CN113109403B - Multichannel biomolecule detection chip based on array nano-holes and manufacturing method thereof - Google Patents
Multichannel biomolecule detection chip based on array nano-holes and manufacturing method thereof Download PDFInfo
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- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
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Abstract
The invention discloses a multichannel biomolecule detection chip based on array nanopores and a manufacturing method thereof, relates to the technical field of nanopore molecule detection, and solves the technical problem of low detection efficiency of the existing biomolecule via holes; meanwhile, the method can develop the simultaneous multichannel via holes of the biomolecules, and realize the rapid detection and high-throughput screening of the biomolecules.
Description
Technical Field
The disclosure relates to the technical field of nanopore molecular detection, in particular to a multichannel biomolecule detection chip based on array nanopores and a manufacturing method thereof.
Background
The invention based on the nanopore technology for detecting biomolecules originates from a Coulter counter and a single-channel current recording technology, and the detection of the via biomolecules can be carried out by adding electrolyte solution into liquid pools at two ends and using electrophoresis technology to drive the biomolecules to pass through the nanopores and measuring the nanopore current change of the biomolecules caused by space occupation in the via process. Therefore, the nanopore detection technology is widely applied to the single-molecule research fields of DNA detection, protein detection and the like, and is also considered as a detection technology for third-generation gene sequencing. However, the current nanopore detection technology only drives biomolecules to pass through a single nanopore, and the problems of easy blocking, low via efficiency and the like exist in the process of via, so that how to improve the via efficiency to realize high-throughput screening of the biomolecules is a problem to be solved currently.
Disclosure of Invention
The present disclosure provides a multichannel biomolecule detection chip based on array nanopores and a manufacturing method thereof, which aims to effectively and simultaneously detect current changes of a plurality of nanopores passing biomolecules, thereby realizing rapid detection and high-throughput screening of the biomolecules.
The technical aim of the disclosure is achieved by the following technical scheme:
the multichannel biomolecule detection chip based on the array nanopores comprises a porous nanopore membrane, at least two nanowires and a packaging layer silicon oxide, wherein each nanowire is independently distributed, the porous nanopore membrane comprises a lower silicon oxide layer, a silicon substrate and an upper silicon oxide layer which are connected from bottom to top, and the packaging layer silicon oxide is connected with the upper silicon oxide layer;
the nano wires are arranged on the upper surface of the upper silicon oxide layer and are encapsulated through the encapsulation layer silicon oxide, one end of each nano wire is provided with a nano hole, the other end of each nano wire is provided with a hole, the nano holes penetrate through the encapsulation layer silicon oxide and the upper silicon oxide layer, the holes penetrate through the encapsulation layer silicon oxide, and the holes serve as leading-out ports to be connected with an external circuit;
wherein the nanopores are arranged in an array on the upper surface of the encapsulation layer silicon oxide.
Further, at corresponding locations of the nanopore bottom in the upper silicon oxide layer: the silicon substrate and the lower silicon oxide layer both form an exposure window, and the exposure window is used for forming a thin film window at the bottom of the nanopore.
A manufacturing method of a multichannel biomolecule detection chip based on array nanopores comprises the following steps:
s1, respectively depositing a lower silicon oxide layer and an upper silicon oxide layer on the lower surface and the upper surface of a silicon substrate by a low-pressure chemical vapor deposition method;
s2: coating photoresist on the upper silicon oxide layer, and hardening to form a first protective layer;
s3: coating photoresist on the lower silicon oxide layer, and carrying out photoetching, developing, cleaning, spin-drying and hardening, wherein the lower silicon oxide layer forms a second protective layer with an exposure window;
s4: removing the silicon oxide at the exposed window by using a reactive ion etching system, and placing the silicon substrate in potassium hydroxide solution to etch the silicon substrate to obtain a film window on the lower surface of the upper silicon oxide layer;
s5: washing the film window obtained in the step S4 by using a piranha solution to obtain a large number of hydroxyl groups on the surface of the film window;
s6: placing the thin film window obtained in the step S5 on a nanometer motion platform, measuring the capacitance change between the nanometer needle head and the nanometer motion platform, and determining the position coordinates of the thin film window;
s7: using near field electrospinning technology to directionally deposit polyamic acid nanowires on the thin film window;
s8: immersing the polyamic acid nanowire obtained in the step S7 into silver salt solution to generate silver carboxylate salt and cleaning, immersing into reducing solution to reduce into silver particles and cleaning, and repeating the step S8 repeatedly until the polyamic acid-silver composite nanowire is formed;
s9: performing inert gas protection sintering on the polyamic acid-silver composite nanowire to obtain a polyamic acid-silver composite nanowire;
s10: covering packaging layer silicon oxide on the polyamide acid-silver composite nano wire by using a magnetron sputtering technology;
s11: punching and thinning the two ends of the polyamide acid-silver composite nano wire by using a focused ion beam to obtain a multichannel biomolecule detection chip;
one end of each polyamide acid-silver composite nanowire is provided with a nano hole, the other end of each polyamide acid-silver composite nanowire is provided with a hole, the nano holes penetrate through the packaging layer silicon oxide and the upper silicon oxide layer, the holes penetrate through the packaging layer silicon oxide, and the holes are used as leading-out ports to be connected with an external circuit;
wherein the nanopores are arranged in an array on the upper surface of the encapsulation layer silicon oxide.
The beneficial effects of the present disclosure are: according to the multichannel biomolecule detection chip based on the array nanopores and the manufacturing method thereof, the opening and closing control of each nanopore can be realized by independently detecting and controlling the current change in the single nanopore through a plurality of nanowires, and the biomolecule through holes can be blocked or allowed; meanwhile, the method can develop the simultaneous multichannel via holes of the biomolecules, and realize the rapid detection and high-throughput screening of the biomolecules.
Drawings
FIG. 1 is a schematic cross-sectional view of a multichannel biomolecule detection chip based on array nanopores according to the present invention;
FIG. 2 is a schematic cross-sectional view of a multichannel biomolecule detection chip based on array nanopores according to the present invention;
FIG. 3 is a schematic diagram showing the distribution of nanowires in a top view of the detection chip according to the present invention;
FIG. 4 is a schematic diagram of step S1 of the method for manufacturing a detection chip according to the present invention;
FIG. 5 is a schematic diagram of step S3 of the method for manufacturing a detection chip according to the present invention;
FIG. 6 is a schematic diagram of step S4 of the method for manufacturing a detection chip according to the present invention;
FIG. 7 is a schematic diagram of step S5 of the method for manufacturing a detection chip according to the present invention;
FIG. 8 is a schematic diagram of step S7 of the method for manufacturing a detection chip according to the present invention;
FIG. 9 is a schematic diagram of step S8 of the method for manufacturing a detection chip according to the present invention;
FIG. 10 is a schematic diagram of step S9 of the method for manufacturing a detection chip according to the present invention;
FIG. 11 is a schematic diagram of step S10 of the method for manufacturing a detection chip according to the present invention;
in the figure: 1-a porous nanoporous membrane; 2-nanowires; 3-encapsulation layer silicon oxide; 4-an extraction port; 5-a first protective layer; 6-a second protective layer; 7-exposing the window; 8-nanopores; 9-holes; 101-a lower silicon oxide layer; 102-a silicon substrate; 103-upper silicon oxide layer; 104-a film window; 201-polyamic acid nanowires; 202-silver particles.
Detailed Description
The technical scheme of the present disclosure will be described in detail below with reference to the accompanying drawings. In the description of the present invention, it should be understood that the terms "first" and "second" are used for descriptive purposes only and are not to be interpreted as indicating or implying any relative importance or number of such features or components in order to distinguish between different components.
In addition, the terms "top-down," "upper," "lower," "upper surface," "lower surface," "one end," "the other end," "display," "bottom," "middle," etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, merely to facilitate description of the invention and to simplify the description, and do not indicate or imply that the devices or elements being referred to must have a specific orientation, be configured and operated in a specific orientation, and therefore should not be construed as limiting the invention. In addition, "inner and outer" refer to inner and outer with respect to the outline of each component itself.
Fig. 1 is a schematic cross-sectional view of a multichannel biomolecule detection chip based on array nanopores according to the present invention, as shown in fig. 1, the detection chip comprises a porous nanopore membrane 1, at least two nanowires 2 and an encapsulation layer of silica 3, each nanowire 2 being independently distributed. The porous nano-pore membrane 1 comprises a lower silicon oxide layer 101, a silicon substrate 102 and an upper silicon oxide layer 103 which are connected from bottom to top, and the packaging layer silicon oxide 3 is connected with the upper silicon oxide layer 103.
The nanowire 2 is disposed on the upper surface of the upper silicon oxide layer 103 and encapsulated by the encapsulation layer silicon oxide 3, that is, the nanowire 2 is disposed in the middle of the upper silicon oxide layer 103 and the encapsulation layer silicon oxide 3, as shown in fig. 2. One end of each nanowire 2 is provided with a nanopore 8, the other end is provided with a hole 9, the nanopore 8 penetrates through the encapsulation layer silicon oxide 3 and the upper silicon oxide layer 103, the hole 9 penetrates through the encapsulation layer silicon oxide 3, and the hole 9 serves as an extraction port 4 to be connected with an external circuit.
Preferably, as can be seen from fig. 2, the nanowire 2 is located in the middle of the upper silicon oxide layer 103 and the encapsulation layer silicon oxide 3, one end of the nanowire 2 is a nanopore 8, and the end of the nanowire 2 is located substantially in the middle of the nanopore 8 or slightly offset from the middle. Thus, the nanowires 2 at both ends of the nanopore 8 and at the middle thereof can serve as electrodes, and the potentials at both ends of the nanopore 8 can be controlled by the nanowires 2, respectively.
As can be seen from fig. 1 and 2, at the corresponding positions of the nanopores 8 on the upper silicon oxide layer 103, the silicon substrate 102 and the lower silicon oxide layer 101 both form an exposure window 7, and the exposure window 7 is formed by etching to make the bottom of the nanopores 8 of the upper silicon oxide layer 103 a dangling thin film (i.e., a thin film window 104), i.e., macropores (exposure window 7) formed by reactive ion etching and chemical wet etching, instead of the punched nanopores.
Fig. 3 is a schematic diagram showing the distribution of the nanowire array of the present invention when the detection chip is in a top view, as shown in fig. 3, at least 2 nanowires are provided, and according to practical needs, a plurality of nanowires 2 may be disposed on the upper surface of the upper silicon oxide layer 103, so that the corresponding nanopores 8 of each nanowire 2 are arranged in an array on the upper surface of the encapsulation layer silicon oxide 3; similarly, the holes at one end of the nanowire 2 are also arranged in an array.
The drawings of the method for manufacturing the multichannel biomolecule detection chip based on the array nano-holes are shown in fig. 4 to 11, and specifically include:
s1, depositing a lower silicon oxide layer 101 and an upper silicon oxide layer 103 on the front side and the back side of a silicon substrate 102 by a low-pressure chemical vapor deposition method, as shown in FIG. 4.
S2: a photoresist is coated on the upper silicon oxide layer 103 and a film is formed to form the first protective layer 5.
S3: photoresist is coated on the lower silicon oxide layer 101, and photolithography, development, cleaning, spin-drying, and film hardening are performed, and the lower silicon oxide layer 101 forms the second protective layer 6 with the exposure window 7, as shown in fig. 5.
S4: the silicon oxide at the exposed window 7 is removed by using a reactive ion etching system, and the silicon substrate 102 is etched by placing the silicon substrate 102 in a potassium hydroxide solution, so as to obtain a thin film window 104 on the lower surface of the upper silicon oxide layer 103, as shown in fig. 6.
S5: the thin film window 104 obtained in step S4 is washed with a piranha solution to obtain a large number of hydroxyl groups on the surface thereof, as shown in fig. 7.
S6: and (5) placing the thin film window 104 obtained in the step (S5) on a nano motion platform, measuring the capacitance change between the nano needle head and the nano motion platform, and determining the position coordinates of the thin film window 104.
In step S6, the nano-motion platform is a part for placing and fixing the whole chip, the nano-motion platform is provided with a substrate, the substrate is generally a metal substrate, the detection chip is fixed on the metal substrate, and the nano-motion platform controls horizontal movement to measure the capacitance change between the nano-needle and the nano-motion platform, and in practice, the capacitance change between the nano-needle and the metal substrate is measured.
S7: using near field electrospinning techniques, polyamic acid nanowires 201 are directionally deposited at the thin film window 104, as shown in FIG. 8.
S8: the polyamic acid nanowire 201 obtained in the step S7 is immersed in a silver salt solution to generate a silver carboxylate salt and washed, then immersed in a reducing solution to reduce the silver carboxylate salt into silver particles 202 and washed, and the step S8 is repeated repeatedly until a polyamic acid-silver composite nanowire is formed, as shown in fig. 9.
S9: the polyamic acid-silver composite nanowire is subjected to inert gas protection sintering to obtain a polyamic acid-silver composite nanowire 2, as shown in fig. 10.
S10: using a magnetron sputtering technique, an encapsulation layer silicon oxide 3 is coated on the polyamic acid-silver composite nanowire 2, as shown in fig. 11.
S11: and punching and thinning the two ends of the polyamide acid-silver composite nanowire 2 by using a focused ion beam to obtain the multichannel biomolecule detection chip.
Step S11, one end of each polyamide acid-silver composite nanowire 2 is provided with a nano hole 8, the other end of each polyamide acid-silver composite nanowire is provided with a hole 9, the nano hole 8 penetrates through the packaging layer silicon oxide 3 and the upper silicon oxide layer 103, the nano hole 8 is positioned above the film window 104, and meanwhile, the nano holes 8 are arranged in an array on the upper surface of the packaging layer silicon oxide 3; the hole 9 penetrates the encapsulation layer silicon oxide 3, and the hole 9 is connected to an external circuit as the extraction port 4.
In step S11, when one end of the polyamic acid-silver composite nanowire 2 is perforated 9, the encapsulation layer silicon oxide 3 at the outlet port 4 is thinned to expose the polyamic acid-silver composite nanowire 2, and then a focused ion beam is used to deposit platinum metal at the hole, so as to obtain the outlet port 4.
In step S4, when the silicon substrate 102 is etched, the silicon substrate 102 should be etched sufficiently and the upper silicon oxide layer 103 cannot be etched through. The full etching is not performed to etch the upper silicon oxide layer 103 as a standard, and the specific etching degree is determined according to the etching thickness, the temperature and the potassium hydroxide concentration.
The foregoing is an exemplary embodiment of the disclosure, the scope of which is defined by the claims and their equivalents.
Claims (4)
1. The multichannel biomolecule detection chip based on array nanopores is characterized by comprising a porous nanopore membrane (1), at least two nanowires (2) and a packaging layer silicon oxide (3), wherein each nanowire (2) is independently distributed, the porous nanopore membrane (1) comprises a lower silicon oxide layer (101), a silicon substrate (102) and an upper silicon oxide layer (103) which are connected from bottom to top, and the packaging layer silicon oxide (3) is connected with the upper silicon oxide layer (103);
the nanowires (2) are arranged on the upper surface of the upper silicon oxide layer (103) and are packaged through the packaging layer silicon oxide (3), one end of each nanowire (2) is provided with a nanopore (8), the other end of each nanowire is provided with a hole (9), the nanopore (8) penetrates through the packaging layer silicon oxide (3) and the upper silicon oxide layer (103), the hole (9) penetrates through the packaging layer silicon oxide (3), and the hole (9) is used as an extraction port (4) to be connected with an external circuit;
wherein the nanopores (8) are arranged in an array on the upper surface of the encapsulation layer silicon oxide (3);
wherein, at corresponding positions of the bottom of the nanopores (8) in the upper silicon oxide layer (103): the silicon substrate (102) and the lower silicon oxide layer (101) both form an exposure window (7), and the exposure window (7) is used for forming a thin film window (104) at the bottom of the nanopore (8).
2. The manufacturing method of the multichannel biomolecule detection chip based on the array nano-holes is characterized by comprising the following steps:
s1, respectively depositing a lower silicon oxide layer (101) and an upper silicon oxide layer (103) on the lower surface and the upper surface of a silicon substrate (102) by a low-pressure chemical vapor deposition method;
s2: coating photoresist on the upper silicon oxide layer (103) and hardening to form a first protective layer (5);
s3: coating photoresist on the lower silicon oxide layer (101), and carrying out photoetching, developing, cleaning, spin-drying and hardening, wherein the lower silicon oxide layer (101) forms a second protective layer (6) with an exposure window (7);
s4: removing silicon oxide at the exposed window (7) by using a reactive ion etching system, and placing the silicon substrate (102) in potassium hydroxide solution to etch the silicon substrate (102) to obtain a film window (104) on the lower surface of the upper silicon oxide layer (103);
s5: washing the film window (104) obtained in the step S4 by using a piranha solution to obtain a large number of hydroxyl groups on the surface of the film window;
s6: placing the thin film window (104) obtained in the step S5 on a nanometer motion platform, measuring the capacitance change between the nanometer needle head and the nanometer motion platform, and determining the position coordinates of the thin film window (104);
s7: using near field electrospinning technique to directionally deposit polyamic acid nanowires (201) on the thin film window (104);
s8: immersing the polyamic acid nanowire (201) obtained in the step S7 into silver salt solution to generate silver carboxylate salt and cleaning, immersing into reducing solution to reduce into silver particles (202) and cleaning, and repeating the step S8 repeatedly until the polyamic acid-silver composite nanowire is formed;
s9: performing inert gas protection sintering on the polyamic acid-silver composite nanowire to obtain a polyamic acid-silver composite nanowire (2);
s10: covering packaging layer silicon oxide (3) on the polyamide acid-silver composite nanowire (2) by using a magnetron sputtering technology;
s11: punching and thinning the two ends of the polyamide acid-silver composite nanowire (2) by using a focused ion beam to obtain a multichannel biomolecule detection chip;
one end of each polyamide acid-silver composite nanowire (2) is provided with a nano hole (8) and the other end is provided with a hole (9), the nano holes (8) penetrate through the packaging layer silicon oxide (3) and the upper silicon oxide layer (103), the holes (9) penetrate through the packaging layer silicon oxide (3), and the holes (9) serve as an extraction port (4) to be connected with an external circuit;
wherein the nanopores are arranged in an array on the upper surface of the packaging layer silicon oxide (3).
3. The method for fabricating the array-nanopore-based multichannel biomolecule detection chip of claim 2, wherein in step S11, when one end of the polyamic acid-silver composite nanowire (2) is perforated, the encapsulation layer silicon oxide (3) at the extraction port (4) is thinned to expose the polyamic acid-silver composite nanowire (2), and then a focused ion beam is used to deposit platinum metal at the hole, thereby obtaining the extraction port (4).
4. The method for fabricating a multi-channel bio-molecular detection chip based on array nano-pores according to claim 3, wherein in step S4, the silicon substrate (102) is etched sufficiently and the upper silicon oxide layer (103) cannot be etched through when the silicon substrate (102) is etched.
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