High-selectivity cytology via lab-on-a-disc western blotting of individual cells.
Cytology of sparingly available cell samples from both clinical and experimental settings would benefit from high-selectivity protein tools. To minimize cell handling losses in sparse samples, we design a multi-stage assay using a lab-on-a-disc that integrates cell handling and subsequent single-cell western blotting (scWestern). As the two-layer microfluidic device rotates, the induced centrifugal force directs dissociated cells to dams, which in turn localize the cells over microwells. Cells then sediment into the microwells, where the cells are lysed and subjected to scWestern. Taking into account cell losses from loading, centrifugation, and lysis-buffer exchange, our lab-on-a-disc device handles cell samples with as few as 200 cells with 75% cell settling efficiencies. Over 70% of microwells contain single cells after the centrifugation. In addition to cell settling efficiency, cell-size filtration from a mixed population of two cell lines is also realized by tuning the cell time-of-flight during centrifugation (58.4% settling efficiency with 6.4% impurity). Following the upstream cell handling, scWestern analysis detects four proteins (GFP, β-TUB, GAPDH, and STAT3) in a glioblastoma cell line. By integrating the lab-on-a-disc cell preparation and scWestern analysis, our platform measures proteins from sparse cell samples at single-cell resolution.
Department of Bioengineering, University of California Berkeley, Berkeley, California 94720, USA. aeh@berkeley.edu and University of California, Berkeley - UCSF Graduate Program in Bioengineering, Berkeley, CA 94720, USA.
John J
0000-0002-4562-3872
JJ
Kim
Department of Bioengineering, University of California Berkeley, Berkeley, California 94720, USA. aeh@berkeley.edu.
Elly
E
Sinkala
Department of Bioengineering, University of California Berkeley, Berkeley, California 94720, USA. aeh@berkeley.edu and University of California, Berkeley - UCSF Graduate Program in Bioengineering, Berkeley, CA 94720, USA.
Amy E
0000-0002-6906-2985
AE
Herr
D015153:Blotting, Western / Q000295:instrumentation*; D045744:Cell Line, Tumor; D002498:Centrifugation / Q000295:instrumentation; D004867:Equipment Design; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods*; D059010:Single-Cell Analysis / Q000295:instrumentation*