Assay miniaturization for the genetic analysis of individual cells enabled by single-cell printing and nanoliter liquid handling

Identification: Riba, Julian


Description

Assay miniaturization for the genetic analysis of individual cells enabled by single-cell printing and nanoliter liquid handling

Julian Riba1, Heiko Becker2, Peter Koltay1, Roland Zengerle1, Stefan Zimmermann1

1Laboratory for MEMS Applications, IMTEK - Department of Microsystems Engineering 2Department of Internal Medicine I, Medical Center

University Freiburg, Freiburg, Germany

Automated single-cell isolation and low volume liquid handling for reagent cost reduction are needed to mature single-cell assays into routine clinical analysis methods. Here, we combine inkjet-like single-cell printing (SCP) for the isolation and deposition of individual cells with non-contact dispensing for reagent dosage in the nanoliter range in order to carry out miniaturized single cell analysis assays

Using standard 384-well plates we successfully performed whole-genome amplification (WGA) on single cells in 10 µl, 5 µl, and 2.5 µl total reaction volumes, resulting in an up to 20-fold reduction of the reaction volume and reagent cost compared to the manufacturer’s protocol. To probe for potentially contaminating free-floating DNA, we used SCP to print empty droplets (n = 1, 3, and 10, respectively) from a suspension of Kasumi-1 cells into individual wells first and then subjected them to WGA. All empty droplets yielded no product in the subsequent PCR on repetitive LINE1 retrotransposons, while droplets generated by SCP containing a single Kasumi-1 cell (n= 33) or a single-cell from an AML patient sample (n=23) were positive.

To achieve the reduction of reaction volumes, a novel concept was used for liquid handling of the WGA reagents: The combination of a Biofluidix PipeJetTMdispenser with a syringe pump allows for aspiration of the reagents and subsequent dosage with droplet volumes down to 30 nl.

In conclusion, we show that the combination of SCP with automated nanoliter liquid handling allows for WGA of individual cells in significantly reduced reaction volumes resulting in up to 20-fold cost savings. Due to the flexibility of both, the SCP and the liquid handling system, the technology can be utilized for the automation and miniaturization of a variety of single-cell assays.

Credits

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