SMILS 2024

Abstract

In this talk, I will describe a novel single-molecule method where we engineer precise spatial and temporal control into the single-molecule experiment. We use a glass nanopore mounted on a 3D nanopositioner to spatially select molecules, deterministically tethered on a glass surface, for controlled translocations. By controlling the distance between the nanopore and the glass surface, we can actively select the region of interest on the molecule and scan it a controlled number of times and at a controlled velocity. Decreasing the velocity and averaging thousands of consecutive readings of the same molecule increases the signal-to-noise ratio (SNR) by two orders of magnitude compared to free translocations. We applied our method to various DNA constructs, achieving down to single nucleotide gap resolution. The spatial multiplexing combined with the sub-nanometer resolution could be used in conjunction with micro-array technologies to enable the screening of DNA, improve point-of-care devices, or enable high-density, addressable DNA data storage.

In the second part of the talk I will introduce two novel types of nanofluidic platforms. The geometry of the first nanofluidic platform combines the benefits of reduced sensing regions typically seen in 2D material nanopores with the asymmetric geometry of capillaries, resulting in ionic selectivity, stability, and scalability. The proposed nature-inspired growing method provides a flexible nanopore platform for various nanofluidic research applications, such as biosensing, energy science, and filtration technologies.

The second nanofluidic platform with a large entrance asymmetry is designed for in-memory processing, which can be mass-produced. Our fabrication process is scalable while the device operates at the second timescale with a conductance ratio in the range 10-60. In-operando optical microscopy unveils the origin of memory, arising from the reversible formation of liquid blisters modulating the device conductance. The combination of features of these mechano-ionic memristive switches permits assembling logic circuits composed of two interactive devices and an ohmic resistor. These results open the way to design multi-component ionic machinery, such as nanofluidic neural networks, and implementing brain-inspired ionic computations

1. Leitao, S.M., Navikas, V., Miljkovic, H., Drake, B., Marion, S., Pistoletti Blanchet, G., Chen, K., Mayer, S.F., Keyser, U.F., Kuhn, A. and Fantner, G.E., Radenovic A.  2023. " Spatially multiplexed single-molecule translocations through a nanopore at controlled speeds. " Nature Nanotechnology, pp.1-7.

2. Chernev, Andrey, Yunfei Teng, Mukeshchand Thakur, Victor Boureau, Lucie Navratilova, Nianduo Cai, Tzu‐Heng Chen, Liping Wen, Vasily Artemov, and Aleksandra Radenovic. 2023 "Nature‐Inspired Stalactite Nanopores for Biosensing and Energy Harvesting." Advanced Materials 2302827.

3. Emmerich, Theo, Yunfei Teng, Nathan Ronceray, Edoardo Lopriore, Riccardo Chiesa, Andrey Chernev, Vasily Artemov, Massimiliano Di Ventra, Andras Kis, and Aleksandra Radenovic. 2024 "Nanofluidic logic with mechano–ionic memristive switches" Nature electronics doi.org/10.1038/s41928-024-01137-9

Optical Genome Mapping: A toolbox for single-molecule epigenetic analysis of DNA in nanochannels

Abstract:

DNA sequencing (NGS) is revolutionizing all fields of biological research. Still, it fails to extract the full range of information associated with genetic material and cannot resolve variations between genomes. The information content of the genome extends beyond the base sequence in the form of chemical modifications such as DNA methylation or chromosomal association with DNA-binding proteins (chromatin). For the last decade, our lab has been developing tools for genomic analysis at the single-cell and single-molecule levels. We show how the physical extension of long DNA molecules in nanofluidic channels reveals this information in the form of a linear, optical “barcode”, like beads threaded on a string, where each bead represents a distinct type of observable.  I’ll present a biochemical and physical toolbox for mapping epigenetic modifications in the genome and demonstrate its application in clinical cancer research.

Abstract

Numerous childhood cancers involve few mutations but are diseases of altered DNA and protein interactions. Multicomponent protein complexes uniquely regulate gene expression and protein folding in numerous cancers. Furthermore, in highly motile metastatic cell subpopulations, protein interactions of cytoskeletal complexes govern cell structure. Thus, we require novel tools to probe the protein complexes in cancer cells at the single-cell level. First, I will describe the design of a microscale fractionation assay (SIFTER) to quantify levels of cytoskeletal protein complexes from single cancer cells. Under diverse stresses from drug treatment to heat shock, we quantify the dysregulation of monomeric and protein complex levels in hundreds of individual cells. Second, I will share the development of a novel single-cell encapsulation technique within bone tumor extracellular matrix-mimicking hydrogel microspheres that is compatible with SIFTER analysis. Third, I will describe the development of a multi-dimensional microscale protein separation method to separate native protein complexes approximately by mass and charge before an orthogonal electrophoretic separation of the denatured protein complex subunits. Altogether, our microscale systems open up new possibilities for characterizing protein complex functions in cancer cell subpopulations that will aid biomarker validation and therapeutic development.