Node-Pore Sensing Enables Label-Free Surface-Marker Profiling of Single Cells

Abstract

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.

High accuracy particle analysis using sheathless microfluidic impedance cytometry

“…However, this causes errors in the measurement signals when particles flow near the electrodes 1 . Using multiple pairs of electrodes, the SNR can be increased 26,27 , and the interaction with immobilised surface antigens can be studied 28 , however the vari- ation in signal with particle position still remains an issue that limits the accuracy of impedance cytometry. In this work, we describe a simple approach to compensate for the variation in signal caused by the random position of a particle.…”

Multiparametric Biomechanical and Biochemical Phenotypic Profiling of Single Cancer Cells Using an Elasticity Microcytometer

“…Future development of the elasticity microcytometer platform could incorporate functional modules for quantitative measurements of single‐cell viscoelasticity and dynamic friction of antigen–antibody bindings . Generating a linear array of confining channels with each channel arranged in series and selectively functionalized with different monoclonal antibodies would further allow simultaneous quantification of expression of multiple cell surface proteins for the same single cells . This future exploration and improvement of the elasticity microcytometer platform will be critical for fulfilling its promise for deep phenotyping of live cells at the single‐cell level in the era of precision medicine.…”

Developments in label‐free microfluidic methods for single‐cell analysis and sorting

“…These devices have been shown to be a robust platform for counting specific subpopulations in blood-from circulating tumor cells to HIV particles (Becker et al, 1995;Watkins, Hassan, & Damhorst, 2013). (Balakrishnan et al, 2015) Three-dimensional tomography…”

“…Each section is functionalized with an antibody, and cells expressing the corresponding surface marker traverse that section more slowly. Adapted with permission from Balakrishnan et al (2015). Further permissions related to the material excerpted should be directed to the American Chemical Society.…”

“…Zhao et al (2014) As recent advances have demonstrated, RPS can also be used to probe other cellular properties beyond size. Balakrishnan et al used a novel method, node-pore sensing (NPS), to screen for multiple cell surface markers ( Figure 2c) (Balakrishnan et al, 2015). The sensing channel was coated with antibodies, and cells expressing surface markers that could specifically interact with these antibodies traversed the channel more slowly.…”