Single-Cell Digital Microfluidic Mass Spectrometry Platform for Efficient and Multiplex Genotyping of Circulating Tumor Cells.

Gene mutation profiling of heterogeneous circulating tumor cells (CTCs) offers comprehensive and real-time molecular information of tumors for targeted therapy guidance, but the lack of efficient and multiplex genotyping techniques for single-CTC analysis greatly hinders its development and clinical application. This paper reports a single-CTC mass spectrometry analysis method for efficient and multiplex mutation profiling based on digital microfluidics. Digital microfluidics affords integrated single-CTC manipulation, from single-CTC isolation to high-performance whole genome amplification, via nanoliter droplet-based wettability trapping and hydrodynamic adjustment of cell distribution. Coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, multiplex mutation information of individual CTCs can be efficiently and accurately identified by the inherent mass differences of different DNA sequences. This platform achieves Kirsten rat sarcoma viral oncogene mutation profiling of heterogeneous CTCs at the single-cell level from cancer patient samples, offering new avenues for genotype profiling of single CTCs and cancer therapy guidance.

Sensitive, Rapid, and Automated Detection of DNA Methylation Based on Digital Microfluidics.

Biomarkers based on DNA methylation have attracted wide attention in biomedical research due to their potential clinical value. Therefore, a sensitive and accurate method for DNA methylation detection is highly desirable for the discovery and diagnostics of human diseases, especially cancers. Here, an integrated, low-cost, and portable point-of-care (POC) device is presented to analyze DNA methylation, which integrates the process of pyrosequencing in a digital microfluidic chip. Without additional equipment and complicated operation, droplets are manipulated by patterned electrodes with individually programmed control. The system exhibited an excellent sensitivity with a limit of detection (LOD) of 10 pg and a comparable checkout down to 5% methylation level within 30 min, which offered a potential substitute for the detection of DNA methylation. With the advantages of portability, ease of use, high accuracy, and low cost, the POC platform shows great potential for the analysis of tumor-specific circulating DNA.

Digital-WGS: Automated, highly efficient whole-genome sequencing of single cells by digital microfluidics.

Single-cell whole-genome sequencing (WGS) is critical for characterizing dynamic intercellular changes in DNA. Current sample preparation technologies for single-cell WGS are complex, expensive, and suffer from high amplification bias and errors. Here, we describe Digital-WGS, a sample preparation platform that streamlines high-performance single-cell WGS with automatic processing based on digital microfluidics. Using the method, we provide high single-cell capture efficiency for any amount and types of cells by a wetted hydrodynamic structure. The digital control of droplets in a closed hydrophobic interface enables the complete removal of exogenous DNA, sufficient cell lysis, and lossless amplicon recovery, achieving the low coefficient of variation and high coverage at multiple scales. The single-cell genomic variations profiling performs the excellent detection of copy number variants with the smallest bin of 150 kb and single-nucleotide variants with allele dropout rate of 5.2%, holding great promise for broader applications of single-cell genomics.

Rapid, real-time chemiluminescent detection of DNA mutation based on digital microfluidics and pyrosequencing.

To explore genome mutation meaningfully, it is in urgent need to develop an automated and inexpensive platform for DNA mutation analysis. Digital microfluidics is a powerful platform for a broad range of applications due to the advantages of high automatization and low reagent consumption. Pyrosequencing enables DNA sequencing based on non-electrophoresis bioluminescence, which is suitable for rapid and sensitive analysis of short sequences. Herein, we describe a palmtop sequencing platform for automatic, real-time and portable analysis of DNA mutations, which is based on the pyrosequencing principle and implemented by digital microfluidics. The portable system can sequence a DNA template with up to 53 bp with 100% accuracy within 2 h. Mutation in the KRAS gene can be detected within 30 min with a LOD as low as 5% mutant level. Portable and accurate gender identification was further demonstrated by sequencing a short amelogenin fragment. With the advantages of portability, ease of use, high accuracy, and low cost, the palmtop sequencing platform shows great potential for portable genetic testing in a variety of circumstances.