Self-digitization chip for quantitative detection of human papillomavirus gene using digital LAMP.

Digital nucleic acid amplification and detection methods provide excellent sensitivity and specificity and allow absolute quantification of target nucleic acids. Isothermal methods such as digital loop-mediated isothermal amplification (digital LAMP) have potential for use in rapid disease diagnosis in low-resource settings due to their speed and lack of thermal cycling. We previously developed a self-digitization (SD) chip, a simple microfluidics device that automatically digitizes a sample into an array of nanoliter wells, for use in digital LAMP. In this work, we improve the SD chip design to increase sample loading efficiency, speed, and completeness, and test a range of well volumes and numbers. We demonstrate the diagnostic capability of this platform by applying it to quantifying human papillomavirus 18 gene.

SD-chip enabled quantitative detection of HIV RNA using digital nucleic acid sequence-based amplification (dNASBA).

Quantitative detection of RNA is important in molecular biology and clinical diagnostics. Nucleic acid sequence-based amplification (NASBA), a single-step method to amplify single-stranded RNA, is attractive for use in point-of-care (POC) diagnostics because it is an isothermal technique that is as sensitive as RT-PCR with a shorter reaction time. However, NASBA is limited in its ability to provide accurate quantitative information, such as viral load or RNA copy number. Here we test a digital format of NASBA (dNASBA) using a self-digitization (SD) chip platform, and apply it to quantifying HIV-1 RNA. We demonstrate that dNASBA is more sensitive and accurate than the real-time quantitative NASBA, and can be used to quantify HIV-1 RNA in plasma samples. Digital NASBA is thus a promising POC diagnostics tool for use in resource-limited settings.

Self-digitization chip for single-cell genotyping of cancer-related mutations.

Cancer is a heterogeneous disease, and patient-level genetic assessments can guide therapy choice and impact prognosis. However, little is known about the impact of genetic variability within a tumor, intratumoral heterogeneity (ITH), on disease progression or outcome. Current approaches using bulk tumor specimens can suggest the presence of ITH, but only single-cell genetic methods have the resolution to describe the underlying clonal structures themselves. Current techniques tend to be labor and resource intensive and challenging to characterize with respect to sources of biological and technical variability. We have developed a platform using a microfluidic self-digitization chip to partition cells in stationary volumes for cell imaging and allele-specific PCR. Genotyping data from only confirmed single-cell volumes is obtained and subject to a variety of relevant quality control assessments such as allele dropout, false positive, and false negative rates. We demonstrate single-cell genotyping of the NPM1 type A mutation, an important prognostic indicator in acute myeloid leukemia, on single cells of the cell line OCI-AML3, describing a more complex zygosity distribution than would be predicted via bulk analysis.

Optical painting and fluorescence activated sorting of single adherent cells labelled with photoswitchable Pdots.

The efficient selection and isolation of individual cells of interest from a mixed population is desired in many biomedical and clinical applications. Here we show the concept of using photoswitchable semiconducting polymer dots (Pdots) as an optical 'painting' tool, which enables the selection of certain adherent cells based on their fluorescence, and their spatial and morphological features, under a microscope. We first develop a Pdot that can switch between the bright (ON) and dark (OFF) states reversibly with a 150-fold contrast ratio on irradiation with ultraviolet or red light. With a focused 633-nm laser beam that acts as a 'paintbrush' and the photoswitchable Pdots as the 'paint', we select and 'paint' individual Pdot-labelled adherent cells by turning on their fluorescence, then proceed to sort and recover the optically marked cells (with 90% recovery and near 100% purity), followed by genetic analysis.

Self-digitization microfluidic chip for absolute quantification of mRNA in single cells.

Quantification of mRNA in single cells provides direct insight into how intercellular heterogeneity plays a role in disease progression and outcomes. Quantitative polymerase chain reaction (qPCR), the current gold standard for evaluating gene expression, is insufficient for providing absolute measurement of single-cell mRNA transcript abundance. Challenges include difficulties in handling small sample volumes and the high variability in measurements. Microfluidic digital PCR provides far better sensitivity for minute quantities of genetic material, but the typical format of this assay does not allow for counting of the absolute number of mRNA transcripts samples taken from single cells. Furthermore, a large fraction of the sample is often lost during sample handling in microfluidic digital PCR. Here, we report the absolute quantification of single-cell mRNA transcripts by digital, one-step reverse transcription PCR in a simple microfluidic array device called the self-digitization (SD) chip. By performing the reverse transcription step in digitized volumes, we find that the assay exhibits a linear signal across a wide range of total RNA concentrations and agrees well with standard curve qPCR. The SD chip is found to digitize a high percentage (86.7%) of the sample for single-cell experiments. Moreover, quantification of transferrin receptor mRNA in single cells agrees well with single-molecule fluorescence in situ hybridization experiments. The SD platform for absolute quantification of single-cell mRNA can be optimized for other genes and may be useful as an independent control method for the validation of mRNA quantification techniques.

Self-digitization of samples into a high-density microfluidic bottom-well array.

This paper describes a sample digitization method that generates tens of thousands of nanoliter-sized droplets in a high-density array in a matter of minutes. We show that the sample digitization depends on both the geometric design of the microfluidic device and the viscoelastic forces between the aqueous sample and a continuous oil phase. Our design avoids sample loss: Samples are split into tens of thousands of discrete volumes with close to 100% efficiency without the need for any expensive valving or pumping systems. We envision this technology will have broad applications that require simple sample digitization within minutes, such as digital polymerase chain reactions and single-cell studies.