Three-dimensional nanochannel electrode confined enhanced crisper / cas12a electrochemical sensor

Abstract

The application discloses a three-dimensional nanochannel electrode confined enhanced CRISPR / Cas12a electrochemical sensor. The electrochemical sensor utilizes micro-nano processing technology and dealloying process to construct a three-dimensional nanochannel electrode, and cooperatively regulates the pore size and specific surface area of the nanochannel through thermal annealing and electrochemical roughening methods. Based on the three-dimensional confined characteristics of the three-dimensional nanochannel electrode, the sensor constructs an electrochemical sensor efficiently coupled with the CRISPR / Cas12a system. The application successfully shortens the Cas12a cleavage reaction time from 2 hours required by a traditional planar electrode to 15 minutes, and realizes the super-sensitive detection of HPV-16 nucleic acid, with the sensitivity being about 20 times higher than that of the planar electrode, and the detection limit being as low as 2.8 pM. The sensor provides an efficient, accurate and portable nucleic acid detection solution for the fields of early tumor screening and instant diagnosis.

Description

Technical Field

[0001] This invention relates to the field of electrochemical biosensing and analytical detection technology, and in particular to a CRISPR / Cas12a electrochemical sensor based on a three-dimensional nanochannel electrically limited domain enhanced CRISPR / Cas12a, which enables rapid and ultrasensitive electrochemical detection of nucleic acid targets; its applications include early screening of malignant tumors and point-of-care testing (POCT). Background Technology

[0002] Electrochemical sensors have important applications in biomedicine, environmental monitoring, and other fields. However, traditional planar electrodes face challenges in signal intensity and sensitivity reduction during miniaturization, particularly in molecular diagnostics where high sensitivity is required. Reduced electrode size leads to decreased surface area, which in turn affects the signal-to-noise ratio and the reaction efficiency of probe molecules. To overcome this limitation, three-dimensional nanochannel electrodes have attracted widespread attention in recent years due to their high specific surface area and good biochemical adaptability. These electrodes can significantly improve sensitivity and lower the detection limit, but their signal enhancement mechanism remains incompletely understood, limiting their application. Meanwhile, the CRISPR / Cas12a system has become an emerging platform for nucleic acid detection due to its high targeting recognition and enzymatic cleavage capabilities. However, at the electrochemical sensor interface, the catalytic efficiency of the Cas12a protein is limited by the diffusion conditions at the solid-liquid interface. Therefore, optimizing the cleavage efficiency of the Cas12a protein in an electrochemical environment to improve detection sensitivity remains a pressing challenge in the field of nucleic acid detection. Therefore, this invention proposes to utilize the confinement effect of three-dimensional nanochannel electrodes and the synergistic effect of CRISPR / Cas12a to construct a high-efficiency nucleic acid detection platform, aiming to improve detection efficiency and sensitivity, and provide strong support for point-of-care diagnosis and early screening. Summary of the Invention

[0003] The purpose of this invention is to address the shortcomings of existing technologies by providing a CRISPR / Cas12a electrochemical sensor based on the three-dimensional nanochannel electric limiting domain enhancement.

[0004] The objective of this invention is achieved through the following technical solutions: constructing a three-dimensional nanochannel electrode using micro-nano fabrication technology and dealloying process; synergistically controlling the pore size and specific surface area of ​​the three-dimensional nanochannel based on thermal annealing and electrochemical roughening methods; and confining the Cas12a protein within the three-dimensional nanochannel by enhancing the confinement diffusion effect through the three-dimensional nanochannel, thereby constructing an electrochemical sensor coupled with the CRISPR / Cas12a system.

[0005] Furthermore, the micro / nano fabrication technology and dealloying process include: after photolithographically forming a seed layer pattern on the substrate surface, a chromium adhesion layer and a gold seed layer are deposited sequentially; after stripping the photoresist, a sensing area is photolithographically formed on the gold seed layer; a gold-silver composite layer is formed by gold-silver co-sputtering; after stripping the photoresist, the gold-silver composite layer is immersed in hot concentrated nitric acid to remove the silver phase, thereby obtaining a three-dimensional nanochannel electrode.

[0006] Furthermore, an insulating layer is formed outside the three-dimensional nanochannel electrode, exposing only the sensing area and electrode leads.

[0007] Furthermore, the pore size and specific surface area of ​​the three-dimensional nanochannels can be synergistically controlled through methods such as thermal annealing and electrochemical roughening.

[0008] Furthermore, thermal annealing enhances the surface diffusion of gold atoms, releases residual stress, and gradually enlarges the pores and thickens the branches; electrochemical roughening promotes the migration and recombination of gold atoms through redox cycles, making the pore structure coarser and more ordered.

[0009] Furthermore, the Cas12a protein is confined within a three-dimensional nanochannel, increasing local concentration and molecular interaction efficiency, thereby improving cleavage efficiency and signal output rate.

[0010] Furthermore, in the CRISPR / Cas12a system of electrochemical sensors, when the target nucleic acid binds to the Cas12a protein, the trans-cleavage activity of the Cas12a protein is activated, which cleaves the signal probe on the surface of the three-dimensional nanochannel, causing the electrochemical signal to decrease, thereby achieving quantitative detection of the nucleic acid target.

[0011] The beneficial effects of this invention are as follows: This invention proposes a CRISPR / Cas12a electrochemical sensor based on a three-dimensional nanochannel electrically limited domain enhancement. A three-dimensional nanochannel electrode is constructed using micro / nano fabrication technology and dealloying processes. Through thermal annealing and electrochemical roughening, the electrode pore size and surface morphology are controllably adjusted. Its unique three-dimensional channel structure effectively extends the range of the electric field, enhances the local concentration and molecular collision probability, and provides a confined diffusion environment for the Cas12a protein, thereby significantly improving the reaction efficiency and shearing activity of the CRISPR / Cas12a system. The electrochemical sensor of this invention has been successfully applied to the accurate detection of HPV-16 nucleic acid in human serum samples, demonstrating its application potential in complex systems. In summary, this invention provides an innovative solution for high-sensitivity, rapid nucleic acid detection, especially suitable for early tumor screening, point-of-care diagnosis, and the detection of other body fluid-related diseases, possessing significant application value and promotion potential. Attached Figure Description

[0012] Figure 1This is a schematic diagram of the fabrication steps for a nanochannel electrode array;

[0013] Figure 2 This is a schematic diagram showing the working principle and performance comparison of the three-dimensional nanochannel electrode synergistic CRISPR / Cas12a system;

[0014] Figure 3 The images are SEM images of nanochannel electrodes with and without thermal annealing treatment. (A) Nanochannel electrode without thermal annealing treatment; (B) to (D) are SEM images of nanochannel electrodes treated at 150°C, 200°C and 250°C for 20 minutes, respectively.

[0015] Figure 4 The images are SEM images of nanochannel electrodes with or without electrochemical roughening treatment. (A) Nanochannel electrode without electrochemical roughening treatment; (B) to (D) are SEM images of nanochannel electrodes after 100, 200 and 300 CV scans in 0.5 M H2SO4 with a voltage range of 0.3 V to 1.2 V, respectively.

[0016] Figure 5 The image shows the CV scans of nanochannel electrodes treated with (A) different thermal annealing temperatures and (B) different electrochemical roughening cycles in 0.05 M H2SO4.

[0017] Figure 6 The effect of nanochannel electrodes under different roughening treatments on the Cas12a protein shearing effect; (A) Nanoporous gold electrode without CV roughening treatment; (B) Nanoporous gold electrode after 100 cycles of CV roughening treatment; (C) Nanoporous gold electrode after 200 cycles of CV roughening treatment.

[0018] Figure 7 The comparison of the shear efficiency of CRISPR / Cas12a at different electrode interfaces is as follows: (A) SWV curves of nanoporous gold electrode before and after 10 minutes of shear reaction; (B) SWV curves of planar electrode before and after 10 minutes of shear reaction.

[0019] Figure 8 The performance comparison of nanochannel electrodes and planar electrodes in HPV-16 target detection is shown in (A) with the percentage change in current (ΔI, %) as the ordinate; and (B) with the absolute value of the change in current (ΔI, μA) as the ordinate. Detailed Implementation

[0020] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0021] This invention proposes a CRISPR / Cas12a electrochemical sensor based on a three-dimensional nanochannel electrically confined domain enhancement. This electrochemical sensor utilizes micro / nano fabrication techniques and dealloying processes to construct a three-dimensional nanochannel electrode; it employs a combination of thermal annealing and electrochemical roughening methods to synergistically control the pore size and specific surface area of ​​the three-dimensional nanochannel; and it enhances the confinement diffusion effect through the three-dimensional nanochannel, thereby confining the Cas12a protein within the three-dimensional nanochannel and constructing an electrochemical sensor coupled with the CRISPR / Cas12a system.

[0022] Specifically, three-dimensional nanochannel electrode fabrication methods include... Figure 1 As shown, firstly, the substrate surface is cleaned using oxygen plasma, and then uniformly coated and pre-baked with photoresist by spin coating to ensure uniform photoresist coverage. Next, the substrate is exposed using a mask (A), followed by post-baking and development to form a seed layer pattern. A chromium adhesion layer and a gold seed layer are sequentially deposited on the seed layer surface using magnetron sputtering. After photoresist stripping, the sensing area is photolithographically patterned on the gold seed layer using a mask (B). A gold-silver composite layer is formed by gold-silver co-sputtering. After photoresist stripping, the gold-silver composite layer is immersed in hot concentrated nitric acid for dealloying to remove the silver phase, yielding a three-dimensional nanochannel electrode. Finally, an insulating layer is formed outside the three-dimensional nanochannel electrode using a mask (C), exposing only the sensing area and electrode leads.

[0023] Specifically, the pore size and specific surface area of ​​the three-dimensional nanochannels are synergistically controlled through methods such as thermal annealing and electrochemical roughening.

[0024] Specifically, thermal annealing enhances the surface diffusion of gold atoms, which helps redistribute gold atoms, releases residual stress, and causes the system to tend towards a lower energy state, thereby gradually increasing the size of the pores and thickening the branches. Electrochemical coarsening promotes the migration and recombination of gold atoms through redox cycles. Gold atoms in high curvature regions migrate to lower curvature regions, causing pores and ligaments to gradually merge and form a larger and more regular pore structure.

[0025] Specifically, the Cas12a protein is confined within a three-dimensional nanochannel, increasing local concentration and molecular interaction efficiency, thereby improving cleavage efficiency and signal output rate.

[0026] Specifically, in the CRISPR / Cas12a system of electrochemical sensors, when the target nucleic acid binds to the Cas12a protein, the trans-cleavage activity of the Cas12a protein is activated, which cleaves the signal probe on the surface of the three-dimensional nanochannel, resulting in a decrease in the electrochemical signal, thus enabling quantitative detection of the nucleic acid target. Figure 2This study demonstrates the working principle and performance differences between three-dimensional nanochannel electrodes, the CRISPR / Cas12a system, and traditional planar electrodes in nucleic acid detection. The high specific surface area of ​​the three-dimensional nanochannel electrode can provide more fixation sites for the signal probe. In addition, the three-dimensional nanochannel electrode enhances the confined diffusion effect through its three-dimensional pore structure, confining the Cas12a protein within the pores, significantly improving local concentration and molecular interaction efficiency, thereby increasing cleavage efficiency and signal output rate. In contrast, planar electrodes lack a confined environment, making reactants prone to diffusion and loss, resulting in lower molecular collision and reaction frequencies and a significantly slower signal decline rate.

[0027] Example 1:

[0028] The surface morphology of the three-dimensional channel electrode was synergistically controlled by thermal annealing and electrochemical roughening. The prepared nanoporous gold electrode was thermally annealed at 150℃, 200℃, and 250℃ for 20 minutes, respectively. Characterization by scanning electron microscopy (SEM) revealed… Figure 3 The unannealed electrode has an average pore size of approximately 9.52 nm, exhibiting a fine and dense nanoporous structure. Figure 3 (A in the text); after treatment at 150℃, the pore size increased to 19.23 nm, and the edges became smooth ( Figure 3 (B in the text); when the annealing temperature rises to 200℃, the average diameter increases to approximately 23.52 nm, the pore size increases significantly and the shape tends to become more regular. Figure 3 (C in the text); when the temperature rises to 250℃, the pore size further expands to approximately 28.57 nm, and the pore and ligament shapes become more regular and uniform. Figure 3 (D in the text). This change reflects the ability of gold atoms to migrate and redistribute more freely at high temperatures, while the system reduces surface energy by decreasing surface area, thus achieving a more pronounced morphological adjustment.

[0029] Example 2:

[0030] Thermal annealing of three-dimensional nanochannel electrodes can adjust the nanopore size to some extent, while electrochemical coarsening can more precisely control the microscopic evolution of the three-dimensional nanochannel electrode by controlling the number of CV scans. The three-dimensional nanochannel electrode was scanned for 100, 200, and 300 cycles in 0.5 M H₂SO₄ with a voltage range of 0.3 V–1.2 V. SEM characterization revealed… Figure 4 The average pore size of the unroughened electrode is approximately 9.97 nm, and the metal ligaments are fine and dense. Figure 4 (A in the text); After 100 CV scans, the average aperture increased to approximately 20.41 nm, and the ligament slightly thickened ( Figure 4 (B in the text); by 200 CV scans, the ligament had further thickened and the aperture had significantly enlarged ( Figure 4 (C in the text); After 300 CV scans, the pores merged and collapsed, and the structure began to transform into a nanocolumnar structure. Figure 4 (D in the diagram). This phenomenon indicates that in the later stages of electrochemical roughening, gold atoms undergo intense migration and rearrangement during repeated redox processes. High-curvature regions are smoothed or filled, and the ligaments between pores break or reorganize, ultimately leading to the gradual disintegration of the porous structure and its transformation into a nanocolumnar morphology. Furthermore, repeated redox processes may consume some gold atoms, reducing structural stability.

[0031] Example 3:

[0032] To quantify the effect of morphological changes on the electrochemical active area, cyclic voltammetry (CV) scans were performed on the three-dimensional nanochannel electrode in 0.05 M H₂SO₄ solution, such as... Figure 5 As shown, the CV reduction peak gradually decreased with increasing thermal annealing temperature and scanning cycles, indicating that both morphology control methods can effectively increase the nanopore size.

[0033] Example 4:

[0034] Three-dimensional nanochannel electrodes provide an ideal microenvironment for CRISPR / Cas12a reactions due to their high specific surface area and confined diffusion effect. Their porous structure effectively enhances the local concentration of reactants and the frequency of molecular collisions, accelerating the reaction kinetics of CRISPR / Cas12a with nucleic acid probes and improving signal output efficiency compared to traditional planar electrodes. However, the performance of this system is limited by the channel size. Too small a pore size physically hinders the diffusion of Cas12a protein, while too large a pore size weakens the confined diffusion effect.

[0035] First, three-dimensional nanochannel electrodes and planar electrodes were prepared using gold-silver sputtering at a volume ratio of 1:2. Subsequently, the electrodes were subjected to 2-hour shearing treatment with target-bound Cas12a protein. However, for the three-dimensional nanochannel electrode prepared using the gold-silver sputtering volume ratio of 1:2, the signal only decreased slightly after shearing. This may be because the pore size of the nanopores is too small, limiting the diffusion of Cas12a protein into the pores. To verify this hypothesis and solve this problem, an electrochemical roughening method was used, performing CV scans of 100 and 200 cycles in H2SO4 solution to expand the nanopore size. Subsequently, the electrode was modified with a 1 µM signal probe and subjected to 2-hour shearing treatment with target-bound Cas12a protein. The results showed that the electrode without electrochemical roughening treatment ( Figure 6 In section A), almost no shearing occurs; after 100 CV scans ( Figure 6 In the B section, the nanopores expanded, improving the diffusion of the Cas12a protein, and the peak current signal decreased to 82.25% of the initial signal; when the number of CV scans increased to 200, ( Figure 6 (C) The nanopores further expand, and the Cas12a protein can diffuse into the pores almost freely. After shearing, the peak current signal drops to 58.01% of the initial signal, and the signal near the planar electrode drops (58.69%), indicating that at this pore size, the diffusion of the Cas12a protein is no longer significantly affected by steric hindrance.

[0036] Example 5:

[0037] To achieve mass production of large-aperture three-dimensional nanochannel electrodes, this invention adjusts the gold-silver sputtering volume ratio to 1:3. Within a 10-minute reaction time, the signal drop of the three-dimensional nanochannel electrode reached 43.32% (…). Figure 7 The signal at point A is close to the signal saturation level reached after a 2-hour shear reaction. In contrast, the signal drop of the planar electrode under the same conditions is only 7.48%. Figure 7 The significant difference (B in the original text) fully demonstrates the synergistic effect of nanoporous gold electrodes with the CRISPR / Cas12a strategy, showcasing their application potential in the field of rapid and ultrasensitive nucleic acid detection.

[0038] Example 6:

[0039] In practical applications, this invention compares the detection performance of three-dimensional nanochannel electrodes and traditional planar electrodes for HPV-16 targets. For example... Figure 8 As shown, within the concentration range of 10 pM to 100 nM, the three-dimensional nanochannel electrode significantly improved the efficiency of the shear reaction within 15 minutes, exhibiting a larger signal change (ΔI %) and an absolute signal change (ΔI μA). In contrast, the planar electrode, although showing a similar trend within a reaction time of 75 minutes, exhibited a significantly smaller signal change amplitude. Linear fitting results indicate that the response sensitivity of the three-dimensional nanochannel electrode is 20 times that of the planar electrode, and it maintains significant signal differences in the low concentration region (10-50 pM), demonstrating excellent low-concentration recognition capability. The detection limit of the three-dimensional nanochannel electrode is 2.8 pM, which is approximately 17.8 times higher than that of the planar electrode (50 pM), demonstrating the application potential of the three-dimensional nanochannel electrode in ultrasensitive nucleic acid detection.

[0040] It should be stated that the content and specific embodiments of this invention are intended to demonstrate the practical application of the technical solutions provided by this invention, and should not be construed as limiting the scope of protection of this invention. Any modifications and changes made to this invention within the spirit and scope of the claims fall within the protection scope of this invention.

Claims

1. A CRISPR / Cas12a electrochemical sensor based on a three-dimensional nanochannel electrically limited domain enhancement, characterized in that, Three-dimensional nanochannel electrodes were constructed using micro / nano fabrication techniques and dealloying processes. The pore size and specific surface area of ​​the three-dimensional nanochannels were synergistically controlled using thermal annealing and electrochemical roughening methods. Too small a pore size would physically hinder the diffusion of Cas12a protein, while too large a pore size would weaken the confined diffusion effect. Therefore, the pore sizes were controlled to 19.23 nm, 23.52 nm, and 28.57 nm. Thermal annealing, by enhancing the surface diffusion of gold atoms, facilitates the redistribution of gold atoms, releases residual stress, and thus causes the system to tend towards a lower energy state, reducing porosity. Gradually increasing in size, the thin branches thicken; electrochemical coarsening promotes the migration and recombination of gold atoms through redox cycles, making the pore structure coarser and more ordered; the confined diffusion effect is enhanced by the three-dimensional nanochannels, and the high specific surface area of ​​the three-dimensional nanochannel electrodes provides more fixation sites for the signal probe, confining the Cas12a protein within the three-dimensional nanochannels, constructing an electrochemical sensor coupled with the CRISPR / Cas12a system, improving local concentration and molecular interaction efficiency, thereby improving cleavage efficiency and signal output rate, and reducing the detection limit to 2.8 pM; The micro / nano fabrication technology and dealloying process include: after photolithographically forming a seed layer pattern on the substrate surface, a chromium adhesion layer and a gold seed layer are deposited sequentially; after stripping the photoresist, a sensing area is photolithographically formed on the gold seed layer; a gold-silver composite layer is formed by gold-silver co-sputtering, with a gold-silver sputtering volume ratio of 1:3; after stripping the photoresist, the gold-silver composite layer is immersed in hot concentrated nitric acid to remove the silver phase, thereby obtaining the three-dimensional nanochannel electrode; The electrochemical sensor of the CRISPR / Cas12a system activates the trans-cleavage activity of the Cas12a protein after the nucleic acid target binds to it, cleaving the signal probe on the surface of the three-dimensional nanochannel, resulting in a decrease in the electrochemical signal, thus enabling the detection of the nucleic acid target in the concentration range of 10 pM to 100 nM.

2. The CRISPR / Cas12a electrochemical sensor based on three-dimensional nanochannel electrically limited domain enhancement according to claim 1, characterized in that, An insulating layer is formed outside the three-dimensional nanochannel electrode, exposing only the sensing area and electrode leads.

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