Real-time analysis and selection of methylated DNA by fluorescence-activated single molecule sorting in a nanofluidic channel.

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.

Bio-Impedance Spectroscopy of Retained Cells Using a Micro-Perforated Sensing Membrane Filtrating Whole Blood Samples under High Flowrate.

Blood filtration using micro-fabricated devices is an interdisciplinary topic of research and innovation driven by clinical applications in cytapheresis, cardiovascular disease monitoring, or liquid biopsy. In this paper, we demonstrate that a micro-perforated membrane can be equipped with sensing microelectrodes for detecting, in situ and in real-time, the capture of cellular material during ex vivo filtration of whole blood under high flow rates. This work describes the fabrication process of the sift and detection microdevice. We demonstrate that reliable electrical signals can be measured in whole blood samples flowing inside a fluidic system at typical flow rates, as large as 11.5 mL/min, hence allowing for large-volume sample processing. The in situ monitoring of the electrical impedance of the microelectrodes is shown to characterize the accumulation of living circulating cells retained by the filtrating membrane, opening interesting applications for monitoring blood filtration processes.

Transfer-printing of single DNA molecule arrays on graphene for high-resolution electron imaging and analysis.

Graphene represents the ultimate substrate for high-resolution transmission electron microscopy, but the deposition of biological samples on this highly hydrophobic material has until now been a challenge. We present a reliable method for depositing ordered arrays of individual elongated DNA molecules on single-layer graphene substrates for high-resolution electron beam imaging and electron energy loss spectroscopy analysis. This method is a necessary step toward the observation of single elongated DNA molecules with single base spatial resolution to directly read genetic and epigenetic information.

Single DNA molecule patterning for high-throughput epigenetic mapping.

We present a method for profiling the 5-methyl cytosine distribution on single DNA molecules. Our method combines soft-lithography and molecular elongation to form ordered arrays estimated to contain more than 250 000 individual DNA molecules immobilized on a solid substrate. The methylation state of the DNA is detected and mapped by binding of fluorescently labeled methyl-CpG binding domain peptides to the elongated dsDNA molecules and imaging of their distribution. The stretched molecules are fixed in their extended configuration by adsorption onto the substrate so analysis can be performed with high spatial resolution and signal averaging. We further prove this technique allows imaging of DNA molecules with different methylation states.

Liquid Biopsy Based on Circulating Cancer-Associated Cells: Bridging the Gap from an Emerging Concept to a Mainstream Tool in Precision Medicine.

The concept of liquid biopsy and the isolation and analysis of circulating biomarkers from blood samples is proposed as a surrogate to solid biopsies and can have the potential to revolutionize the management of patients with cancer. The relevance of circulating tumor cells (CTCs) and the importance of the information they carry is acknowledged by the medical community. But what are the barriers to clinical adoption? This review draws a panorama of the biological implications of CTCs, their physical and biochemical properties, and the current technological bottlenecks for their analysis in relation with the medical needs. Keys and considerations to bridge the technological and clinical gaps that still need to be overcome to be able to introduce CTCs in clinical routine are finally synthesized.

Compact system for in situ laser Doppler velocimetry of blood flow.

This work describes the implementation of a compact system allowing measurement of blood flow velocity using laser Doppler velocimetry in situ. The compact setup uses an optical fiber acting as an emitter and receptor of the signal. The signal is then recovered by a photodiode and processed using a spectrum analyzer. The prototype was successfully tested to measure microbead suspension and whole blood flow velocities in a fluidic chip. Fibers with hemispherical lenses with three different radius of curvature were investigated. This simple yet precise setup would enable the insertion of the fiber via a medical catheter to monitor blood flow velocity in non superficial vessels where previous reported techniques cannot be implemented.

Controlled assembly of bacteria on chemical patterns using soft lithography.

Highly ordered arrays of single living bacteria were obtained by selective adsorption of bacteria onto chemical patterns with micrometric resolution. The chemically engineered template surfaces were prepared with the combination of microcontact printing process and a simple incubation technique. This methodology can be used for fundamental studies of bacterium's inner mechanisms and sub-cellular organization as well as for interfacing living bacteria with artificial microsystems.

Astigmatic multifocus microscopy enables deep 3D super-resolved imaging.

We have developed a 3D super-resolution microscopy method that enables deep imaging in cells. This technique relies on the effective combination of multifocus microscopy and astigmatic 3D single-molecule localization microscopy. We describe the optical system and the fabrication process of its key element, the multifocus grating. Then, two strategies for localizing emitters with our imaging method are presented and compared with a previously described deep 3D localization algorithm. Finally, we demonstrate the performance of the method by imaging the nuclear envelope of eukaryotic cells reaching a depth of field of ~4µm.

Ordered arrays of native chromatin molecules for high-resolution imaging and analysis.

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.

Novel approach for the assembly of highly efficient SERS substrates.

In this paper we present the properties of surface-enhanced Raman scattering (SERS) active substrates elaborated by a low-cost approach. Our methodology relying on capillary assembly and soft lithography allows us to generate periodic two-dimensional (2D) matrixes of 100 nm gold nanoparticle patterns in a very precise, cost-efficient, and large-scale manner. For this study, we assembled nanoparticle aggregates of different sizes (one to six particles) in order to determine the influence of the aggregation on the local electric field enhancement. We further demonstrate that this substrate is greatly efficient not only for SERS but also in metal-enhanced fluorescence (MEF) for local enhancement of conventional fluorescence.

Nanomechanical properties of dead or alive single-patterned bacteria.

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.