CN113109403B - Multichannel biomolecule detection chip based on array nano-holes and manufacturing method thereof - Google Patents

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

Multi-channel biomolecular detection chip based on arrayed nanopores and manufacturing method thereof

technical field

The present disclosure relates to the technical field of nanopore molecular detection, in particular to a multi-channel biomolecular detection chip based on array nanopores and a manufacturing method thereof.

Background technique

The detection of biomolecules based on nanopore technology originated from the invention of the Coulter counter and the single-channel current recording technology. By adding an electrolyte solution to the liquid pool at both ends, and using electrophoresis technology to drive biomolecules through the nanopore, the biological The change of nanopore current caused by molecules due to space occupation can be used to detect through-pore biomolecules. Therefore, nanopore detection technology is widely used in single-molecule research fields such as DNA detection and protein detection, and is also considered to be a detection technology that can be used for third-generation gene sequencing. However, the current nanopore detection technology only drives biomolecules through a single nanopore, and there are problems such as easy clogging and low efficiency during the passage process. How to improve the passage efficiency to achieve high-throughput screening of biomolecules is an urgent need to be solved question.

Contents of the invention

The present disclosure provides a multi-channel biomolecule detection chip based on arrayed nanopores and its manufacturing method. Its technical purpose is to effectively detect the current changes of multiple nanopores passing through biomolecules at the same time, and then realize the rapid detection and high-speed detection of biomolecules. Throughput screening.

The above-mentioned technical purpose of the present disclosure is achieved through the following technical solutions:

A multi-channel biomolecular detection chip based on arrayed nanopores, comprising a porous nanoporous membrane, at least two nanowires and an encapsulation layer of silicon oxide, each of the nanowires is independently distributed, and the porous nanopore membrane includes a bottom-up A lower silicon oxide layer, a silicon substrate, and an upper silicon oxide layer are connected on top, and the silicon oxide of the encapsulation layer is connected to the upper silicon oxide layer;

The nanowires are arranged on the upper surface of the upper silicon oxide layer and encapsulated by the silicon oxide encapsulation layer, each of the nanowires is provided with a nanohole at one end and a hole at the other end, and the nanowires A hole penetrates the silicon oxide of the packaging layer and the upper silicon oxide layer, the hole penetrates the silicon oxide of the packaging layer, and the hole is used as a lead-out port to connect to an external circuit;

Wherein, the nanoholes are arranged in an array on the upper surface of the silicon oxide encapsulation layer.

Further, at the position corresponding to the bottom of the nanopore in the upper silicon oxide layer: both the silicon substrate and the lower silicon oxide layer form an exposure window, and the exposure window is used to allow the bottom of the nanopore to A film window is formed.

A method for making a multi-channel biomolecular detection chip based on array nanopores, comprising:

S1: By low-pressure chemical vapor deposition, deposit a lower silicon oxide layer and an upper silicon oxide layer on the lower surface and the upper surface of the silicon substrate, respectively;

S2: Apply photoresist on the upper silicon oxide layer, and harden the film to form a first protective layer;

S3: Apply photoresist on the lower silicon oxide layer, and perform photolithography, development, cleaning, drying, and hardening, and the lower silicon oxide layer forms a second protective layer with exposed windows;

S4: using a reactive ion etching system to remove the silicon oxide at the exposed window, and place the silicon substrate in a potassium hydroxide solution to etch the silicon substrate to obtain a film window on the lower surface of the upper silicon oxide layer;

S5: cleaning the film window obtained in step S4 with a piranha solution to obtain a large amount of hydroxyl groups on its surface;

S6: placing the film window obtained in step S5 on the nano-motion platform, and measuring the capacitance change between the nano-needle and the nano-motion platform, and determining the position coordinates of the film window;

S7: Directional deposition of polyamic acid nanowires on film windows using near-field electrospinning technology;

S8: Immerse the polyamic acid nanowires obtained in step S7 in a silver salt solution to generate a silver carboxylate salt and wash it, then immerse it in a reducing solution to reduce it to silver particles and wash it, repeat step S8 repeatedly until polyamic acid-silver is formed Composite nanowires;

S9: Sintering the polyamic acid-silver composite nanowire under the protection of an inert gas to obtain a polyamic acid-silver composite nanowire;

S10: Using magnetron sputtering technology, covering the encapsulation layer silicon oxide on the polyamic acid-silver composite nanowire;

S11: using a focused ion beam to punch holes and cut thin at both ends of the polyamic acid-silver composite nanowire to obtain a multi-channel biomolecule detection chip;

Wherein, one end of each polyamic acid-silver composite nanowire is punched with a nanohole, and the other end is punched with a hole, the nanohole runs through the silicon oxide of the packaging layer and the upper silicon oxide layer, and the hole runs through The packaging layer is silicon oxide, and the hole is used as a lead-out port to connect to an external circuit;

Wherein, the nanoholes are arranged in an array on the upper surface of the silicon oxide encapsulation layer.

The beneficial effect of the present disclosure is that: the multi-channel biomolecular detection chip based on arrayed nanopores and its manufacturing method described in the present invention can independently detect and control the current change in a single nanopore through a plurality of nanowires, so that each nanopore can be realized The opening and closing control of biomolecules can be blocked or allowed; at the same time, biomolecules can be simultaneously multi-channeled to achieve rapid detection and high-throughput screening of biomolecules.

Description of drawings

1 is a schematic cross-sectional view of a multi-channel biomolecule detection chip based on arrayed nanopores according to the present invention;

2 is a schematic cross-sectional view of a multi-channel biomolecule detection chip based on arrayed nanopores according to the present invention;

3 is a schematic diagram of the array distribution of nanowires when the detection chip of the present invention is viewed from above;

4 is a schematic diagram of step S1 of the method for manufacturing the detection chip of the present invention;

5 is a schematic diagram of step S3 of the method for manufacturing the detection chip of the present invention;

6 is a schematic diagram of step S4 of the method for manufacturing the detection chip of the present invention;

7 is a schematic diagram of step S5 of the method for manufacturing the detection chip of the present invention;

Fig. 8 is a schematic diagram of step S7 of the manufacturing method of the detection chip of the present invention;

Fig. 9 is a schematic diagram of step S8 of the manufacturing method of the detection chip of the present invention;

Fig. 10 is a schematic diagram of step S9 of the manufacturing method of the detection chip of the present invention;

Fig. 11 is a schematic diagram of step S10 of the manufacturing method of the detection chip of the present invention;

In the figure: 1-porous nanoporous film; 2-nanometer wire; 3-silicon oxide encapsulation layer; 4-exit port; 5-first protective layer; 6-second protective layer; 7-exposed window; 8-nanopore 9-hole; 101-lower silicon oxide layer; 102-silicon substrate; 103-upper silicon oxide layer; 104-film window; 201-polyamic acid nanowire; 202-silver particles.

Detailed ways

The technical solution of the present disclosure will be described in detail below in conjunction with the accompanying drawings. In the description of the present invention, it should be understood that the terms "first" and "second" are only used for descriptive purposes, and cannot be interpreted as indicating or implying relative importance or implicitly indicating the quantity of indicated technical features, Used only to distinguish different components.

In addition, the terms "top-down", "upper", "lower", "upper surface", "lower surface", "one end", "other end", "display", "bottom", "middle", etc. indicate The orientation or positional relationship is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the referred device or element must have a specific orientation or be configured in a specific orientation. and operation, and therefore should not be construed as limiting the invention. In addition, "inside and outside" means inside and outside with respect to the outline of each member itself.

Figure 1 is a schematic cross-sectional view of a multi-channel biomolecular detection chip based on arrayed nanopores according to the present invention. As shown in Figure 1, the detection chip includes a porous nanoporous membrane 1, at least two nanowires 2 and an encapsulation layer of silicon oxide 3. Each nanowire 2 is independently distributed. The porous nanoporous membrane 1 includes a lower silicon oxide layer 101 , a silicon substrate 102 and an upper silicon oxide layer 103 connected from bottom to top, and the packaging layer silicon oxide 3 is connected to the upper silicon oxide layer 103 .

The nanowire 2 is arranged on the upper surface of the upper silicon oxide layer 103 and encapsulated by the packaging layer silicon oxide 3, that is to say, the nanowire 2 is arranged in the middle of the upper silicon oxide layer 103 and the packaging layer silicon oxide 3, as shown in FIG. 2 . One end of each nanowire 2 is provided with a nanohole 8, and the other end is provided with a hole 9, the nanohole 8 penetrates the packaging layer silicon oxide 3 and the upper silicon oxide layer 103, the hole 9 penetrates the packaging layer silicon oxide 3, and the hole 9 Connect to an external circuit as the lead-out port 4.

Preferentially, as can be seen from FIG. 2 , the nanowire 2 is in the middle of the upper silicon oxide layer 103 and the packaging layer silicon oxide 3 , one end of the nanowire 2 is a nanohole 8 , and the end of the nanowire 2 is basically located in the nanohole 8 The middle position or slightly off the middle position. Therefore, both ends of the nanohole 8 and the nanowires 2 in the middle can be used as electrodes, and the potentials at both ends of the nanohole 8 can be controlled through the nanowires 2 .

As can be seen from FIG. 1 and FIG. 2, at the corresponding positions of the nanoholes 8 on the upper silicon oxide layer 103, the silicon substrate 102 and the lower silicon oxide layer 101 all form an exposure window 7, which is to allow the upper The bottom of the nanopore 8 of the silicon oxide layer 103 becomes a suspended film (ie, the film window 104) and is formed by etching, that is, a large hole (exposed window 7) formed by reactive ion etching and chemical wet etching, rather than Nanopores punched out.

Figure 3 is a schematic diagram of the array distribution of nanowires when the detection chip of the present invention is viewed from above, as shown in Figure 3, there are at least two nanowires, and according to actual needs, a plurality of nanowires can be arranged on the upper surface of the upper silicon oxide layer 103 2, then the nanoholes 8 corresponding to each nanowire 2 are arranged in an array on the upper surface of the silicon oxide 3 of the packaging layer; similarly, the holes at one end of the nanowire 2 are also arranged in an array.

The drawings of the manufacturing method of the multi-channel biomolecular detection chip based on the array nanopore described in the present disclosure are shown in Figure 4 to Figure 11, specifically including:

S1: A lower silicon oxide layer 101 and an upper silicon oxide layer 103 are deposited on the front and back sides of the silicon substrate 102 by low-pressure chemical vapor deposition, as shown in FIG. 4 .

S2: Apply photoresist on the upper silicon oxide layer 103, and harden the film to form the first protection layer 5.

S3: Apply photoresist on the lower silicon oxide layer 101, and perform photolithography, development, cleaning, drying, and film hardening, and the lower silicon oxide layer 101 forms a second protective layer 6 with an exposed window 7, as shown in FIG. 5 .

S4: Use a reactive ion etching system to remove the silicon oxide at the exposure window 7, and place the silicon substrate 102 in a potassium hydroxide solution to etch the silicon substrate 102 to obtain the lower surface of the upper silicon oxide layer 103 The film window 104 is shown in FIG. 6 .

S5: Cleaning the film window 104 obtained in step S4 with a piranha solution to obtain a large amount of hydroxyl groups on the surface, as shown in FIG. 7 .

S6: Place the film window 104 obtained in step S5 on the nano-motion platform, measure the capacitance change between the nano-needle and the nano-motion platform, and determine the position coordinates of the film window 104 .

In step S6, the nano-motion platform is the part where the entire chip is placed and fixed. A substrate is installed on the nano-motion platform. The substrate is generally a metal substrate. The detection chip is fixed on the metal substrate, and the horizontal movement is controlled by the nano-motion platform to measure the nano needle. The capacitance change between the nano-motion platform is actually the measurement of the capacitance change between the nano-needle and the metal substrate.

S7: Using near-field electrospinning technology, directionally deposit polyamic acid nanowires 201 on the film window 104 , as shown in FIG. 8 .

S8: Immerse the polyamic acid nanowires 201 obtained in step S7 into a silver salt solution to generate a silver carboxylate salt and wash it, then immerse it in a reducing solution to reduce it to silver particles 202 and wash it, repeat step S8 repeatedly until the polyamic acid is formed - Silver composite nanowires, as shown in FIG. 9 .

S9: Sintering the polyamic acid-silver composite nanowire under the protection of an inert gas to obtain a polyamic acid-silver composite nanowire 2 , as shown in FIG. 10 .

S10: Cover the polyamic acid-silver composite nanowire 2 with an encapsulation layer of silicon oxide 3 by using magnetron sputtering technology, as shown in FIG. 11 .

S11: Using a focused ion beam to punch holes and cut thin at both ends of the polyamic acid-silver composite nanowire 2 to obtain a multi-channel biomolecule detection chip.

Step S11, one end of each polyamic acid-silver composite nanowire 2 is punched with a nanohole 8, and the other end is punched with a hole 9, the nanohole 8 runs through the packaging layer silicon oxide 3 and the upper silicon oxide layer 103, and the nanohole 8 Located above the thin film window 104 , at the same time, the nanoholes 8 are arranged in an array on the upper surface of the packaging layer silicon oxide 3 ; the holes 9 penetrate the packaging layer silicon oxide 3 , and the holes 9 serve as lead-out ports 4 to connect to external circuits.

In step S11, when one end of the polyamic acid-silver composite nanowire 2 is punched 9, the packaging layer silicon oxide 3 at the lead-out port 4 is thinned to expose the polyamic acid-silver composite nanowire 2, Platinum metal is then deposited at the hole using a focused ion beam, resulting in the extraction port 4 .

In step S4, when etching the silicon substrate 102, the silicon substrate 102 should be fully etched and the upper silicon oxide layer 103 cannot be etched through. Here, the standard of sufficient etching is not to etch through the upper silicon oxide layer 103 , and the specific etching degree will be determined according to the etching thickness, temperature, and concentration of potassium hydroxide.

The above are exemplary embodiments of the present disclosure, and the protection scope of the present disclosure 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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