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.

Single molecule epigenetic analysis in a nanofluidic channel.

Epigenetic states are governed by DNA methylation and a host of modifications to histones bound with DNA. These states are essential for proper developmentally regulated gene expression and are perturbed in many diseases. There is great interest in identifying epigenetic mark placement genome wide and understanding how these marks vary among cell types, with changes in environment or according to health and disease status. Current epigenomic analyses employ bisulfite sequencing and chromatin immunoprecipitation, but query only one type of epigenetic mark at a time, DNA methylation, or histone modifications and often require substantial input material. To overcome these limitations, we established a method using nanofluidics and multicolor fluorescence microscopy to detect DNA and histones in individual chromatin fragments at about 10 Mbp/min. We demonstrated its utility for epigenetic analysis by identifying DNA methylation on individual molecules. This technique will provide the unprecedented opportunity for genome wide, simultaneous analysis of multiple epigenetic states on single molecules.

Nanoscale resolution, multicomponent biomolecular arrays generated by aligned printing with parylene peel-off.

We present "Print-and-Peel", a high-throughput method to generate multicomponent biomolecular arrays with sub-100 nm nanoscale feature width. An inkjet printer is first aligned to a parylene template containing nanoscale openings. After printing, the parylene is peeled off to reveal uniformly patterned nanoscale features, despite the imperfect morphologies of the original inkjet spots. We further patterned combinatorial nanoarrays by performing a second print-run superimposed over the first, thereby extending the multiplexing capability of the technique.

Parylene peel-off arrays to probe the role of cell-cell interactions in tumour angiogenesis.

Microenvironmental conditions impact tumour angiogenesis, but the role of cell-cell interactions in modulating the angiogenic capability of tumour cells is not well understood. We have microfabricated a peel-off cell-culture array (PeelArray) chip to spatiotemporally control interactions between tumour cells in a large array format and to analyse angiogenic factor secretion in response to these conditions. The PeelArray chip consists of a polyethylene glycol (PEG) treated glass coverslip coated with a parylene-C template that can be easily peeled off to selectively micropattern biomolecules and cells. We have designed the PeelArray chip to reproducibly deposit large uniform arrays of isolated single cells or isolated cell clusters on fibronectin features of defined surface areas. We have utilised this microfabricated culture system to study the secretion of angiogenic factors by tumour cells, in the presence or absence of cell-cell contact as controlled by micropatterning. Our results indicate that cell-cell interactions play a synergistic role in regulating the expression of angiogenic factors (i.e., vascular endothelial growth factor [VEGF] and interleukin-8 [IL-8]) in various cancer cell lines, independent of other more complex microenvironmental cues (e.g. hypoxia). Our PeelArray chip is a simple and adaptable micropatterning method that enables quantitative profiling of protein secretions and hence, a better understanding of the mechanisms by which cell-cell interactions regulate tumour cell behaviour and angiogenesis.

Prion protein detection using nanomechanical resonator arrays and secondary mass labeling.

Nanomechanical resonators have shown potential application for mass sensing and have been used to detect a variety of biomolecules. In this study, a dynamic resonance-based technique was used to detect prion proteins (PrP), which in conformationally altered forms are known to cause neurodegenerative diseases in animals as well as humans. Antibodies and nanoparticles were used as mass labels to increase the mass shift and thus amplify the frequency shift signal used in PrP detection. A sandwich assay was used to immobilize PrP between two monoclonal antibodies, one of which was conjugated to the resonator's surface while the other was either used alone or linked to the nanoparticles as a mass label. Without additional mass labeling, PrP was not detected at concentrations below 20 microg/mL. In the presence of secondary antibodies the analytical sensitivity was improved to 2 microg/mL. With the use of functionalized nanoparticles, the sensitivity improved an additional 3 orders of magnitude to 2 ng/mL.

Controlling microarray spot morphology with polymer liftoff arrays.

Biological arrays are hindered by the lack of uniformity in the deposition of biomaterials. Efforts aimed at improving this deposition have focused on altering the composition of the solution or the tool used to deposit the material. However, little attention has been paid to controlling material deposition by constraining the physical and chemical topography of the surface. Here we present the use of a hybrid hydrophilic/hydrophobic micropatterned surface to direct the deposition of spotted DNA on microarrays. These polymer "liftoff" arrays combine the hydrophobic surface properties of di-p-xylylene (Parylene) with photolithographically etched hydrophilic openings within the polymer. We show that the flow pattern of solutes on these substrates favors the concentration of dissolved material into the mesoscopic openings underlying the printed spot, resulting in significantly improved uniformity of deposition. Moreover, the micropatterned surface allows for increased replication of spotted materials. Finally, these polymer liftoff arrays display reduced array-to-array variation, improving the reproducibility of data acquisition. We envision that these novel substrates can be generalized to produce more uniform arrays of other patterned biomaterials.