Single molecule techniques have a substantial influence on the developments in the nanobiotechnology field, such as super-resolution microscopy or single molecule DNA sequencing. In this connection, the difficulty is based less on the detection of single molecules, and more on the provision of techniques making it possible not to detect surrounding molecules. A successful approach for excluding such background molecules, i.e. molecules which are not to be detected, is the use of small wells or indentations, for example small wells or indentations in the nanometer range, formed in an approximately 100 nm thick metal layer situated on a glass support. Said wells or indentations having a diameter below approximately half the wavelength of visible light do not allow the diffusion of said light and are therefore referred to as zero-mode waveguides (ZMWs). Said zero-mode waveguides or ZMWs are also referred to as nanoapertures. In said ZMWs, evanescent light occurs near the glass support only to a slight extent, making it possible to observe single fluorescent molecules in biologically relevant concentrations in the nanomolar or micromolar range. These nanophotonic structures allow a breakthrough in single molecule real-time DNA sequencing and in investigating translation. To utilize the full potential of said nanophotonic structures, it is still necessary, however, to resolve various challenges. Firstly, single molecule techniques typically require exactly one molecule, such as a polymerase molecule for sequencing by means of ZMW. If there is more than one polymerase, this rapidly leads to dephasing, and so the sequence information is lost. Currently, said ZMWs or nanoapertures are loaded with a statistical Poisson distribution; this leads to a theoretical maximal distribution of single molecules in said ZMWs of approximately 37%. A second substantial challenge is the fluorescence of the single molecules within the ZMWs, i.e. having a signal as homogeneous as possible. This is not fully understood and most studies focus on FCS analysis, which is formed as an average via the total ZMW volume. A metal coating of the support to form the ZMW can, for example, lead to an altered (plasmon) excitation and the fluorescence process can be altered by quenching because of energy or electron transfer to the metal. Lastly, the radiation rate of the fluorophores may be altered. Furthermore, the radiation rate of the fluorophores may be intensified or reduced. A study which recently appeared showed that these processes are strongly position-dependent and the strongest fluorescence is obtained in nanoapertures having a diameter of 375 nm in the center thereof, Heucke S., et al., Nano Letters, 2014, 391 to 395.
US 2010/009872 describes methods for the targeted loading of biochips with single molecules. It describes DNA origami structures having single-stranded overhangs, to which functional units are coupled, being introduced into array structures. This represents the underlying prior art, specifically in which a distribution of a single structure is present in the ZMWs or nanoapertures with a Poisson distribution.
However, there is still a need to provide methods and arrangements which allow a targeted positioning of structures in the centers of the ZMWs or nanoapertures. More particularly, there is a need for methods and arrangements, where the loading with single molecules not only corresponds to the Poisson distribution, but is also present at a higher percentage in the single indentations or nanoapertures.
USPTO Backgrounds
USPTO Backgrounds