CN111090144B - Preparation method of functionalized zero-mode waveguide hole and waveguide hole structure - Google Patents
Preparation method of functionalized zero-mode waveguide hole and waveguide hole structure Download PDFInfo
- Publication number
- CN111090144B CN111090144B CN201911416151.4A CN201911416151A CN111090144B CN 111090144 B CN111090144 B CN 111090144B CN 201911416151 A CN201911416151 A CN 201911416151A CN 111090144 B CN111090144 B CN 111090144B
- Authority
- CN
- China
- Prior art keywords
- zero
- mode waveguide
- waveguide hole
- nanopore
- hole
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B6/00—Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings
- G02B6/10—Light guides; Structural details of arrangements comprising light guides and other optical elements, e.g. couplings of the optical waveguide type
- G02B6/107—Subwavelength-diameter waveguides, e.g. nanowires
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Abstract
The invention provides a preparation method of a functionalized zero-mode waveguide hole, which comprises the following steps: the transparent substrate and the metal covering layer form the zero-mode waveguide hole; coating a color-sensitive activating group on the side wall of the zero-mode waveguide hole, and irradiating the color-sensitive activating group with light to functionalize the zero-mode waveguide hole; and irradiating the zero-mode waveguide hole from one side of the metal covering layer by using ultraviolet light, so that the DNA polymerase at the zero-mode waveguide hole corresponding to the metal covering layer is inactivated, and the DNA polymerase at the bottom of the zero-mode waveguide hole has activity. The invention also relates to a zero-mode waveguide hole structure with functionalization. According to the invention, different color-sensitive activating groups are coated on the hole walls with different apertures of the zero-mode waveguide hole, and the side walls of different nanopores are selectively modified by using different wavelengths of light, so that the purpose of layered functionalization is achieved; the method improves the single molecule occupancy rate of DNA polymerase in single molecule sequencing and improves the signal-to-noise ratio.
Description
Technical Field
The invention relates to the field of micro-nano machining processes, in particular to a preparation method of a functionalized zero-mode waveguide hole.
Background
The single-molecule real-time DNA sequencing technology is a real-time high-flux DNA sequencing method, and comprises the following steps:
the complex of DNA template/primer/polymerase is surrounded by fluorescently labeled nucleotides; the fluorescently labeled nucleotides bind to the DNA template bases. Fluorescence is continuously emitted during the time of binding to the active site (typically lasting tens of milliseconds), producing a detectable pulse. The nature of the fluorescent dye indicates which bases are bound together; the polymerase incorporates the nucleotide into the nucleic acid strand, releasing the fluorophore; polymerase transfer to the next template position; the previous process is repeated.
Two main techniques of single-molecule real-time DNA sequencing technology are:
one is a Zero Mode Waveguide (ZMW) confinement that allows single molecule detection at enzyme-related labeled nucleotide concentrations, and the other is a fluorescently labeled phosphate-linked nucleotide that allows for uninterrupted DNA polymerization to be observed.
The zero mode waveguide hole array consists of a dense array of holes, the diameter of which is 100 nanometers. The light decays exponentially after entering the ZMWs, only the portion near the bottom is illuminated, each ZMW becomes a nanophotonic visualization chamber for recording a single polymerization reaction, providing a detection volume of only 10-21And (5) rising. This volume is 1000 times higher than diffraction limited confocal microscopy, making it possible to observe single nucleotide incorporation events in the background of the diffusion of fluorescently labeled nucleotides. In addition to reducing the number of labeled nucleotides within the observation volume, the highly confined volume also results in a greatly reduced diffusion visit time. This allows a better temporal distinction between labeled nucleotide diffusion events and enzyme nucleotide incorporation events (typically lasting a few milliseconds) by observing the volume (typically lasting a few microseconds). The second important component is a phosphate-linked nucleotide, whose fluorescent tag is linked to a terminal phosphate, usually by a linker, rather than a base. The unmodified nucleotide is 100% replaced by the phosphate-linked nucleotide because the enzyme breaks down the fluorophore during the binding process, leaving a fully natural double-stranded nucleic acid.
In order to ensure that the sequencing reaction is normally carried out, DNA to be detected, DNA polymerase and primers need to be anchored at the bottoms of ZMW wells, and in order to facilitate the analysis process, the DNA polymerase is anchored at the bottom of each ZMW well to carry out one sequencing reaction.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a preparation method of a functionalized zero-mode waveguide hole.
According to the invention, the zero-mode waveguide hole is set to be multi-aperture, under the irradiation of ultraviolet light, the DNA polymerase at the bottom of the zero-mode waveguide hole has activity, and the DNA polymerase in other areas of the zero-mode waveguide hole is inactivated, so that the occupancy rate of single molecules of the DNA polymerase in the zero-mode waveguide hole is increased.
The invention provides a preparation method of a functionalized zero-mode waveguide hole, which comprises the following steps:
the transparent substrate and the metal covering layer form the zero-mode waveguide hole, and the zero-mode waveguide hole extends through the metal covering layer and extends into the transparent substrate;
coating a color-sensitive activating group on the side wall of the zero-mode waveguide hole, and irradiating the color-sensitive activating group with light to functionalize the zero-mode waveguide hole;
and irradiating the zero-mode waveguide hole from one side of the metal covering layer by using ultraviolet light, so that the DNA polymerase at the zero-mode waveguide hole corresponding to the metal covering layer is inactivated, and the DNA polymerase at the bottom of the zero-mode waveguide hole has activity.
Preferably, the transparent substrate and the metal cover layer form the zero-mode waveguide hole, including:
the aperture of the zero-mode waveguide hole corresponding to the transparent substrate and the metal covering layers is different, and the aperture of the zero-mode waveguide hole corresponding to the transparent substrate from the metal covering layers to the transparent substrate is reduced in sequence;
coating color-sensitivity activating groups on the side walls of the zero-mode waveguide hole, wherein the color-sensitivity activating groups comprise:
and coating different color-sensitive activating groups on the side walls of the zero-mode waveguide holes with different apertures, and irradiating the different color-sensitive activating groups by using light with different wavelengths to functionalize the zero-mode waveguide holes with different apertures.
Preferably, the irradiating different ones of the color-sensitive activating groups with different wavelengths of light includes:
and the wavelengths sequentially irradiate the hole wall of the zero-mode waveguide hole from small to large.
Preferably, a central island is arranged at the bottom of the zero-mode waveguide hole, and the central island is used for connecting DNA polymerase, so that the DNA polymerase is anchored on the central island.
Preferably, the substance of the central island comprises PEG silane of avidin, which anchors DNA polymerase on the central island.
Preferably, the composition of the metal coating is gold.
Preferably, the composition of the metal overlay is aluminum.
The invention also provides a functionalized zero-mode waveguide hole structure, which comprises a transparent substrate and a metal covering layer, wherein the zero-mode waveguide hole extends through the metal covering layer and extends into the transparent substrate; the hole wall of the zero-mode waveguide hole comprises a color sensitivity activating group, and the color sensitivity activating group is used for functionalizing the zero-mode waveguide hole;
when ultraviolet light irradiates the zero-mode waveguide hole from one side of the metal covering layer, DNA polymerase at the position, corresponding to the zero-mode waveguide hole, of the metal covering layer is inactivated, and the DNA polymerase at the bottom of the zero-mode waveguide hole has activity.
Preferably, the zero-mode waveguide hole includes a plurality of different apertures, the apertures of the zero-mode waveguide hole corresponding to the transparent substrate and the plurality of metal covering layers are different, and the apertures of the zero-mode waveguide hole corresponding to the transparent substrate from the plurality of metal covering layers to the transparent substrate are sequentially reduced; the side walls of the zero-mode waveguide holes with different apertures comprise different color-sensitive activating groups, and the different color-sensitive activating groups are irradiated by light with different wavelengths, so that the zero-mode waveguide holes with different apertures are functionalized.
Preferably, the metal covering layer comprises a first metal covering layer and a second metal covering layer, the second metal covering layer is in contact with the transparent substrate, the first metal covering layer and the second metal covering layer respectively correspond to a first nanopore and a second nanopore of the zero-mode waveguide hole, and the aperture of the first nanopore is larger than that of the second nanopore; the transparent substrate corresponds to a third nanopore of the zero-mode waveguide hole, and the aperture of the third nanopore is smaller than that of the second nanopore;
when the ultraviolet light is irradiated from the first metal covering layer along the axial direction of the zero-mode waveguide hole, the DNA polymerase in the first nanopore and the second nanopore is inactivated, and the DNA polymerase in the third nanopore has activity.
Preferably, the metal covering layer includes a third metal covering layer, a fourth metal covering layer and a fifth metal covering layer, the fifth metal covering layer contacts with the transparent substrate, the third metal covering layer, the fourth metal covering layer and the fifth metal covering layer respectively correspond to a fourth nanopore, a fifth nanopore and a sixth nanopore of the zero-mode waveguide hole, and the apertures of the fourth nanopore, the fifth nanopore and the sixth nanopore are sequentially reduced; the transparent substrate corresponds to a seventh nanopore of the zero-mode waveguide hole, and the aperture of the seventh nanopore is smaller than that of the sixth nanopore.
Compared with the prior art, the invention has the beneficial effects that:
the invention discloses a preparation method of a functionalized zero-mode waveguide hole, which coats different color-sensitive activating groups on the hole walls with different apertures of the zero-mode waveguide hole, and selectively modifies the side walls of different nanopores by using different wavelengths of light to achieve the purpose of layered functionalization; the method improves the single molecule occupancy rate of DNA polymerase in single molecule sequencing and improves the signal-to-noise ratio. In addition, the zero-mode waveguide hole is set to be a plurality of apertures, under the irradiation of ultraviolet light, the DNA polymerase in the minimum aperture at the bottom of the zero-mode waveguide hole has activity, and the DNA polymerase in other areas of the zero-mode waveguide hole loses activity, so that the occupancy rate of single molecules of the DNA polymerase in the zero-mode waveguide hole is increased.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings. The detailed description of the present invention is given in detail by the following examples and the accompanying drawings.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the invention without limiting the invention. In the drawings:
FIG. 1 is a schematic diagram of a zero mode waveguide hole as is conventional in the art;
FIG. 2 is a schematic diagram of another zero mode waveguide hole conventional in the art;
FIG. 3 is a schematic diagram of DNA polymerase scattering from a zero mode waveguide pore as is conventional in the art;
FIG. 4 is a schematic diagram of another zero mode waveguide hole DNA polymerase conventional in the art under UV irradiation;
FIG. 5 is an overall flow chart of the method of the present invention for fabricating a zero mode waveguide hole with functionalization;
FIG. 6 is a schematic view of a zero mode waveguide hole with a functionalized zero mode waveguide hole preparation method according to the present invention;
FIG. 7 is a schematic view of a zero-mode waveguide hole wall coated with a color-sensing activating group according to the method for preparing a functionalized zero-mode waveguide hole of the present invention;
FIG. 8 is a schematic diagram of a zero-mode waveguide hole wall coated with a color-sensitive activating group under irradiation of light of different wavelengths according to the method for preparing a functionalized zero-mode waveguide hole of the present invention;
reference numerals: 101. ultraviolet light, 102, DNA polymerase, 103, metal coating, 104, transparent substrate, 105, bottom nanopore of zero mode via, 106, ultraviolet-opaque region, 201, first metal coating, 202, second metal coating, 203, transparent substrate, 204, first color-sensitive activating group, 205, second color-sensitive activating group, 206, third color-sensitive activating group, 207, light of first wavelength, 208, light of second wavelength, 209, light of third wavelength.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.
As shown in fig. 1-4, the metal covering layer 103 and the transparent substrate 104 are fabricated by an electron beam lithography process or a nanoimprint method to form a zero-mode waveguide hole, a portion of the DNA polymerase 102 entering the zero-mode waveguide hole is scattered above and at the side wall of the metal covering layer 103, and a portion of the DNA polymerase 102 is scattered on the transparent substrate 104 at the bottom of the zero-mode waveguide hole, and the light is exponentially attenuated inside the zero-mode waveguide hole according to the optical property of the zero-mode waveguide hole, and when the wavelength of the light is greater than or equal to 1.7 times of the aperture, the light cannot penetrate through the zero-mode waveguide hole. To ensure the single molecule occupancy of the polymerase, the nanopore 105 is fabricated on the bottom of the zero-mode waveguide hole, i.e., the transparent substrate, for example, the nanopore 105 with a pore diameter of about 3 to 5nm is fabricated by bombarding a PET polymer membrane with a field emission electron microscope or heavy metal and performing ultraviolet irradiation. When the DNA polymerase 102 randomly scatters, it will adhere to the top and side walls of the metal cover layer 103, above the transparent substrate 104 and inside the nano-holes 105, and when the zero-mode waveguide hole is irradiated with ultraviolet light with a wavelength of 10-40nm, the ultraviolet light cannot penetrate through the nano-holes 105 with an aperture of 3-5nm, but can penetrate through the upper half of the zero-mode waveguide hole, i.e. above the transparent substrate 104. The DNA polymerase 102 on the top and sidewalls of the metal covering layer 103 and the exposed portion of the transparent substrate 104 is inactivated, and the DNA polymerase 102 scattered at the bottom of the nanopore 105 is still active, i.e., the DNA polymerase 102 in the nanopore 105 is protected, i.e., the region 106 is not irradiated by the ultraviolet light, thereby increasing the single molecule occupancy of the DNA polymerase.
The invention provides a preparation method of a functionalized zero-mode waveguide hole, as shown in fig. 5, comprising the following steps:
s1, the transparent substrate and the metal covering layer form the zero mode wave guide hole, and the zero mode wave guide hole extends through the metal covering layer and extends into the transparent substrate. In one embodiment, as shown in fig. 6, the apertures of the zero mode waveguide holes corresponding to the transparent substrate and the metal covering layers are different, and the apertures of the zero mode waveguide holes corresponding to the metal covering layers to the transparent substrate are sequentially reduced; generally, the zero mode waveguide hole is formed by a metal covering layer and a transparent substrate, and the material of the metal covering layer is metal gold or metal aluminum. In the embodiment, the zero-mode waveguide hole is formed by overlapping the multilayer metal covering layer and the transparent substrate. The plurality of metal covering layers are overlapped to form a plurality of zero-mode waveguide holes with different aperture sizes, and the aperture of each corresponding zero-mode waveguide hole is gradually reduced from the opening of each zero-mode waveguide hole to the transparent substrate, namely the nano-holes in the transparent substrate are the smallest in aperture in the zero-mode waveguide holes.
S2, coating a color-sensitive activating group on the sidewall of the zero-mode waveguide hole, and irradiating the color-sensitive activating group with light to functionalize the zero-mode waveguide hole. In one embodiment, different color-sensitive activating groups are coated on the side walls of different apertures of the zero-mode waveguide hole, and the different color-sensitive activating groups are irradiated by different wavelengths of light, so that the zero-mode waveguide hole with different apertures is functionalized. In one embodiment, as shown in fig. 7 and 8, different color-sensitive activating groups are coated on the inner walls of the zero-mode waveguide holes with different apertures, for example, a first color-sensitive activating group 204, a second color-sensitive activating group 205, and a third color-sensitive activating group 206 are coated on the first nanopore, the second nanopore, and the third nanopore, respectively, and the corresponding color-sensitive activating groups are irradiated with light of different wavelengths to functionalize the color-sensitive activating groups, and the first color-sensitive activating group 204, the second color-sensitive activating group 205, and the third color-sensitive activating group 206 are irradiated with light of a first wavelength 207, a second wavelength 208, and a third wavelength 209, respectively. Such as: ultraviolet light with the wavelength of 15nm activates the color-sensitive activating group coated on the inner wall corresponding to the second metal covering layer with the aperture of 8nm, the activated color-sensitive activating group starts to be coupled with the free fluorescence background, and the group reduces the fluorescence background of the free fluorescence labeled nucleotide in the layer after being activated, so that the signal-to-noise ratio is improved.
When the hole wall of the zero-mode waveguide hole is irradiated by light with different wavelengths, the wavelengths are sequentially irradiated from small to large. The color-sensitive activating group covered on the side wall of the nanopore with the smallest aperture needs to be activated first, so that the activated nanopore with smaller size can be controlled not to be influenced by the subsequently applied longer-wavelength light, namely, the activated nanopore is ensured not to be influenced. Therefore, when the irradiation is performed to the zero-mode waveguide hole having a large aperture, the excitation is performed sequentially from the small aperture to the large aperture. In one embodiment, the walls of the holes corresponding to the first metal cap 201 are functionalized not to bind with the DNA polymerase, so that the DNA polymerase is more scattered inside the holes. The pore walls of the zero-mode waveguide holes corresponding to the second metal cap layer 202 are functionalized to weaken the background fluorescent layer in order to reduce the fluorescent background of the free fluorescently labeled nucleotides in the layer and improve the signal-to-noise ratio. The pore wall of the third nanopore is functionalized to be combined with the DNA polymerase, so that the single molecule occupancy rate of the DNA polymerase is improved, and the purpose of layered functionalization is achieved. For example: two nano holes with different sizes are arranged, wherein the upper layer of the big hole is 30nm corresponding to the zero-mode waveguide hole of the metal covering layer, the lower layer of the small hole is 3nm corresponding to the zero-mode waveguide hole of the substrate, the light wavelength which cannot pass through the big hole is 30 multiplied by 1.7 to 51nm, the light wavelength which cannot pass through the small hole is 3 multiplied by 1.7 to 5.1nm, the small hole at the lowest layer is activated firstly, and the light with the wavelength which is more than 5.1nm is selected from the small hole at the lowest layer, so that only one point above the hole can enter light, the light cannot penetrate through the small hole and can be activated, the enzyme is connected to the point above, but the light of the upper layer of the big hole can also penetrate through the whole hole. And then, activating the light irradiation groups in the upper macropores by using light with the wavelength less than 51nm and the wavelength more than 6nm, so that the light irradiation groups with the wavelength more than 6nm do not influence the activated micropores, and the light irradiation groups with the wavelength less than 51nm can ensure that the light in the macropores can be transmitted and can be modified, so that the enzyme inhibition layers are modified, and the layered functionalization is realized by utilizing the combination of the light wavelength and the pore size.
And S3, irradiating the zero-mode waveguide hole from one side of the metal covering layer by using ultraviolet light, so that the DNA polymerase at the zero-mode waveguide hole corresponding to the metal covering layer is inactivated, and the DNA polymerase at the bottom of the zero-mode waveguide hole has activity. In one embodiment, after irradiation by ultraviolet light, the DNA polymerase in other areas of the zero mode waveguide hole is inactivated, except that the DNA polymerase at the bottom of the zero mode waveguide hole is still active. Generally, when the DNA polymerase is scattered in the zero mode waveguide hole, a small amount of the DNA polymerase will fall into the bottom of the zero mode waveguide hole, i.e. the nanopore of the transparent substrate, and the DNA polymerase is anchored to the bottom of the zero mode waveguide hole by means of a chemical reaction in order to increase the occupancy rate of a single molecule of the DNA polymerase, preferably, the PEG silane is functionalized and then streptavidin is attached, and the DNA polymerase is anchored to the streptavidin, so that the DNA polymerase is fixed to the bottom of the zero mode waveguide hole. The PEG silane is functionalized and then arranged at the bottom center position of the zero-mode waveguide hole, and the DNA polymerase is anchored on the PEG silane through the avidin of the PEG silane, so that the single molecule occupancy rate of the DNA polymerase is improved. And a central island is arranged at the bottom of the zero-mode waveguide hole and used for connecting DNA polymerase, so that the DNA polymerase is anchored on the central island. The material of the central island is PEG silane including a avidin that anchors DNA polymerase on the central island. The size of the central island matches the size of the single DNA polymerase such that the single DNA polymerase is anchored on the central island, increasing DNA polymerase single molecule occupancy; preferably, the central island is located at the center of the bottom of the zero-mode waveguide hole.
The present invention also provides a functionalized zero mode waveguide via structure, as shown in fig. 6-8, comprising a transparent substrate and a metal cap layer, wherein the zero mode waveguide via extends through the metal cap layer and into the transparent substrate; the hole wall of the zero-mode waveguide hole comprises a color sensitivity activating group, and the color sensitivity activating group is used for functionalizing the zero-mode waveguide hole;
when ultraviolet light irradiates the zero-mode waveguide hole from one side of the metal covering layer, DNA polymerase at the position, corresponding to the zero-mode waveguide hole, of the metal covering layer is inactivated, and the DNA polymerase at the bottom of the zero-mode waveguide hole has activity.
In a preferred embodiment, the zero-mode waveguide hole includes a plurality of different apertures, the apertures of the zero-mode waveguide hole corresponding to the transparent substrate and the plurality of metal covering layers are different, and the apertures of the zero-mode waveguide hole corresponding to the transparent substrate from the plurality of metal covering layers to the transparent substrate are sequentially reduced; the side walls of the zero-mode waveguide holes with different apertures comprise different color-sensitive activating groups, and the different color-sensitive activating groups are irradiated by light with different wavelengths, so that the zero-mode waveguide holes with different apertures are functionalized. In the present embodiment, the zero mode waveguide hole is generally formed by a metal covering layer and a transparent substrate, and the material of the metal covering layer is metal gold or metal aluminum. In the embodiment, the zero-mode waveguide hole is formed by overlapping the multilayer metal covering layer and the transparent substrate. The plurality of metal covering layers are overlapped to form a plurality of zero-mode waveguide holes with different aperture sizes, and the aperture of each corresponding zero-mode waveguide hole is gradually reduced from the opening of each zero-mode waveguide hole to the transparent substrate, namely the nano-holes in the transparent substrate are the smallest in aperture in the zero-mode waveguide holes. The transparent substrate and the metal covering layers form a zero-mode waveguide hole of the multilayer structure, the zero-mode waveguide hole of the multilayer structure forms nano holes with different sizes, and the nano holes with different sizes are used for allowing light with different wavelengths to pass or not pass so that the light with different wavelengths has different functions.
In a specific embodiment, the metal covering layers include a first metal covering layer 201 and a second metal covering layer 202, the second metal covering layer 202 is in contact with the transparent substrate 203, the first metal covering layer 201 and the second metal covering layer 202 respectively correspond to a part of a first nanopore and a second nanopore of the zero mode waveguide hole, and the aperture of the first nanopore is larger than that of the second nanopore; the part of the zero-mode waveguide hole extending into the transparent substrate 203 is marked as a third nanopore, and the aperture of the third nanopore is smaller than that of the second nanopore;
and irradiating the ultraviolet light from the first metal covering layer along the axial direction of the zero-mode waveguide hole, wherein the DNA polymerase in the first nanopore and the second nanopore is inactivated, and the DNA polymerase in the third nanopore has activity.
In another embodiment, the metal covering layers include a third metal covering layer, a fourth metal covering layer and a fifth metal covering layer, the fifth metal covering layer is in contact with the transparent substrate, the third metal covering layer, the fourth metal covering layer and the fifth metal covering layer respectively correspond to partial fourth nanopores, partial fifth nanopores and partial sixth nanopores of the zero-mode waveguide hole, and the diameters of the fourth nanopores, the partial fifth nanopores and the partial sixth nanopores are sequentially reduced; and the part of the zero-mode waveguide hole extending into the transparent substrate is a seventh nanopore, and the aperture of the seventh nanopore is smaller than that of the sixth nanopore. In this embodiment, the metal coating forms three-dimensional apertures corresponding to the zero-mode waveguide hole, and the apertures in the zero-mode waveguide hole are decreased in a decreasing manner to increase the single-molecule occupancy of the DNA polymerase when reaching the nanopore at the bottom of the zero-mode waveguide hole.
The number of DNA polymerase dispersed in the nano-pores on the transparent substrate can be minimized by arranging the zero-mode waveguide pore with a multi-pore diameter, so that the single molecule occupancy rate of the DNA polymerase is improved.
The invention discloses a preparation method of a functionalized zero-mode waveguide hole, which coats different color-sensitive activating groups on the hole walls with different apertures of the zero-mode waveguide hole, and selectively modifies the side walls of different nanopores by using different wavelengths of light to achieve the purpose of layered functionalization; the method improves the single molecule occupancy rate of DNA polymerase in single molecule sequencing and improves the signal-to-noise ratio. In addition, the zero-mode waveguide hole is set to be a plurality of apertures, under the irradiation of ultraviolet light, the DNA polymerase in the minimum aperture at the bottom of the zero-mode waveguide hole has activity, and the DNA polymerase in other areas of the zero-mode waveguide hole loses activity, so that the occupancy rate of single molecules of the DNA polymerase in the zero-mode waveguide hole is increased.
The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner; those skilled in the art can readily practice the invention as shown and described in the drawings and detailed description herein; however, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the scope of the invention as defined by the appended claims; meanwhile, any changes, modifications, and evolutions of the equivalent changes of the above embodiments according to the actual techniques of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (8)
1. The preparation method of the functionalized zero-mode waveguide hole is characterized by comprising the following steps of:
forming the zero mode waveguide hole by the transparent substrate and the metal covering layer, wherein the zero mode waveguide hole extends through the metal covering layer and extends into the transparent substrate; wherein the transparent substrate and the metal covering layer form the zero mode waveguide hole, including:
the aperture of the zero-mode waveguide hole corresponding to the transparent substrate and the metal covering layers is different, and the aperture of the zero-mode waveguide hole corresponding to the transparent substrate from the metal covering layers to the transparent substrate is reduced in sequence; coating a color-sensitive activating group on the side wall of the zero-mode waveguide hole, and irradiating the color-sensitive activating group with light to functionalize the zero-mode waveguide hole;
wherein, coating color sensitivity activating groups on the side wall of the zero-mode waveguide hole comprises: coating different color-sensitive activating groups on the side walls of the zero-mode waveguide holes with different apertures, and irradiating the different color-sensitive activating groups by using light with different wavelengths to functionalize the zero-mode waveguide holes with different apertures;
and irradiating the zero-mode waveguide hole from one side of the metal covering layer by using ultraviolet light, so that the DNA polymerase at the zero-mode waveguide hole corresponding to the metal covering layer is inactivated, and the DNA polymerase at the bottom of the zero-mode waveguide hole has activity.
2. The method for preparing the functionalized zero mode waveguide hole according to claim 1, wherein the step of irradiating different color-sensitive activating groups with light of different wavelengths comprises:
and the wavelengths sequentially irradiate the hole wall of the zero-mode waveguide hole from small to large.
3. The method for preparing the functionalized zero mode waveguide hole according to claim 1, wherein a central island is disposed at the bottom of the zero mode waveguide hole, and the central island is used for connecting a DNA polymerase, so that the DNA polymerase is anchored on the central island.
4. The method of claim 3, wherein the material of the central island comprises PEG silane of avidin, which anchors DNA polymerase on the central island.
5. The method for preparing the functionalized zero-mode waveguide hole according to any one of claims 1 to 4, wherein the component of the metal coating layer is gold or aluminum.
6. The structure is characterized by comprising a transparent substrate and a metal covering layer, wherein the zero-mode waveguide hole extends through the metal covering layer and extends into the transparent substrate; the hole wall of the zero-mode waveguide hole comprises a color sensitivity activating group, and the color sensitivity activating group is used for functionalizing the zero-mode waveguide hole;
when ultraviolet light irradiates the zero-mode waveguide hole from one side of the metal covering layer, DNA polymerase at the position, corresponding to the zero-mode waveguide hole, of the metal covering layer is inactivated, and the DNA polymerase at the bottom of the zero-mode waveguide hole has activity;
the zero-mode waveguide holes comprise a plurality of different apertures, the apertures of the zero-mode waveguide holes corresponding to the transparent substrate and the metal covering layers are different, and the apertures of the zero-mode waveguide holes corresponding to the transparent substrate from the metal covering layers to the transparent substrate are reduced in sequence; the side walls of the zero-mode waveguide holes with different apertures comprise different color-sensitive activating groups, and the different color-sensitive activating groups are irradiated by light with different wavelengths, so that the zero-mode waveguide holes with different apertures are functionalized.
7. The functionalized zero-mode waveguide via structure of claim 6, wherein the metal capping layer comprises a first metal capping layer and a second metal capping layer, the second metal capping layer is in contact with the transparent substrate, the first metal capping layer and the second metal capping layer respectively correspond to a first nanopore and a second nanopore of the zero-mode waveguide via, and an aperture of the first nanopore is larger than an aperture of the second nanopore; the transparent substrate corresponds to a third nanopore of the zero-mode waveguide hole, and the aperture of the third nanopore is smaller than that of the second nanopore;
when the ultraviolet light is irradiated from the first metal covering layer along the axial direction of the zero-mode waveguide hole, the DNA polymerase in the first nanopore and the second nanopore is inactivated, and the DNA polymerase in the third nanopore has activity.
8. The functionalized zero-mode waveguide via structure of claim 6, wherein the metal cap layers comprise a third metal cap layer, a fourth metal cap layer and a fifth metal cap layer, the fifth metal cap layer is in contact with the transparent substrate, the third, fourth and fifth metal cap layers respectively correspond to a fourth nanopore, a fifth nanopore and a sixth nanopore of the zero-mode waveguide via, and the pore diameters of the fourth nanopore, the fifth nanopore and the sixth nanopore are sequentially reduced; the transparent substrate corresponds to a seventh nanopore of the zero-mode waveguide hole, and the aperture of the seventh nanopore is smaller than that of the sixth nanopore.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911416151.4A CN111090144B (en) | 2019-12-31 | 2019-12-31 | Preparation method of functionalized zero-mode waveguide hole and waveguide hole structure |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911416151.4A CN111090144B (en) | 2019-12-31 | 2019-12-31 | Preparation method of functionalized zero-mode waveguide hole and waveguide hole structure |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111090144A CN111090144A (en) | 2020-05-01 |
| CN111090144B true CN111090144B (en) | 2022-03-08 |
Family
ID=70398008
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911416151.4A Active CN111090144B (en) | 2019-12-31 | 2019-12-31 | Preparation method of functionalized zero-mode waveguide hole and waveguide hole structure |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111090144B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112680320A (en) * | 2021-01-21 | 2021-04-20 | 季华实验室 | Zero-mode waveguide hole structure, preparation method and use method thereof |
| CN115141819A (en) * | 2021-03-29 | 2022-10-04 | 上海近观科技有限责任公司 | Method for fixing DNA polymerase in gene sequencing |
| CN116626011B (en) * | 2023-07-24 | 2023-10-03 | 中国科学院苏州生物医学工程技术研究所 | Nanometer optical device for single-molecule dynamics detection |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102212515A (en) * | 2006-03-30 | 2011-10-12 | 加利福尼亚太平洋生物科学股份有限公司 | Articles having localized molecules disposed thereon and methods of producing same |
| US20140175052A1 (en) * | 2009-09-11 | 2014-06-26 | Pacific Biosciences Of California, Inc. | Method for preparing zero-mode waveguide arrays with coated walls |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8603741B2 (en) * | 2010-02-18 | 2013-12-10 | Pacific Biosciences Of California, Inc. | Single molecule sequencing with two distinct chemistry steps |
-
2019
- 2019-12-31 CN CN201911416151.4A patent/CN111090144B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102212515A (en) * | 2006-03-30 | 2011-10-12 | 加利福尼亚太平洋生物科学股份有限公司 | Articles having localized molecules disposed thereon and methods of producing same |
| US20140175052A1 (en) * | 2009-09-11 | 2014-06-26 | Pacific Biosciences Of California, Inc. | Method for preparing zero-mode waveguide arrays with coated walls |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111090144A (en) | 2020-05-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111090144B (en) | Preparation method of functionalized zero-mode waveguide hole and waveguide hole structure | |
| US10901145B2 (en) | Method for analyzing luminescent species | |
| Fan et al. | A review on the fabrication of substrates for surface enhanced Raman spectroscopy and their applications in analytical chemistry | |
| JP5127718B2 (en) | Method for measuring one or more analytes in a sample of biological origin having a complex composition and use thereof | |
| JP5222599B2 (en) | Nucleic acid analysis device and nucleic acid analysis apparatus using the same | |
| CN105283560B (en) | The foranalysis of nucleic acids detected by mixed FRET based on nano-pore | |
| US8367159B2 (en) | Methods for producing ZMWs with islands of functionality | |
| EP1274986B1 (en) | Device and method for determining multiple analytes | |
| AU2014369879B2 (en) | Structured substrates for improving detection of light emissions and methods relating to the same | |
| EP1556695B1 (en) | Analytical platform and identification method with analytes, which are to be identified in a sample optionally after fractionation and which serve as immobilized specific binding partners | |
| KR20100085830A (en) | Methods and devices for controlled monolayer formation | |
| JP2021509013A (en) | Detection or quantification of a single molecule by DNA nanotechnology in microwells | |
| EP1390725B1 (en) | Use of optical diffraction elements in detection methods | |
| WO2005107949A1 (en) | Method and apparatus for creating an analysis arrangement comprising discrete, separate test zones used for performing biological, biochemical, or chemical analyses | |
| KR20030040520A (en) | Bio-chip, photoluminescent methods for identifying biological material, and apparatuses for use with such methods and bio-chips | |
| EP1561109A1 (en) | Analytical platform and detection method with analytes which are to be detected in a sample in the form of immobilized specific binding partners | |
| CN111123429B (en) | Preparation method of zero-mode waveguide hole coated with anchoring substance and waveguide hole structure | |
| US10099195B2 (en) | Method for positioning structures in indentations and arrangements thus obtainable | |
| US20230129378A1 (en) | Systems and methods for detection of analytes | |
| CN112680320A (en) | Zero-mode waveguide hole structure, preparation method and use method thereof | |
| AT501110A1 (en) | ARRAYS TO BIND MOLECULES | |
| US20180135118A1 (en) | Pinhole Zero-Mode Waveguides | |
| DE102013000682B3 (en) | Detecting molecules, involves immobilizing mixtures of two or more different biochemical capture molecules on two or more analytical array positions, and binding two or more different analytes on each array position | |
| WO2023106372A1 (en) | Enzyme measurement method, microchamber array, kit, method for capturing raman scattering image, and method for measuring target molecule | |
| Martin | Characterization and Application of Hybrid Nanostructures for Enhanced Biological Imaging Using Fluorescence Microscopy Techniques |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |