Fabrication and Application of a DNA Hydrogel-Based SERS-FL Dual-Mode Sensor

Abstract

This invention discloses the fabrication and application of a DNA hydrogel-based SERS-FL dual-mode sensor, comprising a DNA framework-based hydrogel and Au@Ag nanoparticles and quantum dots attached to cavities within the hydrogel. This sensor offers advantages such as stable properties, simple fabrication, convenience, and high efficiency. It exhibits high specificity, sensitivity, and accuracy, enabling practical application in the in-situ quantitative detection of biomarkers in serum and cellular biological samples. This, in turn, helps improve the identification of biomarkers in cancer patients and facilitates the development of an early SERS-FL predictive analysis method for cancer.

Description

Technical Field

[0001] This invention relates to the field of biosensor technology, specifically to the fabrication and application of a DNA hydrogel-based SERS-FL dual-mode sensor. Background Technology

[0002] With societal development, people's material living standards have greatly improved compared to the past. However, the emergence of numerous food and environmental problems has led to a prolonged state of sub-health, increasing the risk of cancer in certain groups. Therefore, raising public awareness of sub-health and effectively monitoring human health are crucial for early warning, diagnosis, and treatment of cancer. Currently, one of the clinically accepted diagnostic criteria for cancer is monitoring the levels of relevant tumor biomarkers. However, existing detection methods have limitations in early cancer diagnosis, and inaccurate quantitative analysis can easily lead to false positives / negatives.

[0003] Therefore, there is an urgent need to develop a novel, sensitive, accurate, and highly selective dual-mode sensor for direct quantitative analysis of serum and cells, thereby providing important research value and technical support for constructing an early predictive analysis method for cancer biomarkers using SERS-FL. Summary of the Invention

[0004] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the objective of this invention is to provide the preparation and application of a SERS-FL dual-mode sensor based on DNA hydrogel. The SERS-FL dual-mode sensor of this invention can simultaneously perform quantitative analysis of specific biomarkers in a sample (such as serum) using two different signals, SERS and fluorescence. It offers convenient and efficient detection, high specificity, high sensitivity, and high accuracy, which is beneficial for improving the identification of early-stage latent cancers.

[0005] In a first aspect, the present invention provides a surface-enhanced Raman scattering-fluorescence (SERS-FL) dual-mode sensor, the SERS-FL sensor comprising a hydrogel with a DNA framework as its backbone and Au@Ag nanoparticles and NQDs (nanoquant dots) attached to the cavity structure of the hydrogel.

[0006] In some embodiments of the present invention, the DNA framework is a three-dimensional structural framework obtained by hybridization of DNA-1, DNA-2, DNA-3, aptamers and cDNA.

[0007] In this invention, the DNA framework is a Y-shaped DNA three-dimensional backbone precursor, wherein DNA-1, DNA-2, and DNA-3 are semi-complementary sequences, and the aptamer and cDNA can form complementary double strands (Apt-cDNA). Thus, DNA-1, DNA-2, DNA-3, and Apt-cDNA will form a Y-shaped structure through base complementary pairing.

[0008] In some embodiments of the present invention, the hybridization is annealed hybridization, and the annealing temperature is 92-95°C.

[0009] In some embodiments of the present invention, the nucleotide sequence of said DNA-1 is shown in SEQ ID NO:3.

[0010] In some embodiments of the present invention, the nucleotide sequence of the DNA-2 is shown in SEQ ID NO:4.

[0011] In some embodiments of the present invention, the nucleotide sequence of the DNA-3 is shown in SEQ ID NO:5.

[0012] In some embodiments of the present invention, the nucleotide sequence of the aptamer is shown in SEQ ID NO:1.

[0013] In some embodiments of the present invention, the aptamer specifically targets the biomarker epithelial cell adhesion molecule (EpCAM).

[0014] In some embodiments of the present invention, the nucleotide sequence of the cDNA is shown in SEQ ID NO:2.

[0015] In some embodiments of the present invention, the length of each nucleotide sequence is required to be 30 to 60 bases.

[0016] In some embodiments of the present invention, the hydrogel also contains graphene oxide.

[0017] In some embodiments of the present invention, the Au@Ag nanoparticles have a particle size of 15–40 nm.

[0018] In some embodiments of the present invention, the Au@Ag nanoparticles include an Ag shell and an Au nanoparticle core.

[0019] In some embodiments of the present invention, the thickness of the Ag shell is 5 to 10 nm.

[0020] In this invention, the working principle of the SERS-FL dual-mode sensor is as follows: Under specific laser irradiation, NQDs encapsulated in the hollow structure inside the hydrogel of the SERS-FL dual-mode sensor undergo energy transfer through GO electron mass transfer, allowing Au@Ag NPs to gain more electrons. At this time, the SERS signal is significantly enhanced while the NQDs are in a quenched state. After the SERS-FL dual-mode sensor captures the target object in the sample, the DNA hydrogel lyses after a certain period of incubation (releasing NQDs, and their fluorescence is restored). The Au@Ag NPs (precipitate) and NQDs (supernatant) are collected by centrifugation. The Au@Ag NPs are used as the SERS substrate for SERS detection, and the supernatant is used for FL testing, thus completing the detection of dual signals of specific biomarkers in human serum and cancer cells.

[0021] In some embodiments of the present invention, the SERS-FL dual-mode sensor detects specific substances based on Raman detection (for SERS results) and fluorescence detection (for FL results), respectively.

[0022] Surface-enhanced Raman spectroscopy (SERS), as a highly sensitive, photostable spectroscopy technique that also possesses molecular "fingerprint" characteristics, has extremely high application value in the detection field. By further combining SERS with fluorescence (FL) spectroscopy, rapid response and imaging characteristics are achieved, resulting in a complementary dual-modal biosensor that significantly improves the accuracy and sensitivity of the analytical method in this invention.

[0023] A second aspect of the present invention provides a method for fabricating the SERS-FL dual-mode sensor described in the first aspect of the present invention, comprising the following steps:

[0024] The DNA-1, DNA-2, DNA-3, aptamer and cDNA described in the first aspect of the present invention are mixed and annealed and hybridized at 92-95°C. Then, silver-coated Au nanoparticles and NQDs are added separately and incubated for 20-40 min. An equal volume of the mixture containing silver-coated Au nanoparticles and NQDs is added separately, and then acrylamide monomer and graphene oxide are added. The mixture is incubated for another 1-5 h to obtain the SERS-FL dual-mode sensor.

[0025] In some embodiments of the present invention, the silver-coated Au nanoparticles may be nanoparticles obtained by coating an Ag shell using commercially available Au NPs or commercially available Au@Ag nanoparticles.

[0026] In this invention, the silver-coated Au nanoparticles are prepared by: heating an aqueous solution of chloroauric acid to boiling and then adding trisodium citrate dropwise; when the solution turns a pale wine red, stopping the heating and cooling to room temperature; centrifuging to obtain Au NPs; adding ascorbic acid under continuous stirring, and then adding AgNO3 to obtain Au@Ag nanoparticles (i.e., silver-coated Au nanoparticles).

[0027] In some embodiments of the present invention, the thickness of the silver coating on Au@Ag nanoparticles is adjusted by regulating the ratio of Au NPs to AgNO3.

[0028] In some embodiments of the present invention, the molar ratio of Au NPs to AgNO3 is 1:20 to 1:40.

[0029] In some embodiments of the present invention, the preparation method of NQD is as follows: deionized water and ammonia are added to a mixed solution of citric acid and penicillamine, and the mixture is placed in an oven at 150-200°C for 2-6 hours. After cooling to room temperature, the mixture is dialyzed for 22-26 hours to obtain NQD.

[0030] In some embodiments of the present invention, the molecular weight cutoff for dialysis is 1 kDa.

[0031] Of course, those skilled in the art can also directly use commercially available NQDs, with a particle size requirement of 5–20 nm for the NQDs.

[0032] In some embodiments of the present invention, a catalyst is added after the acrylamide monomer and graphene oxide are added.

[0033] In some embodiments of the present invention, the catalyst comprises tetramethylethylenediamine. Of course, those skilled in the art can also reasonably adjust the selection of the catalyst according to actual application requirements to obtain specific effects.

[0034] In some embodiments of the present invention, the molar concentrations of silver-coated Au nanoparticles, NQDs, acrylamide monomer, graphene oxide, and catalyst are 0.2–0.5 mmol / L, 1.5–3.0 μmol / L, 1.4–1.8 μmol / L, 85–125 μmol / L, and 8.0–10.0 nmol / L, respectively.

[0035] A third aspect of the invention provides the use of the SERS-FL dual-mode sensor described in the first aspect of the invention in the preparation of cell and / or cell lysate capture products.

[0036] In some embodiments of the present invention, the cells are cancer cells.

[0037] In some embodiments of the present invention, the cancer includes, but is not limited to, lung cancer, colorectal cancer, and pancreatic cancer.

[0038] A fourth aspect of the present invention provides the application of the SERS-FL dual-mode sensor described in the first aspect of the present invention in the preparation of disease detection products.

[0039] In some embodiments of the present invention, the disease includes cancer.

[0040] In some embodiments of the present invention, the cancer includes, but is not limited to, lung cancer, colorectal cancer, and pancreatic cancer.

[0041] In some embodiments of the present invention, the test sample includes serum and cell products.

[0042] The Au@Ag-NQDs / GO DNA hydrogel SERS-FL dual-mode sensor of this invention is based on DNA hydrogel-guided SERS-FL dual signal conversion. Compared with conventional sensors, it has advantages such as stable properties, simple preparation, and controllable particle size. It has been successfully applied to the detection of trace biomarkers EpCAM in human serum and cell samples. It uses both SERS and fluorescence methods for detection simultaneously, which is convenient, efficient, specific, sensitive, and accurate. It is beneficial to improve the identification of cancer and to construct a precise SERS-FL analysis method for cancer biomarkers. It has great application prospects in the early prediction analysis of diseases.

[0043] It should be noted that the target protein in this invention is not limited to a certain biomarker used in this invention. Appropriate target proteins can be selected according to actual needs, and corresponding DNA sequences can be designed, which has extremely high flexibility.

[0044] The beneficial effects of this invention are:

[0045] (1) The Au@Ag-NQDs / GO DNA hydrogel SERS-FL dual-mode sensor in this invention has the advantages of stable properties, simple preparation, convenience and high efficiency, and can be practically applied to the in situ quantitative detection of biomarkers in serum and cell biological samples.

[0046] (2) This invention has for the first time prepared an Au@Ag-NQDs / GO DNA hydrogel SERS-FL dual-mode sensor, which is made by encapsulating Au@Ag and NQDs in the internal cavity structure of the hydrogel and preparing it by DNA complementary pairing, providing a technical reference for the dual signal detection of biomarkers in human serum and cell samples.

[0047] (3) The detection method of Au@Ag-NQDs / GO DNA hydrogel SERS-FL dual-mode sensor in this invention is convenient, efficient, specific, sensitive and accurate, which is conducive to improving the identification of biomarkers in cancer patients and constructing an early prediction analysis method for cancer SERS-FL. Attached Figure Description

[0048] Figure 1 This is a schematic diagram of the experimental process for the fabrication (A) and application (B) of the SERS-FL dual-mode sensor of the present invention.

[0049] Figure 2 The image shows the TEM characterization of the Au@Ag-NQDs / GO DNA hydrogel of the SERS-FL dual-mode sensor of this invention, where A is Au@Ag NPs; B is pure DNA hydrogel; C is DNA hydrogel encapsulated with nanomaterials; and D is Au@Ag-NQDs / GO DNA hydrogel.

[0050] Figure 3 Gel electrophoresis characterization of the three-dimensional structural DNA used in the SERS-FL dual-mode sensor of this invention; wherein lanes a to f are, in order, marker, L1, L1-L2 structure, L1-L2-L3 structure, S1-S2 structure, and L1-L2-L3-S1-S2 structure.

[0051] Figure 4 The present invention provides a SERS-FL dual-mode sensor for the application of SERS-FL linear equations with respect to EpCAM standard solutions; wherein A and B are SERS detection results, and C and D are FL detection results.

[0052] Figure 5 This invention investigates the biocompatibility of the SERS-FL dual-mode sensor on Hela, A549, and MCF-7 cell lines. Specifically, A represents the effect of different concentrations of Au@Ag NPs on Hela, A549, and MCF-7 cell lines; B represents the effect of different concentrations of NQDs on Hela, A549, and MCF-7 cell lines; and C represents the effect of different incubation times of the SERS-FL dual-mode sensor on Hela, A549, and MCF-7 cell lines.

[0053] Figure 6 The results show the in situ capture of three cell types—Hela (A), A549 (B), and MCF-7 (C)—by the SERS-FL dual-mode sensor of this invention.

[0054] Figure 7 The present invention provides a SERS-FL dual-mode sensor for the quantitative analysis of EpCAM in actual serum (B, D, F) and cell lysate samples (A, C, E). Detailed Implementation

[0055] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0056] Construction of Au@Ag-NQDs / GO DNA hydrogel SERS-FL dual-mode sensor

[0057] The Au@Ag-NQDs / GO DNA hydrogel SERS-FL dual-mode sensor (hereinafter referred to as the SERS-FL dual-mode sensor) in this embodiment of the invention is obtained by complementary pairing of a double-stranded DNA containing a specific aptamer modified with Au@Ag at a specific particle size with another single-stranded DNA containing the same aptamer. This SERS-FL dual-mode sensor can be used for the simultaneous detection of dual signals of biomarkers in human serum and cell samples.

[0058] The method for fabricating the SERS-FL dual-mode sensor in this embodiment of the invention specifically includes the following steps:

[0059] (1) Preparation of Au@Ag nanoparticles (Au@Ag NPs):

[0060] A 0.1 mmol / L aqueous solution of chloroauric acid was heated to boiling, and then 1.5 mL of the reducing agent trisodium citrate was added dropwise. Heating was stopped and the solution was cooled to room temperature when it turned a pale wine-red color. Au NPs were obtained by centrifugation. Under continuous stirring, 2.5 mL of ascorbic acid was added to a beaker containing 10.0 mL of the prepared Au NPs solution. AgNO3 was added to adjust the thickness of the Ag shell coating on the Au NPs (the thickness of the Ag shell can be changed by adding different volumes of AgNO3; in this example, the concentration of AgNO3 used was 5–10 mmol / L, and the thickness of the Ag shell was 5–10 nm). Pure water was added, and the mixture was centrifuged twice to obtain Au@AgNPs.

[0061] Of course, commercially available Au NPs can be used directly for Ag shell coating, or commercially available Au@Ag nanoparticles can be used, provided that the particle size of the final particles is between 15 and 40 nm.

[0062] (2) Preparation of NQDs (nano quantum dots):

[0063] Add 0.5–0.8 mg of citric acid and 0.2–0.5 mg of penicillamine to a beaker, pour in deionized water and a small amount of ammonia, stir well, and then transfer to a polytetrafluoroethylene reactor. Then place it in a 175°C oven and react for 4 hours. After cooling to room temperature, dialyze continuously for 24 hours using a 1 kDa dialysis bag to obtain the NQDs solution.

[0064] (3) Fabrication of SERS-FL dual-mode sensor:

[0065] Prepare 20 μM stock solutions of the DNA in Table 1 below using TE buffer (pH = 8.0), and then dilute them to different concentrations using Tris-HCl buffer (pH = 7.4).

[0066] Table 1. DNA information used to prepare the SERS-FL dual-mode sensor

[0067]

[0068] The DNA-1, DNA-2, DNA-3, Apt (aptamer), and cDNA solutions obtained in the above steps were mixed and annealed at 95°C for 5 min to form a DNA precursor. Then, 500 μL of the obtained precursor was mixed with the Au@Ag NPs and NQDs solutions prepared in the above steps, and incubated on a shaker at room temperature for 30 min. Then, the two incubated mixtures were mixed in equal volumes (specifically 1.0 mL in this example). 0.4 mL of acrylamide monomer, 0.05 mL of tetramethylethylenediamine (as a catalyst), and 0.03 mL of GO (graphene oxide) were added sequentially, vortexed twice, and incubated for another 3 h to obtain the Au@Ag-NQDs / GO DNA hydrogel SERS-FL dual-mode sensor.

[0069] The fabrication and application flowchart of the Au@Ag-NQDs / GO DNA hydrogel SERS-FL dual-mode sensor is shown below. Figure 1 As shown. Characterization of the Au@Ag-NQDs / GO DNA hydrogel SERS-FL dual-mode sensor.

[0070] The Au@Ag NPs prepared in step (1) of the above embodiments, the hydrogel containing only DNA obtained based on step (3) of the above embodiments, the DNA hydrogel encapsulating only Au@Ag NPs (i.e., the DNA hydrogel prepared without adding NQDs in step (2)), and the Au@Ag-NQDs / GO DNA hydrogel SERS-FL dual-mode sensor were characterized by scanning electron microscopy and transmission electron microscopy, respectively.

[0071] The results are as follows Figure 2 As shown.

[0072] from Figure 2As can be seen from A in the above examples, the Au@Ag NPs prepared have a uniform structure, and the particle size of the obtained particles is between 15 and 40 nm. Figure 2 As can be seen from B, hydrogels containing only DNA exhibit a distinct cavity structure. Figure 2 As can be seen from C, the morphology of the DNA hydrogel encapsulating only the nanomaterials is slightly rough, and the internal cavity structure is clearly visible. From Figure 2 As can be seen from D, in the Au@Ag-NQDs / GO DNA hydrogel SERS-FL dual-mode sensor, Au@Ag NPs are uniformly distributed on the surface of the internal cavity of the hydrogel, indicating that the Au@Ag-NQDs / GO DNA hydrogel SERS-FL dual-mode sensor was successfully constructed.

[0073] The three-dimensional structure of DNA in the SERS-FL dual-mode sensor was characterized using gel electrophoresis. The samples included SERS-FL dual-mode sensors composed of DNA containing only L1, only L1 and L2, only L1-L2-L3, only S1 and S2, and only L1-L2-L3-S1-S2.

[0074] The results are as follows Figure 3 As shown.

[0075] It can be observed that L1-L2-L3-S1-S2 can form a three-dimensional DNA backbone structure (with the expected length) through layer-by-layer base complementarity hybridization, indicating that the DNA backbone design is reasonable and proving the feasibility of preparing hydrogels with the DNA backbone as the core.

[0076] Performance verification of the Au@Ag-NQDs / GO DNA hydrogel SERS-FL dual-mode sensor

[0077] In this embodiment, EpCAM (epithelial cell adhesion molecule) standard solutions of 0.5-60.0 pg / mL were used as test samples to test the SERS-FL dual-mode sensor prepared in the above embodiment.

[0078] The results are as follows Figure 4 As shown.

[0079] It can be observed that within the concentration range of 0.5-60.0 pg / mL of EpCAM standard solution, the SERS-FL dual-mode sensor prepared in the embodiments of this invention exhibits a good linear relationship with EpCAM, and the linear equation is as follows:

[0080] Corresponding SERS intensity: I 862cm -1 = -5025.9lgC + 10617; where, R 2 =0.9952.

[0081] Corresponding FL intensity: I 433nm = 423.9lgC + 174.7; where, R 2 =0.9939.

[0082] The above results show that the LOD of the SERS and FL modes of the SERS-FL dual-mode sensor prepared in the embodiments of the present invention are 0.17 pg / mL and 0.35 pg / mL, respectively (S / N = 3).

[0083] Practical Application Effects of Au@Ag-NQDs / GO DNA Hydrogel SERS-FL Dual-Mode Sensor

[0084] In this embodiment, HeLa, A549, and MCF-7 cells were used as test samples to test the SERS-FL dual-mode sensor prepared in the above embodiment.

[0085] (1) Biocompatibility of Au@Ag-NQDs / GO DNA hydrogel SERS-FL dual-mode sensor:

[0086] The specific testing method is as follows:

[0087] The biotoxicity of different concentrations of Au@Ag NPs (0–100.0 μg / mL) and NQDs (0–25.0 μg / mL) and different incubation times (0.5–24 h) on HeLa, A459, and MCF-7 cells was investigated. 100.0 μL of 10 4 Cells were cultured at a concentration of 1 / mL, corresponding to different concentrations or incubation times, and then 10.0 μL of CCK8 reagent was added for 4 h of further culture. Finally, the relative viability of cells before and after 405 nm laser irradiation was determined using the standard MTT assay, and the relevant absorbance (A) at 450 nm was recorded using a TECAN Spark microplate reader (Tecan, Switzerland). All samples were measured in triplicate. Relative cell viability was assessed using the following cell viability formula:

[0088] Cell viability (%) = (A test -A CCK8 / A control -A CCK8 )×100%.

[0089] Among them, A test Indicates the absorbance of the experimental group; A CCK8 Indicates the absorbance of CCK8; A control This represents the absorbance of the control group.

[0090] The results are as follows Figure 5 As shown.

[0091] The results showed that Au@Ag NPs had no effect on HeLa, A459, and MCF-7 cells within the concentration range of 0–100.0 μg / mL, and the relative cell viability of HeLa, A459, and MCF-7 cells co-incubated with Au@Ag NPs all exceeded 85%. Figure 5 (A) NQDs had no effect on HeLa, A459, and MCF-7 cells in the concentration range of 0–25.0 μg / mL, and the relative cell viability of HeLa, A459, and MCF-7 cells co-incubated with them all exceeded 85%. Figure 5 (B) The results show that the Au@Ag NPs and NQDs prepared separately in the above examples have good biocompatibility. Further, the experimental results of the SERS-FL dual-mode sensor show that within an incubation time range of 0.5–24 h, HeLa, A459, and MCF-7 cell lines all maintained stable and good cell viability in the SERS-FL dual-mode sensor solution, indicating that the SERS-FL dual-mode sensor has good biocompatibility.

[0092] (2) Capture effect of Au@Ag-NQDs / GO DNA hydrogel SERS-FL dual-mode sensor:

[0093] The SERS-FL dual-mode sensor prepared in the above embodiments was used to capture HeLa, A549, and MCF-7 cells. The specific testing method is as follows:

[0094] To achieve in situ detection of target proteins in cells, nuclear-stained cells were first encapsulated in a three-dimensional DNA hydrogel, and then FL imaging was used to count the cells embedded in the DNA hydrogel. The specific procedures are as follows: Cells were cultured to a height of 1×10⁶ cells / year. 6 Cells were cultured at a density of 1 / mL, the old culture medium was removed, and trypsin digestion was performed. The reaction was terminated by adding 4 mL of DMEM medium, followed by centrifugation to collect the cells. After careful pipetting, the nuclei were stained with molecular dyes. 2 μL of DAPI solution was added, and the cells were incubated in the dark for 5 min, followed by washing three times with PBS. 10.0 μL of these cells were added to a confocal microarray containing a DNA hydrogel dual-mode sensor and cultured overnight. After incubation for 3–6 h, the cells were washed twice with PBS to remove excess cells. Finally, FL confocal imaging analysis was performed. The FL imaging conditions were set as follows: 3D imaging was set to Z-stock stacking mode, the excitation wavelength was set to 405 nm (DAPI molecules), and the emission wavelength range was between 440 and 490 nm.

[0095] To avoid signal interference from DAPI dye molecules and contamination during imaging, subsequent SERS-FL bimodal analysis was performed under the same procedures without DAPI treatment. Following the steps described above, a DNA hydrogel encapsulating cells was obtained. By adding a certain amount of cell lysis buffer, the cells were lysed, releasing the target protein, which was specifically recognized by the aptamers in the DNA hydrogel, thus generating a bimodal stimulus response. The gel transitioned to a sol state, releasing Au@Ag NPs and NQDs, respectively. The precipitate and supernatant were collected by centrifugation and used for SERS-FL bimodal analysis, respectively.

[0096] When performing in situ imaging-quantitative analysis of EpCAM in cells, the SERS analysis conditions were as follows: excitation wavelength of 785 nm, integration time of 10 s, laser power of 5 mM, and 3 tests; the FL detection conditions were as follows: excitation wavelength of 543 nm, emission wavelength of 580 nm, and 3 tests.

[0097] The results are as follows Figure 6 As shown.

[0098] It can be observed that the SERS-FL dual-mode sensor has good in-situ capture ability for three types of cells. Calculations based on the number of cells per unit volume show that the density of HeLa, A549, and MCF-7 cells captured in situ by the DNA hydrogel is 50–54 cells / cm³. 3 46-48 per cm 3 and 48-50 per cm 3 .

[0099] (3) Actual test results of the Au@Ag-NQDs / GO DNA hydrogel SERS-FL dual-mode sensor:

[0100] In this embodiment, human serum (12 groups) and cell lysis samples (EpCAM, 3 groups each from Hela, A549 and MCF-7 cells, respectively) were used as test subjects to test the actual testing effect of the Au@Ag-NQDs / GO DNA hydrogel SERS-FL dual-mode sensor in a real-world scenario.

[0101] The specific testing method is as follows: 200 μL of diluted sample solution is added to the pre-prepared DNA hydrogel dual-mode sensor and incubated in a 37℃ shaker for 2–4 hours to allow for sufficient reaction and smooth transition from a gel state to a sol state. Then, the sample is centrifuged and washed twice with PBS. The bottom sediment is collected and placed on a clean silicon wafer for SERS detection. The supernatant collected in the first step is used for FL detection. The SERS analysis conditions are as follows: excitation wavelength 785 nm, integration time 10 s, laser power 5 mM, and 3 tests. The FL detection conditions are as follows: excitation wavelength 543 nm, emission wavelength 580 nm, and 3 tests.

[0102] The results are as follows Figure 7 As shown.

[0103] Dual-mode quantitative analysis was performed on three cell types (HeLa, A549, and MCF-7, 3 groups each) and serum samples (12 groups). The DNA hydrogel dual-mode sensor utilized aptamers to specifically recognize target analytes in the samples, causing DNA backbone breakage and subsequent DNA disintegration. Separation was achieved by centrifugation, followed by SERS and fluorescence detection to obtain SERS and fluorescence signals. Figure 7 In this context, A, C, and E represent the SERS and fluorescence signals obtained after dual-mode analysis of three cell samples (3 groups each) of HeLa, A549, and MCF-7 using a dual-mode sensor, as well as the content of the target compound EpCAM in the cell samples calculated according to a linear formula. Figure 7 In the figure, B, D, and F represent the SERS and fluorescence signals obtained after dual-mode analysis of 12 groups of serum samples using a dual-mode sensor, and the target analyte EpCAM in the serum samples calculated according to a linear formula. These results demonstrate that the SERS-FL dual-mode sensor can be practically applied to the quantitative analysis of EpCAM in human serum and cell lysate samples, providing valuable technical support and basic data for developing efficient, accurate, and rapid analytical methods for complex samples.

[0104] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A surface-enhanced Raman-fluorescence dual-mode sensor, characterized in that, The surface-enhanced Raman-fluorescence dual-mode sensor comprises a hydrogel with a DNA framework as its backbone and Au@Ag nanoparticles and nano-quantum dots (NQDs) attached to the cavity structure of the hydrogel. The DNA framework is a three-dimensional structural framework obtained by hybridization of DNA-1, DNA-2, DNA-3, aptamers, and cDNA. The nucleotide sequence of the DNA-1 is shown in SEQ ID NO:3; The nucleotide sequence of the DNA-2 is shown in SEQ ID NO:4; The nucleotide sequence of the DNA-3 is shown in SEQ ID NO:5; The nucleotide sequence of the aptamer is shown in SEQ ID NO:1; The nucleotide sequence of the cDNA is shown in SEQ ID NO:

2.

2. The surface-enhanced Raman-fluorescence dual-mode sensor according to claim 1, characterized in that, The hydrogel also contains graphene oxide.

3. The surface-enhanced Raman-fluorescence dual-mode sensor according to claim 1, characterized in that, The Au@Ag nanoparticles have a particle size of 15~40 nm.

4. A method for preparing the surface-enhanced Raman-fluorescence dual-mode sensor according to any one of claims 1 to 3, comprising the following steps: mixing DNA-1, DNA-2, DNA-3, aptamer and cDNA as described in claim 1 and annealing and hybridizing at 92 to 95°C, then adding silver-coated Au nanoparticles and NQDs respectively, and incubating for 20 to 40 min; adding an equal volume of the mixture containing silver-coated Au nanoparticles and NQDs respectively, then adding acrylamide monomer and graphene oxide, and continuing incubation for 1 to 5 h to obtain the surface-enhanced Raman-fluorescence dual-mode sensor.

5. The preparation method according to claim 4, characterized in that, After adding acrylamide monomer and graphene oxide, a catalyst is also added; the catalyst includes tetramethylethylenediamine.

6. The preparation method according to claim 5, characterized in that, The molar concentrations of the silver-coated Au nanoparticles, NQDs, acrylamide monomer, graphene oxide, and catalyst are 0.2–0.5 mmol / L, 1.5–3.0 μmol / L, 1.4–1.8 μmol / L, 85–125 μmol / L, and 8.0–10.0 nmol / L, respectively.

7. The use of the surface-enhanced Raman-fluorescence dual-mode sensor according to any one of claims 1 to 3 in the preparation of products that capture cells and / or cell lysates.

8. The application according to claim 7, characterized in that, The cells in question are cancer cells.

9. The application of the surface-enhanced Raman-fluorescence dual-mode sensor according to any one of claims 1 to 3 in the preparation of disease detection products, wherein the diseases include lung cancer, colorectal cancer, and pancreatic cancer.

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