Abstract
Arrays of individual molecules can combine the advantages of microarrays and single-molecule studies. They miniaturize assays to reduce sample and reagent consumption and increase throughput, and additionally uncover static and dynamic heterogeneity usually masked in molecular ensembles. However, realizing single-DNA arrays must tackle the challenge of capturing structurally highly dynamic strands onto defined substrate positions. Here, we create single-molecule arrays by electrostatically adhering single-stranded DNA of gene-like length onto positively charged carbon nanoislands. The nanosites are so small that only one molecule can bind per island. Undesired adsorption of DNA to the surrounding non-target areas is prevented via a surface-passivating film. Of further relevance, the DNA arrays are of tunable dimensions, and fabricated on optically transparent substrates that enable singe-molecule detection with fluorescence microscopy. The arrays are hence compatible with a wide range of bioanalytical, biophysical, and cell biological studies where individual DNA strands are either examined in isolation, or interact with other molecules or cells.
Highlights
Arrays of individual molecules can combine the advantages of microarrays and single-molecule studies
The nanoislands are written onto an optically transparent substrate which is covered with a surface passivating poly(ethylene glycol) (PEG) film (Fig. 1, green area)
To generate nanoislands via e-beam writing (Fig. 1, step 1), we used glass slides coated with a 17 nm-thin layer of indium tin oxide (ITO)
Summary
Arrays of individual molecules can combine the advantages of microarrays and single-molecule studies They miniaturize assays to reduce sample and reagent consumption and increase throughput, and uncover static and dynamic heterogeneity usually masked in molecular ensembles. Binding individual strands in 1:1 stoichiometry has not yet been achieved due to the structurally dynamic nature of DNA strands and the resulting geometric mismatch to the defined and static nanosites. In this regard, flexible single-stranded DNA is more difficult than compacted DNA particles[2,35,36] even though the former are of greater relevance in many biomolecular assays. The generic fabrication route can vary the diameter of the nanoislands and is designed to be compatible with other DNA strands of different length
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