Precision cancer monitoring using a novel, fully integrated, microfluidic array partitioning digital PCR platform.

A novel digital PCR (dPCR) platform combining off-the-shelf reagents, a micro-molded plastic microfluidic consumable with a fully integrated single dPCR instrument was developed to address the needs for routine clinical diagnostics. This new platform offers a simplified workflow that enables: rapid time-to-answer; low potential for cross contamination; minimal sample waste; all within a single integrated instrument. Here we showcase the capability of this fully integrated platform to detect and quantify non-small cell lung carcinoma (NSCLC) rare genetic mutants (EGFR T790M) with precision cell-free DNA (cfDNA) standards. Next, we validated the platform with an established chronic myeloid leukemia (CML) fusion gene (BCR-ABL1) assay down to 0.01% mutant allele frequency to highlight the platform's utility for precision cancer monitoring. Thirdly, using a juvenile myelomonocytic leukemia (JMML) patient-specific assay we demonstrate the ability to precisely track an individual cancer patient's response to therapy and show the patient's achievement of complete molecular remission. These three applications highlight the flexibility and utility of this novel fully integrated dPCR platform that has the potential to transform personalized medicine for cancer recurrence monitoring.

Hydrodynamic resettability for a microfluidic particulate-based arraying system.

Precision hydrodynamic controls of microparticles (e.g., microbeads and cells) are critical to diverse lab-on-a-chip applications. Microfluidic particulate-based arraying techniques are widely used; however, achieving full microarray resettability without sacrificing trapping performance has remained a significant challenge. Here we present a single-layer hydrodynamic methodology for releasing high numbers of microparticles after a microfluidic arraying process. Experiments with suspended streptavidin-coated polystyrene microbeads (15 µm in diameter) revealed resetting efficiencies of 100%, with trapping and loading efficiencies of 99% and 99.8%, respectively. Experiments with suspended endothelial cells (13-17 µm in diameter) revealed trapping efficiencies of 65% and 93% corresponding to arraying of one cell or at least one cell per trap, respectively, with loading efficiencies of 78%. Full cell-based resettability was also observed, with the caveat that reagents that promote cellular detachment from the substrate were required. The presented resettable microarray could be readily integrated into bead-based or cell-based microfluidic platforms to enable: (i) the retrieval of high numbers of microparticles (e.g., for subsequent analyses and/or use in additional experiments), and (ii) microarray reusability.

A self-loading microfluidic device for determining the minimum inhibitory concentration of antibiotics.

This article describes a portable microfluidic technology for determining the minimum inhibitory concentration (MIC) of antibiotics against bacteria. The microfluidic platform consists of a set of chambers molded in poly(dimethylsiloxane) (PDMS) that are preloaded with antibiotic, dried, and reversibly sealed to a second layer of PDMS containing channels that connect the chambers. The assembled device is degassed via vacuum prior to its use, and the absorption of gas by PDMS provides the mechanism for actuating and metering the flow of fluid in the microfluidic channels and chambers. During the operation of the device, degas driven flow introduces a suspension of bacterial cells, dissolves the antibiotic, and isolates cells in individual chambers without cross contamination. The growth of bacteria in the chambers in the presence of a pH indicator produces a colorimetric change that can be detected visually using ambient light. Using this device we measured the MIC of vancomycin, tetracycline, and kanamycin against Enterococcus faecalis 1131, Proteus mirabilis HI4320, Klebsiella pneumoniae, and Escherichia coli MG1655 and report values that are comparable to standard liquid broth dilution measurements. The device provides a simple method for MIC determination of individual antibiotics against human pathogens that will have applications for clinical and point-of-care medicine. Importantly, this device is designed around simplicity: it requires a single pipetting step to introduce the sample, no additional components or external equipment for its operation, and provides a straightforward visual measurement of cell growth. As the device introduces a novel approach for filling and isolating dead-end microfluidic chambers that does not require valves and actuators, this technology should find applications in other portable assays and devices.

A computer-based, image-analysis method to quantify HIV-1 infection in a single-cycle infectious center assay.

Rapid and precise quantitation of the infectivity of HIV is important for molecular virologic studies as well as for measuring the activities of antiviral drugs and neutralizing antibodies. In the present study, an indicator cell line and image-analysis software were used to quantify HIV infectivity. Cells of the P4R5 line, which express the receptors for HIV infection as well as beta-galactosidase under the control of the HIV-1 long terminal repeat, were infected either with CXCR4- or CCR5-using viruses, including primary isolates, then stained 2 days later with X-gal to turn infected cells blue. Digital images of monolayers of the infected cells were captured using a high resolution CCD video camera and a macro video zoom lens. A software program was developed to process the images and to count the blue-stained foci of infection. The assay was applied to assess the infectivity of site-directed viral mutants, and to measure the activity of antiviral drugs and neutralizing antibody. The results indicate that the described method allows for the rapid quantitation of infected cells over a wide range of viral inocula with reproducibility, accuracy and relatively low cost.