Nanopore gene sequencing structure and preparation method thereof
By designing passivated layer pores as central through-holes and surface area expansion structures in nanopore gene sequencing structures, the problem of reduced lifetime and accuracy caused by the accumulation of organic phase in electrode pores was solved, achieving long lifetime and high accuracy of gene sequencing structures.
Patent Information
- Application Number
- CN202511072741.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-31
- Publication Date
- 2025-10-28
Smart Images
Figure CN120843253A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of semiconductor technology, specifically relating to a nanopore gene sequencing structure and its preparation method. Background Technology
[0002] With the successful completion of the Human Genome Project, genome sequencing technology has made great strides. Over the past decade, the continuous advancement of next-generation sequencing (NGS) technology has led to a continuous reduction in the cost of genome sequencing and a continuous improvement in sequencing throughput and accuracy, providing important technical support for deconstructing complex genome structures and answering questions about phenotypic changes and disease mechanisms caused by genome variations.
[0003] However, the amplification errors introduced by PCR during library construction in NGS sequencing, along with read length limitations (typically less than 500 bp), make it difficult for NGS technology to meet the higher requirements of some modern biological problems, such as the determination of long repetitive fragments on DNA and the determination of DNA / RNA methylation modifications. Therefore, nanopore sequencing, a third-generation sequencing technology capable of determining longer reads (up to 10 kbp) without complex library construction, has emerged and received widespread attention in recent years. Nanopore sequencing can achieve sequencing speeds of approximately 10 deoxynucleoside triphosphates (dNTPs) per second, but its error rate is relatively high, reaching 15%. Because errors are randomly generated, it does not exhibit the biased errors common in second-generation sequencing technologies, allowing for effective error correction through multiple sequencing runs. However, due to the concentration gradient of the organic phase during testing, the organic phase in third-generation nanopores slowly spreads through the sidewalls to the bottom during prolonged use, becoming non-conductive. This significantly reduces electrode lifespan and accuracy, essentially limiting its use to single-use testing. To address this anomaly, short-duration testing is often employed, which introduces some error into both electrode well lifespan and accuracy. Another approach is to increase the electrode well area to mitigate coverage, but this increases the well area and reduces the number of wells per unit area, thus impacting the cost of gene sequencing.
[0004] To address the aforementioned issues, a nanopore gene sequencing structure and its preparation method are needed to extend the lifespan of gene sequencing chips and ensure the accuracy of gene sequencing. Summary of the Invention
[0005] This invention addresses all or part of the problems of the prior art by providing a nanopore gene sequencing structure and its preparation method. By modifying the structure of the passivation layer in the nanopore gene sequencing structure, the passivation layer pores are designed as a central through-hole and a surface area expansion structure surrounding the central through-hole. While maintaining the effective area of the electrode pores essentially unchanged, the specific surface area of the passivation layer pores is increased, thus balancing sequencing sensitivity and reliability while delaying pore blockage and extending the device's lifespan.
[0006] This invention provides a nanopore gene sequencing structure, comprising: an electrode layer, a passivation layer, and a biofilm layer stacked sequentially; through-holes, coaxial vias, are formed in the electrode layer, passivation layer, and biofilm layer, respectively corresponding to electrode pores, passivation layer pores, and biofilm layer pores; the passivation layer pores include a central via and surface area extension structures surrounding the central via. By increasing the specific surface area of the passivation layer pores without substantially changing the electrode pore area, the accumulation of organic phase is slowed down, thereby improving the accuracy and lifetime of third-generation gene sequencing electrode pores.
[0007] The surface area expansion structure is a continuous closed polygonal structure. By increasing the specific surface area of the passivation layer pores through the polygonal ring structure, the organic phase flows downward along the outer polygonal ring structure, effectively reducing the accumulation of organic phase in the electrode pores.
[0008] The surface area expansion structure is composed of uniformly arranged polygonal or circular toothed units. This disperses the organic phase flow path and reduces its accumulation in the electrode orifices.
[0009] The diameter of the electrode hole is 50-70 μm to ensure sufficient detection sensitivity.
[0010] The central through-hole of the passivation layer has the same diameter as the electrode hole, and the organic phase flows along the sidewall surface area expansion structure, inhibiting its accumulation in the electrode hole.
[0011] The thickness of the electrode layer is Ensure good conductivity and stability of the electrode layer to guarantee the signal sensitivity of the test.
[0012] The biofilm layer has a thickness of 90-130 μm, which facilitates the effective capture of biological signals.
[0013] A method for preparing a nanopore gene sequencing structure is also provided, comprising the following steps: S1: providing an electrode layer formed of conductive metal, and forming electrode pores in the electrode layer; S2: forming a passivation layer on the electrode layer, and forming passivation layer pores in the passivation layer, wherein the passivation layer pores include a central through-hole and a surface area extension structure surrounding the central through-hole; S3: depositing a biofilm layer on the passivation layer, and forming biofilm layer pores in the biofilm layer, thereby achieving controllable fabrication of a high-performance nanopore gene sequencing structure.
[0014] In step S2, the passivation layer is formed using plasma-enhanced chemical vapor deposition. The thickness of the passivation layer is precisely controlled, ensuring the morphological integrity of the subsequent passivation layer pore surface area expansion structure.
[0015] The passivation layer is a silicon oxide layer or a silicon nitride layer, which achieves effective isolation.
[0016] Compared with the prior art, the beneficial effects of the present invention mainly include the following: by modifying the structure of the passivation layer in the nanopore gene sequencing structure, the passivation layer pore is designed as a structure composed of a central through hole and a surface area extension structure surrounding the central through hole. While keeping the effective area of the electrode pore unchanged, the specific surface area of the passivation layer pore is increased, which takes into account both sequencing sensitivity and reliability, delays pore blockage, and extends the service life of the device. Attached Figure Description
[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0018] Figure 1-2 This is a schematic diagram of the accumulation of organic phases in existing nanopore gene sequencing structures.
[0019] Figure 3 This is a schematic diagram of the nanopore gene sequencing structure provided by the present invention.
[0020] Figure 4 This is a schematic diagram of organic phase accumulation for nanopore gene sequencing structure sequencing provided by the present invention.
[0021] Figure description: 1-substrate, 2-electrode layer, 21-electrode hole, 3-passivation layer, 31-passivation layer hole, 4-biofilm layer, 41-biofilm layer hole. Detailed Implementation
[0022] The following description and accompanying drawings fully illustrate specific embodiments of the invention to enable those skilled in the art to practice them. Other embodiments may include structural, logical, electrical, procedural, and other changes. The embodiments represent only possible variations. Individual components and functions are optional unless explicitly required, and the order of operation may vary. Some portions and features of some embodiments may be included in or replace portions and features of other embodiments.
[0023] Third-generation gene sequencing technology relies on electrode pore structures to convert biological signals into electrical signals. Its core principle is to drive DNA / RNA molecules through nanopores immersed in an aqueous buffer solution using an electric field, detecting the changes in ionic current caused by the molecules' passage to achieve base recognition and gene sequencing. However, some drawbacks exist in practical applications. The main issue is that during long sequencing processes, organic phase can slowly permeate through the sidewalls of the electrode pores to the bottom electrode pores due to concentration gradients or structural defects. This leads to the accumulation of organic molecules in the bottom electrode pores, resulting in a lack of conductivity at the bottom. Figure 1-2 As shown, this reduces electrode well lifespan and sequencing accuracy.
[0024] Example 1
[0025] This embodiment provides a nanopore gene sequencing structure, such as Figure 3 As shown, it includes: a substrate 1, which is a semiconductor chip structure, including a substrate and integrated signal processing circuitry; an electrode layer 2 is formed on the substrate 1, and the electrode layer 2 is typically made of a metal with low resistivity, such as Pt, Al, or Cu, with a thickness generally of [missing information]. Electrode pores 21 are formed in electrode layer 2. Electrode pores 21 are conductive channels for converting the corresponding biological signals of third-generation gene sequencing into electrical signals. The diameter of the electrode pores is generally 50-70 μm. In this embodiment, the diameter of the electrode pores is 60 μm.
[0026] A passivation layer 3 is formed on the electrode layer 2, and a passivation layer hole 31 coaxially connected to the electrode hole 21 is formed on the passivation layer 3. The passivation layer hole 31 includes a central through hole and a surface area expansion structure surrounding the central through hole. The surface area expansion structure is a continuous closed polygonal structure composed of uniformly arranged polygonal tooth units or circular tooth units. In this embodiment, the surface area expansion structure is composed of uniformly arranged triangular tooth units. During sequencing, when the organic phase slowly spreads downward along the sidewall of the passivation layer hole 31 under the action of the concentration gradient, it will first spread along the outer polygonal ring structure, thereby reducing the accumulation of organic phase in the electrode hole 21. The central through hole of the passivation layer hole 31 has the same diameter as the electrode hole 21, and the larger size of the outer periphery of the passivation layer hole 31 can disperse the flow path of the organic phase and inhibit the accumulation of organic phase in the electrode hole 21.
[0027] A biofilm layer 4 is formed on the passivation layer 3. The passivation layer 3 can be a silicon oxide layer, silicon nitride layer, etc., and is typically 2 μm thick. It is used to separate the lower electrode layer 2 from the upper biofilm layer 4. Biofilm layer 4 contains biofilm layer pores 41 that are coaxially connected to the passivation layer pores 31. The biofilm layer 4 plays a crucial role in third-generation gene sequencing and is the actual functional layer for capturing biological signals; it is generally a membrane layer composed of organic matter, and its thickness is typically 90 μm-130 μm.
[0028] Example 2
[0029] This embodiment provides a method for preparing a nanopore gene sequencing structure, used to prepare the nanopore gene sequencing structure described in Example 1, including the following steps:
[0030] S1: A substrate 1 is provided, which is a semiconductor chip structure, including a substrate and an integrated signal processing circuit; an electrode layer 2 is formed by depositing a conductive metal on the substrate 1. The conductive metal can be a low-resistance metal such as Pt, Al, or Cu, and an electrode hole 21 is formed in the electrode layer 2.
[0031] S2: A passivation layer 3 is formed on the electrode layer 21 by plasma-enhanced chemical vapor deposition. Then, through processes such as coating, exposure, development, etching, resist removal, and cleaning, a passivation layer hole 31 with an extended surface area structure is formed in the passivation layer 3.
[0032] S3: Deposit a biofilm layer 4 on the passivation layer 3, and form biofilm pores 41 on the biofilm layer 4.
[0033] The prepared nanopore gene sequencing structure, without significantly altering the area of electrode pore 21, improves the accuracy and lifespan of the third-generation gene sequencing electrode pores by increasing the specific surface area of the passivation layer pore 31, thereby reducing the accumulation of organic phase in the bottom electrode pore 21. Figure 4 As shown, during the sequencing process, the organic phase slowly spreads along the sidewall due to the concentration gradient. When it flows through the passivation layer pore 31, it disperses the flow path of the organic phase, causing the organic phase to spread along the sidewall of the polygonal ring structure of the passivation layer pore 31. This inhibits the accumulation of the organic phase in the bottom electrode pore 21, improves the sequencing structure accuracy, and extends the structure lifetime.
[0034] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.
Claims
1. A nanopore gene sequencing structure, characterized in that, include: The electrode layer, passivation layer, and biofilm layer are stacked in sequence. Through-holes are formed in the electrode layer, passivation layer and biofilm layer, which correspond to electrode holes, passivation layer holes and biofilm layer holes, respectively. The passivation layer hole includes a central through-hole and a surface area extension structure surrounding the central through-hole.
2. The nanopore gene sequencing structure according to claim 1, characterized in that, The surface area expansion structure is a continuously closed polygonal structure.
3. The nanopore gene sequencing structure according to claim 2, characterized in that, The surface area expansion structure is composed of uniformly arranged polygonal tooth units or circular tooth units.
4. The nanopore gene sequencing structure according to claim 1, characterized in that, The diameter of the electrode hole is 50-70 μm.
5. The nanopore gene sequencing structure according to claim 1, characterized in that, The central through-hole of the passivation layer is the same diameter as the electrode hole.
6. The nanopore gene sequencing structure according to claim 1, characterized in that, The thickness of the electrode layer is 7. The nanopore gene sequencing structure according to claim 1, characterized in that, The thickness of the biofilm layer is 90-130 μm.
8. A method for preparing nanopore gene sequencing structures, characterized in that, The preparation of the nanopore gene sequencing structure according to any one of claims 1-7 includes the following steps: S1: Provide an electrode layer formed of conductive metal, in which electrode holes are formed; S2: A passivation layer is formed on the electrode layer, and a passivation layer hole is formed in the passivation layer, the passivation layer hole including a central through hole and a surface area extension structure surrounding the central through hole; S3: Deposit a biofilm layer on the passivation layer and form biofilm pores in the biofilm layer.
9. The method for preparing nanopore gene sequencing structures according to claim 8, characterized in that, In step S2, the passivation layer is formed using a plasma-enhanced chemical vapor deposition process.
10. The method for preparing nanopore gene sequencing structures according to claim 8, characterized in that, The passivation layer is a silicon oxide layer or a silicon nitride layer.