A SERS sensor for detecting FEN1 enzyme activity

Abstract

This invention discloses a SERS sensor for detecting FEN1 enzyme activity, its preparation method, and its application. The SERS sensor comprises a gold disk temperature-controlled electrode HAuE, magnetic beads MB, the skin flap endonuclease FEN1, double-stranded DNA S1 / S2 / S3 with a skin flap, the exonuclease Exo III, a substrate probe H1, a trigger probe triDNA, a signal probe S4, a signal molecule 4-MBA, and a SERS tag modified with MCH on the surface of a gold nanocube. By increasing the surface temperature of HAuE, FEN1 activity significantly increases, leading to the rapid generation of the trigger probe triDNA. During Exo III-assisted amplification, triDNA binds to MB / H1, generating a large amount of MBs / ssDNA. MBs / ssDNA binds to the SERS tag, thereby detecting the SERS signal. The SERS intensity is positively correlated with FEN1 activity, achieving highly sensitive detection of FEN1 activity.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology, specifically relating to a SERS sensor for detecting the activity of FEN1 enzyme (Flapendonuclease 1). Background Technology

[0002] Flap endonuclease 1 (FEN1) plays a crucial role in DNA replication and repair. Studies have shown that upregulation of FEN1 is associated with the progression of various tumors, including hepatocellular carcinoma, breast cancer, and prostate cancer. Therefore, in recent years, FEN1 has been identified as an important biomarker for cancer diagnosis and monitoring, and accurate and sensitive detection of FEN1 activity is of great significance. Methods for detecting FEN1 activity include fluorescence, electrochemistry, colorimetry, and photoelectrochemistry, but there are no reports on a SERS method for detecting FEN1 activity.

[0003] Surface-enhanced Raman scattering (SERS) is a rapid and sensitive analytical method. Using noble metal nanoparticles as a substrate and modifying their surfaces with Raman reporter molecules to construct SERS tags can significantly enhance the Raman signal of the reporter molecules. Besides the metallic composition, the size and shape of the nanostructure also greatly influence the Raman enhancement activity of the substrate. Compared to spherical nanoparticles, the sharp edges of gold nanocubes (AuNCs) can strongly enhance the electromagnetic field, providing Raman enhancement activity. Therefore, AuNCs are suitable as substrate materials for assembling SERS tags and show promise for applications in biosensing.

[0004] In recent years, many amplification strategies to improve detection sensitivity have been applied in the field of biosensing. Among them, the exonuclease III (ExoIII)-assisted cyclic amplification strategy has attracted much attention due to its high efficiency and ease of operation. Exo-III can progressively remove single nucleotides from the 5'-hydroxyl terminus of double-stranded DNA without a specific recognition site, and the Exo-III-assisted amplification strategy has been widely used in the field of biosensing.

[0005] Temperature-controlled electrodes are a technique that heats the electrode surface by applying an electric current, and their advantage lies in the ability to rapidly increase the electrode temperature. Currently, there are no reported SERS biosensors that combine an Exo-III-assisted amplification strategy with a temperature-controlled electrode to detect FEN1 activity. Summary of the Invention

[0006] The present invention aims to provide a novel SERS biosensor that combines a temperature-controlled thermoelectrode, Exo III-assisted amplification, and AuNC-based SERS enhancement for highly sensitive detection of FEN1 activity. Specifically, the SERS biosensor comprises a gold disk temperature-controlled electrode (HAuE), magnetic beads (MB), a flap endonuclease FEN1, double-stranded DNA S1 / S2 / S3 with a flap, an exonuclease Exo III, a substrate probe H1, a trigger probe triDNA, a signal probe S4, a signal molecule 4-MBA, and a SERS tag modified with MCH on the surface of a gold nanocube. By increasing the surface temperature of the HAuE, FEN1 activity is significantly increased, leading to the rapid generation of the trigger probe triDNA. During Exo III-assisted amplification, triDNA binds to MB / H1, generating a large amount of MBs / ssDNA. MBs / ssDNA binds to the SERS tag, thereby detecting the SERS signal. The SERS intensity is positively correlated with FEN1 activity, thus achieving highly sensitive detection of FEN1 activity.

[0007] The objective of this invention is achieved through the following technical solution: A method for preparing a SERS sensor for detecting FEN1 enzyme activity includes the following steps: (1) Preparation of SERS tag 5 μL of 10 μM signal probe S4 was activated with 5 μL of 10 mM TCEP for 1 h to obtain activated signal probe S4. 25 μL of AuNCs solution and 165 μL of 0.01% SDS were added to the activated signal probe S4, and the mixture was incubated at 37 °C for 12 h. After incubation, 5 μL of 2 M NaCl was added every 0.5 h for a total of two additions, followed by overnight incubation at 37 °C. 2 μL of 2 mM 4-MBA ethanol solution was added, and incubation continued for 1 h. 1.5 μL of 2 mM MCH was added, and incubation continued for 2 h. After incubation, the mixture was centrifuged three times at 3000 rpm for 10 min each time, and the precipitate was collected and redispersed in 75 μL Tris Buffer 2 to obtain the SERS tag solution, which was stored at 4 °C. (2) FEN1 activity detection The temperature-controlled gold disk electrode HAuE was polished with Al2O3 powder, then ultrasonically cleaned with ethanol and ultrapure water respectively, and electrochemically cleaned by immersion in 0.5M H2SO4 solution. Finally, it was cleaned with ultrapure water and dried with nitrogen to obtain the activated HAuE electrode. A mixture of 5 μL of 10 μM oligonucleotide chain S1, 5 μL of 10 μM oligonucleotide chain S2, and 5 μL of 10 μM oligonucleotide chain S3 was heated to 95°C and held for 5 min, then slowly cooled to 37°C. It was then activated for 1 h in the dark with 5 μL of 10 mM TCEP containing 0.4 M NaCl to obtain a comDNA solution. 10 μL of the comDNA solution was added dropwise to the surface of the activated HAuE electrode and incubated at room temperature for 1 h, followed by Tris Buffer. After rinsing, the electrode was dried in nitrogen to obtain the HAuE / comDNA electrode. The HAuE / comDNA electrode was then blocked in 2 mM MCH for 30 min to obtain the HAuE / comDNA / MCH electrode. The HAuE / comDNA / MCH electrode was then immersed in FEN1 reaction buffer containing FEN1 enzyme, heated with DC current at 57°C for 2 h, and then transferred to a centrifuge tube to inactivate at 80°C for 20 min to obtain a solution containing triDNA, which was stored at 4°C. 100 μL of streptavidin-modified magnetic beads (1 mg / mL) were washed with Tris Buffer 3 and dispersed in 85 μL Tris Buffer 3. 15 μL of 10 μM substrate probe H1 was added, and the mixture was incubated at 37°C for 2 h. After washing with Tris Buffer 3 again, the mixture was dispersed in 60 μL Tris Buffer 3 to obtain the MB / H1 assembly. 30 μL of a triDNA-containing solution was mixed with 15 μL of the MB / H1 assembly, 5 μL of 10×NE Buffer, and 1 μL of 15 U / μL Exo III enzyme, and incubated at 37°C for 2 h to obtain the MBs / ssDNA assembly. 51 μL of the MBs / ssDNA assembly was washed with Tris Buffer 3 and dispersed in 25 μL Tris Buffer 3, then mixed with 75 μL of LERS tag solution, and incubated at 37°C for 6 h to obtain the MB / ssDNA / MCH / 4-MBA / S4 / AuNC assembly. 51 μL of the mixture was then magnetically dispersed... The MB / ssDNA / MCH / 4-MBA / S4 / AuNC module was washed 6 times with Tris buffer 3 and concentrated to 2.5 μL. It was then dropped onto a silicon wafer, dried at 45°C, and subjected to SERS detection. The sequence of the signal probe S4 is: 5'-SH-(CH2)6-TTTTTTTTTCACTCGTG-3'. The oligonucleotide chain S1 sequence is: 5'-AAAAAGCGTGTCATTCCTGTCCGATGCTCGTCATTGCATCGTGAAGGG-3', and the oligonucleotide chain S2 sequence is: 5'-TGATCCTTGATCCTTAGTAGATGC-3'. The oligonucleotide chain S3 sequence is: 5'-SH-TTTTTTTTTTCCCTTCACGATGCAATGACGCATCTACTAAGGATCAAGGATCA-3', and the triDNA sequence is: 5'-AAAAAGAGTGTCATTCCTGTCCGATGCTC-3'. The sequence of the substrate probe H1 is as follows: 5'-biotin-TTTTTTCACGAGTGTCATTCCCTGCATCGGACAGGAATGACACTCTTTTT-3'.

[0008] Further, the preparation method of the above AuNCs solution is as follows: 0.6 mL of 0.01 M NaBH4 is injected into a mixed solution of 10 mL of 0.1 M CTAB and 0.25 mL of 0.01 M HAuCl4, stirred at 600 rpm for 1 min, and then allowed to stand at 28 °C for 3 h to obtain a gold nanoparticle seed solution with a particle size of 3 nm; while stirring, 1.5 mL of 0.1 M ascorbic acid, 2 mL of 0.2 MCTAC, and 50 μL of the gold nanoparticle seed solution with a particle size of 3 nm are mixed, and then 2 mL of 0.5 mM HAuCl4 is injected, and allowed to stand for 15 min to obtain an AuNPs solution with a particle size of 10 nm; the obtained AuNPs solution with a particle size of 10 nm is centrifuged, the supernatant is removed, and the precipitate is redispersed in 1 mL of 0.02 M CTAC to obtain a resuspension of AuNPs with a particle size of 10 nm; 25 mL of 0.1 M CTAC and 1.625 mL of 0.01 M HAuCl4 are added to obtain a gold nanoparticle seed solution with a particle size of 3 nm. Mix 0.01M ascorbic acid with 125μL of AuNPs resuspension with a particle size of 10nm, add 25mL of 0.5mM HAuCl4, centrifuge, remove the supernatant, and disperse the precipitate in 3mL of 0.01M CTAC to obtain AuNCs solution.

[0009] Furthermore, the above Tris Buffer 1 formulation is: 10mM tris-HCl, 0.1M NaCl; pH 8.0.

[0010] Furthermore, the above Tris Buffer 2 formulation is: 10mM Tris-HCl, 0.1M NaCl, 0.01% SDS; pH 8.0.

[0011] Furthermore, the FEN1 reaction buffer containing the FEN1 enzyme is composed of the FEN1 enzyme and 1×ThermoPol Buffer, wherein the 1×ThermoPol Buffer is formulated as follows: 20mM Tris-acetate, 10mM (NH4)2SO4, 10mM KCl, 10mM MgSO4, 0.1% Triton X-100; pH 8.8.

[0012] Furthermore, the above Tris Buffer 3 formulation is as follows: 10mM Tris-HCl, 0.1M NaCl, 0.05% Tween-20; pH 8.0.

[0013] Furthermore, the above 10×NEBuffer formulation is: 100mM Bis-Tris-Propane-HCl, 100mM MgCl2, 10mM DTT; pH 7.0.

[0014] A SERS sensor prepared using the above-described preparation method.

[0015] The above-mentioned SERS sensor is used for the detection of FEN1 enzyme activity for non-disease diagnosis and treatment purposes.

[0016] The detection principle of the SERS sensor for FEN1 enzyme activity detection in this invention is as follows: Figure 1 As shown. The SERS tag is obtained by modifying the AuNC surface with S4, 4-MBA, and MCH. The S1 / S2 / S3 DNA complex (comDNA) is fixed to the HAuE surface by Au-S bonds. The 5' flap structure in the comDNA allows the FEN1 enzyme to cleave the 5' flap and generate trigger DNA (triDNA). As the electrode temperature increases, the activity of the FEN1 enzyme increases, thus generating more triDNA. The hairpin DNA (H1) modified on the MB surface can be opened and hybridized with the triDNA. The H1 in the resulting double-stranded DNA is cleaved from its 3' end by the Exo III enzyme. The released triDNA hybridizes with another H1 on the MB for cyclic amplification. Finally, the short single-stranded DNA (ssDNA) remaining on the MB surface hybridizes with S4 on the SERS tag MCH / 4-MBA / S4 / AuNC, thus obtaining the MB / ssDNA / MCH / 4-MBA / S4 / AuNC assembly, and the SERS signal can be measured. The SERS intensity is proportional to the concentration of the FEN1 enzyme. Therefore, the proposed SERS sensor can be used to detect the activity of the FEN1 enzyme. Furthermore, the detection sensitivity increases significantly with increasing electrode temperature. In summary, this invention successfully developed a SERS sensor for detecting FEN1 activity.

[0017] The significant advantages of this invention are: This invention proposes a novel AuNC-enhanced SERS biosensor for detecting FEN1 activity by combining HAuE and Exo III enzyme-assisted cyclic amplification. The AuNC substrate in the SERS tag strongly enhances the 4-MBA SERS signal. As the electrode surface temperature increases, the FEN1 enzyme activity also increases, allowing for the cleavage of more triDNA and initiation of Exo III enzyme-assisted cyclic amplification. The combination of heated electrodes, Exo III-assisted cyclic amplification, and AuNC-based SERS enhancement endows this biosensor with excellent selectivity and high sensitivity. The detection limit reaches 1.33 × 10⁻⁶. -5 U / μL. Furthermore, this method has also been used to measure the activity of FEN1 in real cell extracts, demonstrating its promising application in disease diagnosis. Attached Figure Description

[0018] Figure 1 : Schematic diagram of the SERS biosensor of this invention.

[0019] Figure 2 : Sensor detection performance optimization diagram. Where A represents the optimization of FEN1 enzyme digestion temperature, B represents the optimization of SERS tag and MBs / ssDNA reaction time, and C represents the optimization of Exo III enzyme dosage. Figure 3 The sensor's sensitivity detection results are shown in the image. A represents the Raman spectrum of 4-MBA, and B represents the Raman spectrum of 4-MBA at 1585 cm⁻¹. -1 Peak intensity and FEN1 enzyme concentration (1.67 × 10⁻⁶) -5 –1.67×10 -2 The curve of U / μL), C is 4-MBA at 1585cm. -1 Peak intensity and FEN1 enzyme concentration (1.67 × 10⁻⁶) -5 –3.33×10 -3 Logarithmic linear relationship curve (U / μL).

[0020] Figure 4 Figure 1 shows the results of FEN1 enzyme activity detection in cancer cell extracts. A represents the FEN1 enzyme activity measured in cell extracts from cervical cancer cells (HeLa) and human breast cancer cells (MCF-7), and B is the logarithmic linear relationship curve between SERS intensity and the number of HeLa cells. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] The Tris Buffer 1 used in this embodiment of the invention has the following formulation: 10 mM tris-HCl, 0.1 M NaCl; pH 8.0.

[0023] The Tris Buffer 2 formulation used in this embodiment of the invention is: 10mM tris-HCl, 0.1M NaCl, 0.01% (wt / vol) SDS; pH 8.0.

[0024] The Tris Buffer 3 formulation used in this embodiment of the invention is: 10mM tris-HCl, 0.1M NaCl, 0.05% (wt / vol) Tween-20; pH 8.0.

[0025] The formulation of 1×ThermoPol Buffer used in this embodiment of the invention is as follows: 20mM tris-acetate, 10mM (NH4)2SO4, 10mM KCl, 10mM MgSO4, 0.1% Triton X-100; pH 8.8.

[0026] The 10×NEBuffer used in this embodiment of the invention TM Formula 1 is: 100mM bis-tris-propane-HCl, 100mM MgCl2, 10mM DTT; pH 7.0.

[0027] The preparation method of the gold nanocube (AuNCs) solution used in this embodiment of the invention is as follows: 0.6 mL of NaBH4 (0.01 M) was added to a mixed solution of 10 mL CTAB (0.1 M) and 0.25 mL HAuCl4 (0.01 M), stirred at 600 rpm for 1 minute, and then allowed to stand at 28 °C for 3 hours to obtain a gold nanoparticle seed solution with a particle size of 3 nm. While stirring, 1.5 mL of ascorbic acid (0.1 M), 2 mL of CTAC (0.2 M), and 50 μL of the 3 nm gold nanoparticle seed solution were mixed, and then 2 mL of HAuCl4 (0.5 mM) was added. The mixture was allowed to stand at 27 °C for 15 minutes to obtain a gold nanosphere (AuNPs) solution with a particle size of 10 nm. The obtained 10 nm AuNPs solution was centrifuged at 14400 rpm for 30 minutes, the supernatant was removed, and the precipitate was redispersed in 1 mL of... In CTAC (0.02M), a resuspension of AuNPs with a particle size of 10 nm was obtained; 25 mL CTAC (0.1M), 1.625 mL ascorbic acid (0.01M) and 125 μL of the AuNPs resuspension with a particle size of 10 nm were mixed, and 25 mL HAuCl4 (0.5 mM) was added. After centrifugation at 8000 rpm for 20 minutes, the supernatant was removed, and the precipitate was dispersed in 3 mL CTAC (0.01M) to obtain a solution of AuNCs with a side length of 41 nm.

[0028] The nucleotide sequences used in the embodiments of this invention are shown in the table below: Example 1: Sensor Fabrication (1) Preparation of SERS tag 5 μL of 10 μM signal probe S4 was activated in the dark for 1 h with 5 μL of 10 mM TCEP solution to reduce SS bonds, resulting in activated signal probe S4. 25 μL of AuNCs solution and 165 μL of 0.01% (wt / vol) SDS were added to the activated signal probe S4, and the mixture was incubated at 37 °C for 12 h in a shaker. After incubation, 5 μL of NaCl (2 M) was added every 0.5 h for a total of two additions, and the mixture was incubated overnight in a shaker (37 °C, 300 rpm). 2 μL of 4-MBA ethanol solution (2 mM) was added, and incubation continued for 1 h. 1.5 μL of MCH solution (2 mM) was added, and incubation continued for 2 h. After incubation, the mixture was centrifuged three times at 3000 rpm for 10 min each time to remove unmodified S4, 4-MBA, and MCH. The precipitate was collected and redispersed in 75 μL TrisBuffer 2 to obtain the SERS tag solution, which was stored at 4 °C.

[0029] (2) FEN1 activity detection The temperature-controlled gold disk electrode HAuE was polished sequentially with 1 μm and 0.05 μm Al2O3 powder, and then ultrasonically cleaned with ethanol and ultrapure water for 2 min each. The treated HAuE electrode was then electrochemically cleaned in 0.5 M H2SO4 solution, followed by rinsing with ultrapure water and drying with nitrogen to obtain the activated HAuE electrode for later use. A mixture of oligonucleotide chains S1 (10 μM, 5 μL), S2 (10 μM, 5 μL), and S3 (10 μM, 5 μL) was heated to 95 °C and held for 5 min, then slowly cooled to 37 °C, and then activated with 5 μL of TCEP (10 mM) containing 0.4 M NaCl in the dark for 1 h to form double-stranded DNA S1 / S2 / S3 with skin flap (denoted as comDNA). Next, 10 μL of comDNA was added to the activated HAuE electrode surface and incubated at room temperature for 1 h. The electrode was then rinsed with Tris Buffer 1 and dried under nitrogen to obtain the HAuE / comDNA electrode. The HAuE / comDNA electrode was immersed in 300 μL of MCH (2 mM) for 30 min (25 °C) to block non-specific binding sites on the electrode surface, resulting in the HAuE / comDNA / MCH electrode. The HAuE / comDNA / MCH electrode was then immersed in 300 μL of FEN1 reaction buffer (composed of 1×ThermoPol Buffer and FEN1 enzyme). The electrode was heated with DC current and incubated at different temperatures for 2 h to allow the 5' flap of comDNA to be cleaved by FEN1 and release the initiator probe triDNA into the reaction system. After incubation, the reaction solution was transferred to a centrifuge tube and inactivated at 80 °C for 20 min to obtain a solution containing triDNA, which was stored at 4 °C for further use.

[0030] Take 100 μL of streptavidin-modified magnetic beads at a concentration of 1 mg / mL, wash with 100 μL of Tris Buffer 3, and then disperse the magnetic beads in 85 μL of Tris Buffer 3 to obtain a magnetic bead dispersion. Add 15 μL of biotin-modified substrate probe H1 (10 μM) to the obtained magnetic bead dispersion, incubate at 37 °C for 2 h, then wash the magnetic beads three times with Tris Buffer 3 and disperse the magnetic beads in 60 μL of Tris Buffer 3 to obtain the MB / H1 assembly. Mix 30 μL of triDNA solution with 15 μL of MB / H1 assembly and 5 μL of 10×NE Buffer. TM1 μL of Exo III enzyme (different concentrations) was mixed and incubated at 37 °C for 2 h to obtain MBs / ssDNA modules. The obtained MBs / ssDNA modules were washed three times with Tris Buffer 3, and then magnetic beads were dispersed in 25 μL Tris Buffer 3. This was then mixed with 75 μL SERS tag solution and incubated at 37 °C for different times to obtain MB / ssDNA / MCH / 4-MBA / S4 / AuNC modules. 51 μL of MB / ssDNA / MCH / 4-MBA / S4 / AuNC modules were washed six times with 100 μL Tris Buffer 3 using a magnet, concentrated to 2.5 μL, dropped onto a silicon wafer, dried at 45 °C for 20 min, and then subjected to SERS detection. The SERS detection conditions were: 633 nm laser, laser power 7.5 mW, integration time 10 s * 25 times, grating 1800 l / mm, and objective lens 50×.

[0031] Example 2: Optimization of Experimental Conditions To achieve optimal detection performance of the sensor designed in Example 1, three experimental parameters were optimized in this example: the temperature of FEN1 enzyme digestion was optimized (37, 47, 57, 65, 70℃), the incubation time of SERS tag with MBs / ssDNA was optimized (2, 4, 6, 8, 10 h), and the amount of Exo III enzyme was optimized (5, 10, 15, 20, 25 U).

[0032] like Figure 2 As shown, Figure 2 A represents the optimization of the FEN1 enzyme digestion temperature. As the electrode temperature increased from 37℃ to 57℃, the Raman intensity continued to increase. However, when the temperature was higher than 57℃, the Raman intensity decreased. Excessively high temperatures may cause changes in the structure of the FEN1 enzyme, leading to a decrease in activity. Therefore, 57℃ was selected as the optimal temperature for the FEN1 enzyme digestion process. Figure 2 B represents the optimization of the reaction time between the SERS tag and MBs / ssDNA. As the reaction time between the SERS tag and MBs / ssDNA increases, the Raman intensity also increases, reaching a plateau after 6 hours. Therefore, 6 hours was chosen as the optimal time for the experiment. Figure 2 C represents the optimization of the Exo III enzyme dosage. As the Exo III dosage increases, the SERS intensity gradually increases and reaches a plateau at 15 U. Therefore, 15 U was selected as the optimal Exo III dosage for subsequent experiments.

[0033] Example 3: Sensor sensitivity to FEN1 activity detection Under the optimized experimental conditions of Example 2, the sensitivity of the SERS biosensor proposed in this invention was explored by using different concentrations of FEN1 enzyme.

[0034] like Figure 3 As shown, with the increase of FEN1 enzyme concentration, at 1585 cm⁻¹... -1 The SERS intensity gradually increased at the location. The FEN1 enzyme concentration (from 1.67 × 10⁻⁶) increased. -5 Up to 3.33×10 -3 There is a good linear relationship between the logarithm of U / μL and Raman intensity. The corresponding regression equation is y = 16776.08lgC. FEN1 +82649.28 (U / μL, R) 2 =0.992). The limit of detection (LOD) was calculated to be 1.33 × 10⁻⁶. -5 U / μL (S / N = 3). This indicates that the sensor can achieve highly sensitive detection of FEN1 enzyme activity.

[0035] Example 4: Measurement of FEN1 activity in cell extracts from real-world cancer cells. To evaluate the applicability of the proposed SERS biosensor to detect FEN1 activity in real biological samples, we measured the activity of the FEN1 enzyme in cell extracts from cervical cancer cells (HeLa) and human breast cancer cells (MCF-7). Figure 4 A). A significantly stronger SERS signal than that against a blank background was observed in the extract containing 10,000 cells. Furthermore, as... Figure 4 As shown in Figure B, the SERS intensity increases with increasing cell number. From 100 to 10,000 cells, the SERS intensity (y) is linearly correlated with the logarithm of the HeLa cell number (N), as expressed by the formula: y = 14194.47lgN - 26673.61(R) 2 =0.995), and the LOD was calculated to be 86 cells. These results indicate that this method can be used to detect FEN1 enzyme activity in real samples and has broad application prospects in clinical diagnosis.

[0036] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A method for preparing a SERS sensor for detecting FEN1 enzyme activity, characterized in that: Includes the following steps: (1) Preparation of SERS tag 5 μL of 10 μM signal probe S4 was activated with 5 μL of 10 mM TCEP for 1 h to obtain activated signal probe S4. 25 μL of AuNCs solution and 165 μL of 0.01% SDS were added to the activated signal probe S4, and the mixture was incubated at 37 °C for 12 h. After incubation, 5 μL of 2 M NaCl was added every 0.5 h for a total of two additions, followed by overnight incubation at 37 °C. 2 μL of 2 mM 4-MBA ethanol solution was added, and incubation continued for 1 h. 1.5 μL of 2 mM MCH was added, and incubation continued for 2 h. After incubation, the mixture was centrifuged three times at 3000 rpm for 10 min each time, and the precipitate was collected and redispersed in 75 μL Tris Buffer 2 to obtain SERStag solution, which was stored at 4 °C. (2) FEN1 activity detection The temperature-controlled gold disk electrode HAuE was polished with Al2O3 powder, then ultrasonically cleaned with ethanol and ultrapure water respectively, and electrochemically cleaned by immersion in 0.5 M H2SO4 solution. Finally, it was washed with ultrapure water and dried with nitrogen to obtain the activated HAuE electrode. A mixture of 5 μL of 10 μM oligonucleotide chain S1, 5 μL of 10 μM oligonucleotide chain S2, and 5 μL of 10 μM oligonucleotide chain S3 was heated to 95℃ and held for 5 min, then slowly cooled to 37℃, and then activated with 5 μL of 10 mM TCEP containing 0.4 M NaCl in the dark for 1 h to obtain a comDNA solution. 10 μL of comDNA solution was added dropwise to the surface of the activated HAuE electrode, incubated at room temperature for 1 h, rinsed with Tris Buffer 1, and dried in nitrogen to obtain the HAuE / comDNA electrode. The HAuE / comDNA electrode was then blocked by immersion in 2 mM MCH for 30 minutes. min, obtain HAuE / comDNA / MCH electrode; immerse HAuE / comDNA / MCH electrode in FEN1 reaction buffer containing FEN1 enzyme, heat electrode with DC power at 57℃ for 2 h, then transfer to centrifuge tube at 80℃ for 20 min to inactivate, obtain solution containing triDNA and store at 4℃. 100 μL of streptavidin-modified magnetic beads were washed with Tris Buffer 3 and dispersed in 85 μL Tris Buffer 3. 15 μL of 10 μM substrate probe H1 was added and incubated at 37 °C for 2 h. After washing with Tris Buffer 3, the beads were dispersed in 60 μL Tris Buffer 3 to obtain the MB / H1 component. 30 μL of a solution containing triDNA was mixed with 15 μL of LMB / H1 component, 5 μL of 10×NE Buffer, and 1 μL of 15 U / μL Exo III enzyme, and incubated at 37°C for 2 h to obtain the MBs / ssDNA component; 51 μL of the MBs / ssDNA component was washed with Tris Buffer 3 and dispersed in 25 μL of Tris Buffer 3, then mixed with 75 μL of SERS tag solution, and incubated at 37°C for 6 h to obtain the MB / ssDNA / MCH / 4-MBA / S4 / AuNC component; The 51 μL MB / ssDNA / MCH / 4-MBA / S4 / AuNC assembly was washed 6 times with Tris buffer 3 using a magnet, concentrated to 2.5 μL, dropped onto a silicon wafer, dried at 45°C, and then subjected to SERS detection. The sequence of the signal probe S4 is: 5'-SH-(CH2)6-TTTTTTTTTCACTCGTG-3'. The oligonucleotide chain S1 sequence is as follows: 5'-AAAAAGCGTGTCATTCCTGTCCGATGCTCGTCATTGCATCGTGAAGGG-3', The S2 sequence of the oligonucleotide chain is: 5'-TGATCCTTGATCCTTAGTAGATGC-3'. The S3 sequence of the oligonucleotide chain is as follows: 5'-SH-TTTTTTTTTTCCCTTCACGATGCAATGACGCATCTACTAAGGATCAAGGATCA-3', The triDNA sequence is: 5'-AAAAAGAGTGTCATTCCTGTCCGATGCTC-3'. The sequence of the substrate probe H1 is as follows: 5'-biotin-TTTTTTCACGAGTGTCATTCCCTGCATCGGACAGGAATGACACTCTTTTT-3'.

2. The preparation method according to claim 1, characterized in that: The AuNCs solution was prepared as follows: 0.6 mL of 0.01 M NaBH4 was added to a mixed solution of 10 mL of 0.1 M CTAB and 0.25 mL of 0.01 M HAuCl4, stirred at 600 rpm for 1 min, and then allowed to stand at 28℃ for 3 h to obtain a gold nanoparticle seed solution with a particle size of 3 nm. While stirring, 1.5 mL of 0.1 M ascorbic acid, 2 mL of 0.2 M CTAC, and 50 μL of the 3 nm gold nanoparticle seed solution were mixed, and then 2 mL of 0.5 mM HAuCl4 was added. After standing for 15 min, a 10 nm AuNPs solution was obtained. The 10 nm AuNPs solution was centrifuged, the supernatant was removed, and the precipitate was redispersed in 1 mL of 0.02 M CTAC to obtain a 10 nm AuNPs resuspension. 25 mL of 0.1 M CTAC and 1.625 mL of 0.01 M HAuCl4 were added to the solution. Mix M ascorbic acid and 125 μL of AuNPs resuspension with a particle size of 10 nm, add 25 mL of 0.5 mM HAuCl4, centrifuge, remove the supernatant, and disperse the precipitate in 3 mL of 0.01 M CTAC to obtain AuNCs solution.

3. The preparation method according to claim 1, characterized in that: The Tris Buffer 1 formulation is: 10 mMtris-HCl, 0.1 M NaCl; pH 8.

0.

4. The preparation method according to claim 1, characterized in that: The Tris Buffer 2 formulation is: 10 mM Tris-HCl, 0.1 M NaCl, 0.01% SDS; pH 8.

0.

5. The preparation method according to claim 1, characterized in that: The FEN1 reaction buffer containing FEN1 enzyme is composed of FEN1 enzyme and 1×ThermoPol Buffer. The 1×ThermoPol Buffer has the following formula: 20 mM Tris-acetate, 10 mM (NH4)2SO4, 10 mM KCl, 10 mM MgSO4, 0.1% Triton X-100; pH 8.

8.

6. The preparation method according to claim 1, characterized in that: The Tris Buffer 3 formulation is as follows: 10 mM Tris-HCl, 0.1 M NaCl, 0.05% Tween-20; pH 8.

0.

7. The preparation method according to claim 1, characterized in that: The 10×NEBuffer formulation is: 100 mM Mbis-Tris-Propane-HCl, 100 mM MgCl2, 10 mM DTT; pH 7.

0.

8. The SERS sensor prepared by the preparation method according to any one of claims 1 to 7.

9. The application of the SERS sensor as described in claim 8, characterized in that: The activity detection of FEN1 enzyme is applied for purposes other than disease diagnosis and treatment.

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