阅读说明：本技术 一种用于单分子荧光限域激发的纳米微阵列近场结构 (Nanometer microarray near-field structure for monomolecular fluorescence limited-domain excitation ) 是由 赵祥伟 崔玉军 于 2019-09-18 设计创作，主要内容包括：本发明公开了一种用于单分子荧光限域激发的纳米微阵列近场结构,包括基底和电介质填充部分,所述电介质填充部分均匀的阵列在基底中,所述电介质填充部分均在高度H方向上贯穿基底,所述基底材料为导电金属,所述电介质填充部分为透明材质。本发明的近场机构将单个荧光分子的激发限制在一个开放的平面上的纳升体积内,利用了介质填充等离激元纳米孔将激发限域到开放表面,有效降低了生物大分子与进入激发体积的空间位阻,与零模波导相比,该结构具有更优良的性能,且可以从上下两个方向采集单分子荧光信号,测试中应用灵活。(The invention discloses a nano micro-array near-field structure for monomolecular fluorescence confinement excitation, which comprises a substrate and a dielectric filling part, wherein the dielectric filling part is uniformly arrayed in the substrate, the dielectric filling part penetrates through the substrate in the height H direction, the substrate is made of conductive metal, and the dielectric filling part is made of transparent material. The near-field mechanism limits the excitation of a single fluorescent molecule in a nanoliter volume on an open plane, utilizes a medium to fill a plasmon nanopore to limit the excitation to an open surface, effectively reduces the steric hindrance of biomacromolecules and the entrance of biomacromolecules to the excitation volume, has better performance compared with zero-mode waveguide, can acquire single-molecule fluorescent signals from the upper direction and the lower direction, and is flexible to apply in testing.)

1. The nano microarray near field structure for the excitation of the single molecule fluorescence confinement comprises a substrate and dielectric filling parts, wherein the dielectric filling parts are distributed in the substrate, the dielectric filling parts penetrate through the substrate in the height H direction, the substrate is made of conductive metal, and the dielectric filling parts are made of solid-phase transparent materials.

2. The nano-microarray near-field structure for single-molecule fluorescence confinement excitation of claim 1, wherein the substrate material is one of aluminum, copper, gold and silver.

3. The nano-microarray near-field structure for single-molecule fluorescence confinement excitation of claim 1, wherein the solid-phase transparent material is one of silicon dioxide, sapphire, titanium dioxide, PE, PMMA, and PC.

4. The nanomicro-array near-field structure for single-molecule fluorescence confinement excitation of claim 1, wherein the dielectric filling portion is a uniform array in the substrate.

5. The nano-microarray near-field structure for single-molecule fluorescence confinement excitation of claim 1, wherein the dielectric filling portion is a truncated cone with a top end diameter Ds of 10-100nm, a bottom end diameter DB of 50-500nm, and the top end diameter is smaller than the bottom end diameter.

6. The near field structure of claim 1, wherein the dielectric filling portion is pyramid-shaped, the top end surface of the pyramid-shaped portion is a square with a side length of 10-100nm, the bottom end surface of the pyramid-shaped portion is a square with a side length of 50-500nm, and the side length Ws of the top end surface is smaller than the side length WB of the bottom end surface.

7. The near-field structure of nano-microarray for single-molecule restricted fluorescence domain excitation according to claim 1, wherein the height H of the near-field structure of nano-microarray is 100-1000 nm.

8. The nano-microarray near-field structure for single-molecule fluorescence confinement excitation of claim 1, wherein a substrate is disposed at the bottom of the nano-microarray near-field structure, and the substrate is made of the same material as the dielectric filling material.

Technical Field

The invention relates to a near field structure for monomolecular fluorescence confinement excitation, in particular to a nano microarray near field structure for monomolecular fluorescence confinement excitation.

Background

DNA sequencing technologies developed over the last decade have revolutionized bioscience and have the potential to revolutionize many aspects of medical practice in the next few years. However, to achieve this possibility, there are still many challenges that must be addressed, including reducing the cost of sequencing per run, simplifying sample preparation, reducing run time, increasing sequence read length, improving data analysis, and so forth. Single molecule sequencing on nanofabricated arrays, such as nanopore arrays, can address some of these challenges, however, these methods have their own set of technical difficulties, e.g., reliable nanostructure fabrication, control of the rate of translocation of DNA through the nanopore, nucleotide discrimination, detection of electrical signals from large arrays of nanopore sensors, etc., optical detection of nucleotides has been proposed as a potential solution to some of the technical difficulties in the field of nanopore sequencing; and has been implemented in the field of single molecule sequencing using Zero Mode Waveguide (ZMW) arrays by reducing the excitation volume to 10 at the bottom of the metal nanopore^-18To 10^-21ZMWs can be used to observe single fluorescent molecules even in biologically relevant concentrations up to the micromolar to millimolar range and to obtain Fluorescence Correlation Spectra (FCS) thus, ZMWs and their derivatives have been widely used in a variety of applications including high concentration single molecule analysis, single cell and single enzyme analysis, polymerase molecule DNA sequencing, nanofluidic channel DNA sequencing, oligomerization kinetics, redox reactions, and the like.

However, in a ZMW, the nanopores are tens of nanometers in diameter and are approximately 100 nanometers deep, and only the volume 10-20 nanometers above the bottom is excited. Due to the steric effect and the static bilayer structure on the surface of the nanopore, biomolecules such as enzyme or DNA are difficult to fall into the nanopore. To overcome this, the ZMW is constructed with sloped sidewalls to allow the mouth of the ZMW to be wider than its bottom, thereby facilitating the introduction of analyte into the ZMW. Alternatively, during the redox reaction, potentials are applied to different layers of the structure to perform spectroelectrochemical analysis. In another study, a nanopore is embedded at the bottom of the nanopore and a voltage is applied to introduce DNA strands into the ZMW, which are then captured by a DNA polymerase for subsequent use. However, the complexity and cost of ZMWs is increasing. Excitation and collection of single molecule fluorescent signals remains a significant challenge.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a nano microarray near-field structure for monomolecular fluorescence limited domain excitation, which is more flexible in application and better in performance.

The technical scheme is as follows: the nano microarray near field structure for the excitation of the single molecule fluorescence confinement comprises a substrate and dielectric filling parts, wherein the dielectric filling parts are distributed in the substrate, the dielectric filling parts penetrate through the substrate in the height H direction, the substrate is made of conductive metal, and the dielectric filling parts are made of solid-phase transparent materials.

Further, the substrate material is one of metal aluminum, copper, gold and silver.

Further, the material of the dielectric filling part is one of silicon dioxide, sapphire, titanium dioxide, PE, PMMA and PC.

Further, the dielectric filling portion is uniformly arrayed in the substrate.

Further, the dielectric filling part is in a truncated cone shape, the diameter Ds of the top end face of the truncated cone shape is 10-100nm, the diameter DB of the bottom end face of the truncated cone shape is 50-500nm, and the diameter of the top end face is smaller than that of the bottom end face.

Furthermore, the dielectric filling part is pyramid-shaped, the top end face of the pyramid-shaped part is a square with the side length of 10-100nm, the bottom end face of the pyramid-shaped part is a square with the side length of 50-500nm, and the side length Ws of the top end face is smaller than the side length WB of the bottom end face.

Further, the height H of the nano microarray near field structure is 100-1000 nm.

Furthermore, a substrate is arranged at the bottom of the nano micro array near field structure, and the substrate material is the same as the dielectric filling part material.

The preparation method comprises the following steps: because the invention needs to make fine patterns on the nanometer scale, although the etching efficiency of the laser is obviously higher than that of the focused plasma beam etching, the etching precision is far lower than that of the focused plasma beam etching, so that the near-field structure meeting different inclination angle requirements can be prepared by utilizing the focused plasma beam direct writing technology and adjusting parameters such as the ion beam energy, the incident current, the ion beam residence time and the like.

Has the advantages that: the nano-microarray near-field structure for single-molecule fluorescence limited-field excitation according to the present invention is a device for assisting efficient optical detection and analysis of polymers because it is a planar structure, provides more freedom to improve performance, can introduce desired molecules into the surface of the nano-microarray structure for detection without other mechanisms, can obtain better electric field strength than ZMW, and has been proved by numerical simulation that its performance can be not only comparable to ZMW but also better than the corresponding ZMW at a given ratio of height to top end-face diameter (Ds). Meanwhile, the width ratio of the upper end face and the lower end face can be adjusted according to requirements to adjust the electric field intensity at the vertex, and compared with the traditional ZMW, the improved freedom degree can obtain better electric field intensity at the vertex.

Wherein the physical quantities represented by the respective parameters: wave vector Kspp of surface plasmon SPP, plasmon incident wavelength λ p, incident light wavelength λ 0, ∈_mDielectric constant of metal, ∈_dThe dielectric constant of the dielectric.

The incident field strength of the microarray structure can be understood by the metal-dielectric boundary condition 0 and the kinetics of plasmon modes (surface plasmons-SPP) at the metal-dielectric interface. These surface plasmons SPP carry evanescent modes within the cavity that move with the wave vector (kspp), the plasmon entrance wavelength (λ p), for a cut-off wavelength λ_cut-_offThe metal-insulator-metal system (MIM) of (A) are related to each other as shown in equation (1a)Here e_mAnd e_dWhich are the dielectric constants of metal and dielectric, respectively, it is clear from equation (1a) that the wave vector of the generated plasma is inversely related to the incident wavelength, which relationship is further mediated by the dielectric constant of the metal as well as the dielectric constant of the dielectric material. Equation (1b) emphasizes the relationship between the wavelength of a surface plasmon and its wave vector. The near-field structure is considered advantageous over a zero-mode waveguide because it is a planar structure that does not require a mechanism to introduce the desired molecules into the nanopores of a ZMW without significantly degrading device performance. Furthermore, the near-field structure provides more freedom to improve performance, and better electric field strength can be obtained than a ZMW.

The NFS was numerically simulated using a standard FDTD method (time domain finite difference method) with the near-field structure having a refractive index of 1.33 on the top surface and 1 on the bottom in air. The refractive index of silica was chosen to be 1.45 and the optical constants for aluminum were taken from Palik. For comparison, we created a simulation model of a conventional ZMW, and for the two models to be comparable, the same near-field structure (NFS) tip face diameter and ZMW width were selected, and the ZMW thickness equivalent to the near-field structure was selected. Finally, it was investigated by placing a dipole source in the analysis region directly above the near-field structure. The electric field emitted by the dipoles is then collected from the bottom of the structure to determine the proportion of the electric field that can be received by the viewer.

The simulation results obtained from the near-field structure with a height of 100nm and the ZMW are shown in fig. 5. When the structure height (near field structures and zmw) is increased at 100nm, the electric field strength along the z-axis increases for all structures. But the effect on near field structure performance is much greater than on ZMWs. As the height increases, the decrease in electric field strength can be attributed to increased attenuation due to long-term interaction with the aluminum sidewalls. Due to the increased height, the peak intensity of a ZMW is much less than that of an NFS for two reasons. The first reason is that ZMWs confine light to a small space surrounded by metal walls, which is not the case in NFS. In ZMWs, the metal walls cause rapid decay of the electric field, increasing the strength at the interface. For these reasons, the electric field strength of the ZMW remains almost constant at the interface, while the electric field strength of the near-field structure is greatly reduced. Second, in the case of a ZMW, the incoming electric field does not interact with the metal wall before reaching the cavity. In the case of a ZMW, this is also a field of view. Whereas for near-field structures, a metal-walled cavity is used for confinement and focusing of the electric field, with the viewing region above the cavity. Therefore, at the interface, the strength of the near-field structure is smaller than that of the ZMW even if DB ═ 100nm, and this advantage of the ZMW is quickly smoothed.

In the test, the near-field structure limits the excitation of a single fluorescent molecule in a nanoliter volume on an open plane, and utilizes a medium to fill a plasmon nanopore to limit the excitation domain to an open surface, thereby effectively reducing the steric hindrance between biological macromolecules and the molecules entering the excitation volume. Compared with zero-mode waveguide, the structure has better performance, can acquire single-molecule fluorescence signals from the upper direction and the lower direction, and is flexible to apply in testing.

Drawings

FIG. 1 is a schematic front cross-sectional view of a truncated cone-shaped nano-microarray near-field structure;

FIG. 2 is a schematic top view of a near-field structure of a truncated cone shaped nano-microarray;

FIG. 3 is a schematic front cross-sectional view of a pyramidal nano-microarray near-field structure;

FIG. 4 is a schematic top view of a pyramidal nano-microarray near-field structure;

FIG. 5 is a graph showing the variation of electric field intensity with distance from the surface of a truncated cone-shaped nano-microarray near-field structure;

FIG. 6 schematic diagram of real-time sequencing of single-molecule DNA.

Detailed Description

For a further understanding of the present invention, reference is made to the following detailed description taken in conjunction with the accompanying drawings 1-5 and examples.