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.

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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 size—AP-1060 and NALM-1—based 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.

The G protein-coupled GABA B receptors, constituted from GABA B1 and GABA B2 subunits, are important regulators of neuronal excitability by mediating long-lasting inhibition. One factor that determines receptor availability and thereby the strength of inhibition is regulated protein degradation. GABA B receptors are constitutively internalized from the plasma membrane and are either recycled to the cell surface or degraded in lysosomes. Lys-63-linked ubiquitination mediated by the E3 ligase Mind bomb-2 (MIB2) is the signal that sorts GABA B receptors to lysosomes. However, it is unknown how Lys-63-linked ubiquitination and thereby lysosomal degradation of the receptors is regulated. Here, we show that Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) promotes MIB2-mediated Lys-63-linked ubiquitination of GABA B receptors. We found that inhibition of CaMKII in cultured rat cortical neurons increased cell surface GABA B receptors, whereas overexpression of CaMKIIβ, but not CaMKIIα, decreased receptor levels. This effect was conveyed by Lys-63-linked ubiquitination of GABA B1 at multiple sites mediated by the E3 ligase MIB2. Inactivation of the CaMKII phosphorylation site on GABA B1 (Ser-867) strongly reduced Lys-63-linked ubiquitination of GABA B receptors and increased their cell surface expression, whereas the phosphomimetic mutant GABA B1 (S867D) exhibited strongly increased Lys-63-linked ubiquitination and reduced cell surface expression. Finally, triggering lysosomal degradation of GABA B receptors by sustained activation of glutamate receptors, a condition occurring in brain ischemia, was accompanied with a massive increase of GABA B1 (Ser-867) phosphorylation-dependent Lys-63-linked ubiquitination of GABA B receptors. These findings indicate that CaMKIIβ-dependent Lys-63-linked ubiquitination of GABA B1 at multiple sites controls sorting of GABA B receptors to lysosomes for degradation under physiological and pathological condition.