Node-pore sensing enables label-free surface-marker profiling of single cells.

Flow cytometry is a ubiquitous, multiparametric method for characterizing cellular populations. However, this method can grow increasingly complex with the number of proteins that need to be screened simultaneously: spectral emission overlap of fluorophores and the subsequent need for compensation, lengthy sample preparation, and multiple control tests that need to be performed separately must all be considered. These factors lead to increased costs, and consequently, flow cytometry is performed in core facilities with a dedicated technician operating the instrument. Here, we describe a low-cost, label-free microfluidic method that can determine the phenotypic profiles of single cells. Our method employs Node-Pore Sensing to measure the transit times of cells as they interact with a series of different antibodies, each corresponding to a specific cell-surface antigen, that have been functionalized in a single microfluidic channel. We demonstrate the capabilities of our method not only by screening two acute promyelocytic leukemia human cells lines (NB4 and AP-1060) for myeloid antigens, CD13, CD14, CD15, and CD33, simultaneously, but also by distinguishing a mixture of cells of similar size­AP-1060 and NALM-1­based on surface markers CD13 and HLA-DR. Furthermore, we show that our method can screen complex subpopulations in clinical samples: we successfully identified the blast population in primary human bone marrow samples from patients with acute myeloid leukemia and screened these cells for CD13, CD34, and HLA-DR. We show that our label-free method is an affordable, highly sensitive, and user-friendly technology that has the potential to transform cellular screening at the benchside.

Node-pore sensing: a robust, high-dynamic range method for detecting biological species.

Resistive-pulse sensing (RPS), which is based on measuring the current pulse produced when a single particle transits a pore or channel, is an extremely versatile technique used to determine the size and concentration of cells and viruses and to detect single molecules. A major challenge to RPS is dynamic range: smaller particles in a heterogeneous sample can go undetected because of low signal-to-noise ratios (SNRs) and the fact that the pore size must be commensurate with that of the largest particles. Here, we describe a fundamentally different pore that provides an unprecedented dynamic detection range, from tens of nanometers to several microns in size, without the need for pre-sorting or filtration. Because of its unique geometry--nodes inserted along the channel--our pore produces distinct electronic signatures that overcome low SNRs. We demonstrate the power of our device by directly detecting and enumerating human immunodeficiency virus (HIV) in human plasma.

Sorting single satellite cells from individual myofibers reveals heterogeneity in cell-surface markers and myogenic capacity.

Traditional cell-screening techniques such as FACS and MACS are better suited for large numbers of cells isolated from bulk tissue and cannot easily screen stem or progenitor cells from minute populations found in their physiological niches. Furthermore, these techniques rely upon irreversible antibody binding, potentially altering cell properties, including gene expression and regenerative capacity. To address these challenges, we have developed a novel, label-free stem-cell analysis and sorting platform capable of quantifying cell-surface marker expression of single functional organ stem cells directly isolated from their micro-anatomical niche. Using our unique platform, we have discovered a remarkable heterogeneity in both the regenerative capacity and expression of CXCR4, ß1-integrin, Sca-1, M-cadherin, Syndecan-4, and Notch-1 in freshly isolated muscle stem (satellite) cells residing on different, single myofibers and have identified a small population of Sca-1(+)/Myf5(+) myogenic satellite cells. Our results demonstrate the utility of our single-cell platform for uncovering and functionally characterizing stem-cell heterogeneity in the organ microniche.