Intracellular delivery of mRNA to human primary T cells with microfluidic vortex shedding.

Intracellular delivery of functional macromolecules, such as DNA and RNA, across the cell membrane and into the cytosol, is a critical process in both biology and medicine. Herein, we develop and use microfluidic chips containing post arrays to induce microfluidic vortex shedding, or µVS, for cell membrane poration that permits delivery of mRNA into primary human T lymphocytes. We demonstrate transfection with µVS by delivery of a 996-nucleotide mRNA construct encoding enhanced green fluorescent protein (EGFP) and assessed transfection efficiencies by quantifying levels of EGFP protein expression. We achieved high transfection efficiency (63.6 ± 3.44% EGFP + viable cells) with high cell viability (77.3 ± 0.58%) and recovery (88.7 ± 3.21%) in CD3 + T cells 19 hrs after µVS processing. Importantly, we show that processing cells via µVS does not negatively affect cell growth rates or alter cell states. We also demonstrate processing speeds of greater than 2.0 × 106 cells s-1 at volumes ranging from 0.1 to 1.5 milliliters. Altogether, these results highlight the use of µVS as a rapid and gentle delivery method with promising potential to engineer primary human cells for research and clinical applications.

Photoactivated bioconjugation between ortho-azidophenols and anilines: a facile approach to biomolecular photopatterning.

Methods for the surface patterning of small molecules and biomolecules can yield useful platforms for drug screening, synthetic biology applications, diagnostics, and the immobilization of live cells. However, new techniques are needed to achieve the ease, feature sizes, reliability, and patterning speed necessary for widespread adoption. Herein, we report an easily accessible and operationally simple photoinitiated reaction that can achieve patterned bioconjugation in a highly chemoselective manner. The reaction involves the photolysis of 2-azidophenols to generate iminoquinone intermediates that couple rapidly to aniline groups. We demonstrate the broad functional group compatibility of this reaction for the modification of proteins, polymers, oligonucleotides, peptides, and small molecules. As a specific application, the reaction was adapted for the photolithographic patterning of azidophenol DNA on aniline glass substrates. The presence of the DNA was confirmed by the ability of the surface to capture living cells bearing the sequence complement on their cell walls or cytoplasmic membranes. Compared to other light-based DNA patterning methods, this reaction offers higher speed and does not require the use of a photoresist or other blocking material.

Direct attachment of microbial organisms to material surfaces through sequence-specific DNA hybridization.

A new technique is reported for the attachment of synthetic DNA strands to the surfaces of microbial organisms. This gives algal, bacterial, and fungal cells the ability to bind to complementary strands extending from patterned surfaces that can be produced on platforms such as microfluidic devices. The ability of this method to establish complex 2- and 3-dimensional cocultures comprising multiple organism types is also presented.