The main goal of this paper is to probe mechanical properties of living and dead bacteria via atomic force microscopy (AFM) indentation experimentations. Nevertheless, the prerequisite for bioAFM study is the adhesion of the biological sample on a surface. Although AFM has now been used in microbiology for 20 years, the immobilization of micro-organisms is still challenging. Immobilizing a single cell, without the need for chemical fixation has therefore constituted our second purpose. Highly ordered arrays of single living bacteria were generated over the millimeter scale by selective adsorption of bacteria onto micrometric chemical patterns. The chemically engineered template surfaces were prepared with a microcontact printing process, and different functionalizations of the patterns by incubation were investigated. Thanks to this original immobilization strategy, the Young moduli of the same cell were measured using force spectroscopy before and after heating (45 degrees C, 20 min). The cells with a damaged membrane (after heating) present a Young modulus twice as high as that of healthy bacteria.

Epigenetic modifications, such as DNA and histone methylation, are responsible for regulatory pathways that affect disease. Current epigenetic analyses use bisulfite conversion to identify DNA methylation and chromatin immunoprecipitation to collect molecules bearing a specific histone modification. In this work, we present a proof-of-principle demonstration for a new method using a nanofluidic device that combines real-time detection and automated sorting of individual molecules based on their epigenetic state. This device evaluates the fluorescence from labeled epigenetic modifications to actuate sorting. This technology has demonstrated up to 98% accuracy in molecule sorting and has achieved postsorting sample recovery on femtogram quantities of genetic material. We have applied it to sort methylated DNA molecules using simultaneous, multicolor fluorescence to identify methyl binding domain protein-1 (MBD1) bound to full-duplex DNA. The functionality enabled by this nanofluidic platform now provides a workflow for color-multiplexed detection, sorting, and recovery of single molecules toward subsequent DNA sequencing. I n chromatin, chemical modifications to histone proteins and DNA alter the status of the epigenome and influence gene regulation and normal development. Their aberrant placement has been linked to the onset of cancer (1, 2) and other diseases. Bisulfite conversion and immunoprecipitation (IP) have been used extensively to examine these modifications on locus-specific and genome-wide scales. These approaches have limitations in terms of material handling or multiplexed detection. Conventional chromatin immunoprecipitation (ChIP) requires an abundance of input material, often 10 3 -10 6 cells for genome-wide studies, to compensate for >99% material loss during processing (3, 4). This problem compounds for sequential re-ChIP reactions, limiting the study of multivalent modifications (4), which could provide a clear view of epigenetic coordination. Whereas DNA methylation analysis using bisulfite conversion can operate on picogram quantities of DNA (5-7), the conversion causes degradation of >90% of the input DNA. Methods that combine ChIP and bisulfite sequencing in a sequential process (8) have demonstrated progress in multiplexed epigenetic analysis. There continues to be active research in reducing the input material requirements and in automation of the processes (9-11) for epigenetic analysis. Furthermore, there is interest in additional capability for simultaneous detection of multiple epigenetic modifications in the same material.Miniaturized fluidic devices offer a compelling toolset for multiplexed detection and efficient sample handling in analytical and preparatory systems. Microfluidics have performed complicated workflows that include nanoliter sample handling (12) and incorporate electrodes (13-16) or valves (12, 17) for sophisticated processing. Nanofluidics have achieved attoliter-scale fluid volume confinement to isolate and quantify the attributes of individual molecules that can ...

Individual chromatin molecules contain valuable genetic and epigenetic information. To date, there have not been reliable techniques available for the controlled stretching and manipulation of individual chromatin fragments for high-resolution imaging and analysis of these molecules. We report the controlled stretching of single chromatin fragments extracted from two different cancerous cell types (M091 and HeLa) characterized through fluorescence microscopy and atomic force microscopy (AFM). Our method combines soft-lithography with molecular stretching to form ordered arrays of more than 250,000 individual chromatin fragments immobilized into a beads-on-a-string structure on a solid transparent support. Using fluorescence microscopy and AFM, we verified the presence of histone proteins after the stretching and transfer process.