10−16), and the ΔΔCt values from HT RT-qPCR correlated significantly with those from qPCR. The control genes were differentially expressed across the eight leukocyte subsets in the control subjects (p = 1.3 × 10−5, analysis of variance). Two analytical methods, absolute and relative, gave concordant results and were significantly correlated (p = 1.9 × 10−9). HT RT-qPCR permits the rapid, reproducible, and quantitative measurement of multiple transcripts using minimal sample amounts. The protocol described yielded leukocyte subsets of high purity and identified two analytic methods for use.

A vertically-stacked, polymer, microfluidic point mutation analyzer: Rapid, high accuracy detection of low-abundance K-ras mutations

Recognition of point mutations in the K-ras gene can be used for the clinical management of several types of cancers. Unfortunately, several assay and hardware concerns must be addressed to allow users not well trained in performing molecular analyses the opportunity to undertake these measurements. To provide for a larger user base for these types of molecular assays, a vertically stacked microfluidic analyzer with a modular architecture and process automation was developed. The analyzer employs a primary polymerase chain reaction (PCR) coupled to an allele-specific ligase detection reaction (LDR). Each functional device, including continuous flow thermal reactors for the PCR and LDR, passive micromixers, and ExoSAP-IT purification, was designed and tested. Individual devices were fabricated in polycarbonate using hot embossing and were assembled using adhesive bonding for system assembly. The system produced LDR products from a DNA sample in approximately 1h, an 80% reduction in time compared with conventional benchtop instrumentation. Purifying the post-PCR products with the ExoSAP-IT enzyme led to optimized LDR performance, minimizing false-positive signals and producing reliable results. Mutant alleles in genomic DNA were quantified to the level of 0.25 ng of mutant DNA in 50 ng of wild-type DNA for a 25-μl sample, equivalent to DNA from 42 mutant cells.

Development of a diffuser/nozzle-type micropump based on magnetohydrodynamic (MHD) principle

This paper reports a research effort to microfabricate a nozzle-diffuser type of micropumps based on the magnetohydrodynamic (MHD) principle using LIGA technologies. The micropump is driven using the Lorentz force and can be used to deliver electrically conductive fluids. The major advantage of a MHD-based micropump is that it does not contain any moving parts. It may have potential applications in medicine delivery, biological and biomedical studies. Prototypes of MHD micropumps have been fabricated and tested. Significant bubble generation was observed due to electrolysis effect. These bubbles made the flow two-phase one and resulted in flow rate reduction. To overcome bubble generation, a new generation of MHD micropumps is currently under development. This new, diffuser/nozzle type of the MHD micropumps is based on the similar design as widely used in the diffuser/nozzle pumps with diaphragm.