Carcino-embryonic antigen detection kit based on magnetic bead-DNA self-assembly and SERS probe rapid response as well as preparation method and application of carcino-embryonic antigen detection kit

Abstract

The invention discloses a carcino-embryonic antigen detection kit based on magnetic bead-DNA self-assembly and SERS probe rapid response and a preparation method and application thereof, and belongs to the technical field of biological detection. The preparation method of the kit comprises the following steps: constructing a magnetic bead double-stranded DNA probe and a surface enhanced Raman scattering (SERS) probe; the target protein is combined with the aptamer, so that the protected DNA chain is dissociated, the conformation of the aptamer is opened, and the capture site is exposed. The site is hybridized with a captured DNA chain on the SERS probe, so that the SERS probe is connected to the magnetic bead. After the compound is enriched through magnetic separation, the Raman signal intensity is detected, so that accurate quantification of the target protein is realized. Through signal conversion and enrichment amplification, the method has the advantages of high sensitivity, high specificity and quick response.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and more specifically to a carcinoembryonic antigen detection kit based on magnetic bead-DNA self-assembly and rapid SERS probe response, its preparation method, and its application. Background Technology

[0002] In modern clinical medicine, accurate and rapid detection of specific protein biomarkers is crucial for early disease diagnosis, disease monitoring, and treatment efficacy evaluation. Carcinoembryonic antigen (CEA) is a recognized broad-spectrum tumor marker, and its concentration level in body fluids is closely related to the occurrence and development of various malignant tumors. Therefore, establishing a highly sensitive, highly specific, and easy-to-operate CEA detection method has significant clinical value for improving cancer diagnosis and treatment.

[0003] Traditional protein detection methods, such as enzyme-linked immunosorbent assay (ELISA), are widely used, but they often have limitations such as cumbersome operation procedures, long detection cycles, and the need to improve sensitivity, making it difficult to fully meet the clinical needs for early and trace biomarker detection.

[0004] Chinese invention patent application CN201711272662.4 proposes a CEA detection kit based on bimolecular complementary fluorescence (BiFC) technology. It achieves homogeneous detection, eliminating the washing step required by traditional ELISA, simplifying the operation to a "mix-read" process, making it rapid and convenient, suitable for rapid clinical screening. However, it requires the preparation of two different antibody-protein fragment conjugates, making the process relatively complex and increasing cost and quality control difficulty. Furthermore, as a fluorescence detection method, the background fluorescence of complex samples such as serum may interfere with the sensitivity and accuracy of the detection.

[0005] Chinese invention patent application CN202310844120.9 proposes a CEA detection kit based on immunoturbidimetry. It innovatively uses gold-silver composite modified colloidal gold as a label, significantly improving detection sensitivity and reagent stability, thus solving the problem of insufficient sensitivity in traditional colloidal gold methods. However, the core "modified colloidal gold" preparation process is complex, involving multiple chemical synthesis steps. This not only increases production costs and technical difficulty but also poses a challenge to ensuring the consistency of performance across different batches of products.

[0006] To overcome the aforementioned shortcomings, emerging detection technologies are constantly being developed. Among them, surface-enhanced Raman scattering (SERS) technology shows great potential in the field of biosensing due to its ability to provide molecular "fingerprint" spectra, its ultra-high sensitivity, and its excellent resistance to photobleaching. This technology typically utilizes the plasmon resonance effect of noble metal nanoparticles to greatly enhance the signal of Raman reporter molecules adsorbed on their surface, thereby enabling the detection of trace substances.

[0007] In molecular recognition, DNA aptamers, as single-stranded nucleic acid molecules obtained through in vitro screening techniques, can bind to targets with high affinity and high specificity, functionally comparable to antibodies. Compared to antibodies, DNA aptamers have advantages such as simple chemical synthesis, ease of modification, good stability, and lower cost, making them ideal recognition elements for constructing novel biosensors.

[0008] In addition, magnetic microtechnology, which enables rapid separation, enrichment and purification of target substances through an external magnetic field, has been widely used to simplify biological detection processes, reduce background interference and improve detection sensitivity.

[0009] However, how to cleverly integrate the specific recognition capability of DNA aptamers, the ultra-high sensitivity of SERS technology, and the convenient separation and enrichment function of magnetic beads to design a sensing strategy that can efficiently convert protein binding events into detectable SERS signals remains a pressing technical problem in this field. Existing technologies lack a novel, rapid detection method that integrates signal conversion and enrichment amplification by inducing a "switch" in the DNA double-strand conformation through a target protein, thereby triggering the adsorption of SERS probes onto the surface of magnetic beads. Summary of the Invention

[0010] This invention discloses a carcinoembryonic antigen (CEA) detection kit based on magnetic bead-DNA self-assembly and rapid SERS probe response, along with its preparation method and applications. The invention aims to address the problems existing in current protein detection technologies, particularly for important biomarkers such as CEA, overcoming the drawbacks of traditional methods (such as ELISA) which are cumbersome, time-consuming, and have limited sensitivity. The specific technical problem this invention addresses is the development of a novel biosensing strategy that organically combines the high specificity of DNA aptamer recognition, the convenient separation and enrichment of magnetic beads, and the ultra-high sensitivity of surface-enhanced Raman scattering (SERS) detection, thereby achieving rapid, simple, highly sensitive, and highly specific quantitative detection of target proteins.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A carcinoembryonic antigen detection kit based on magnetic bead-DNA self-assembly and rapid response of SERS probe, comprising magnetic bead double-stranded DNA probe and SERS probe; The magnetic bead double-stranded DNA probe comprises a magnetic bead, a protein aptamer DNA strand (CEA-aptamer-12) connected to the magnetic bead, and an aptamer-protective DNA strand (CEA-aptamer-12-half) that partially hybridizes with the CEA-aptamer-12. The CEA-aptamer-12 is a DNA aptamer of the target protein, and its sequence includes a recognition portion for recognizing and binding the target protein and a capture portion for subsequent capture. The CEA-aptamer-12-half is complementary to the recognition and capture portions of the CEA-aptamer-12 to form a double-stranded structure. The SERS probe comprises noble metal nanoparticles, Raman signal molecules modified on their surface, and a capture DNA strand (CEA-cDNA-12) complementary to the capture portion of the CEA-aptamer-12.

[0012] Furthermore, the target protein is carcinoembryonic antigen (CEA).

[0013] Furthermore, the CEA-aptamer-12 is connected to the surface of the magnetic beads via a biotin-avidin system, specifically by modifying the 5' end of the first DNA strand with biotin and coating the surface of the magnetic beads with avidin.

[0014] Furthermore, the noble metal nanoparticles are gold nanoparticles (AuNPs), and the Raman signal molecule is TAMRA dye.

[0015] Furthermore, the CEA-cDNA-12 and the Raman signaling molecule TAMRA are linked to the gold nanoparticle surface through Au-S bonds formed by thiol groups.

[0016] Furthermore, the aptamer CEA-aptamer-12 has the sequence (Biotin-AAA AAA AAA AAT ACCAGC TTA TTC AAT TTT GGT GTG TTG G); the aptamer protective DNA strand (CEA-aptamer-12-half) sequence is (CCA ACA CAC CAA AAT TGA ATA AGC TGG TA); the capture DNA (CEA-cDNA-12) sequence on the SERS probe is (SH - CCA TCC AAC ACA CCA A); and the Raman tag molecule sequence (TAMRA-DNA) sequence on the SERS probe is (TAMRA - TTT TTT CCT AGC GAC - SH).

[0017] A method for preparing a carcinoembryonic antigen detection kit based on magnetic bead-DNA self-assembly and rapid SERS probe response includes the following steps: (1) Magnetic bead double-stranded DNA probe: The probe includes a magnetic bead, a protein aptamer DNA strand (CEA-aptamer-12) connected to the magnetic bead, and an aptamer protective DNA strand (CEA-aptamer-12-half) that hybridizes with the CEA-aptamer-12; the CEA-aptamer-12 is a DNA aptamer of the target protein, and its sequence includes a recognition portion for recognizing and binding the target protein and a capture portion for subsequent capture; the CEA-aptamer-12-half is complementary to the recognition portion and the capture portion of the CEA-aptamer-12 to form a double-stranded structure; (2) SERS probe: The SERS probe includes noble metal nanoparticles, Raman signal molecules modified on their surface, and a capture DNA strand (CEA-cDNA-12) complementary to the capture portion of the CEA-aptamer-12. (3) The sample to be tested is mixed with magnetic bead double-stranded DNA probe and SERS probe and incubated: When the target protein is present in the sample, the target protein binds to the recognition part of CEA-aptamer-12, causing CEA-aptamer-12-half to dissociate from CEA-aptamer-12, thereby exposing the capture part of CEA-aptamer-12. (4) The exposed capture portion hybridizes with CEA-cDNA-12 on the SERS probe to form a complex containing magnetic beads, target protein and SERS probe; (5) Magnetic separation and enrichment: Detect the Raman signal intensity of the magnetic bead complex and determine the concentration of the target protein in the sample based on the Raman signal intensity.

[0018] Furthermore, the incubation conditions in step (3) are 30-40 ℃ and the incubation time is 30-60 minutes.

[0019] An application of a carcinoembryonic antigen (CEA) detection kit based on magnetic bead-DNA self-assembly and rapid SERS probe in the preparation of products for detecting CEA, which detects the Raman signal intensity of the magnetic bead complex and determines the concentration of the target protein in the sample based on the Raman signal intensity.

[0020] Compared with the prior art, the present invention has the following beneficial effects: This invention constructs a magnetic bead double-stranded DNA probe and a surface-enhanced Raman scattering (SERS) probe. The target protein binds to the aptamer, causing the protective DNA strand to dissociate, thus "opening" the aptamer conformation and exposing the capture site. This site hybridizes with the capture DNA strand on the SERS probe, thereby attaching the SERS probe to the magnetic bead. After enriching the complex through magnetic separation, the Raman signal intensity is detected, enabling precise quantification of the target protein. This invention, through signal conversion and enrichment amplification, offers advantages such as high sensitivity, high specificity, and rapid response. Attached Figure Description

[0021] Figure 1 This is a flowchart of the present invention; Figure 2 This is a schematic diagram of lung cancer biomarker protein detection using a SERS probe based on signal conversion in this invention; Figure 3 (a) is the UV absorption curve of gold nanosol; Figure 3 (b) TEM image of gold nanoparticles; Figure 3 (c) Particle size distribution of gold nanoparticles; Figure 4 These are ultraviolet spectra, including: (a) AuNPs-cDNA; (b) AuNPs-ROX; (c) AuNPs; (d) SERS-Probe; Figure 5 These are the laser confocal Raman spectra of the control experiment, where: (a) no CEA was added during incubation; (b) CEA was added during incubation. Figure 6 (a) shows the SERS Raman spectra under different concentration conditions; Figure 6 (b) is a graph showing the linear relationship between Raman intensity and different CEA concentrations. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0023] Example 1 A carcinoembryonic antigen detection kit based on magnetic bead-DNA self-assembly and rapid response of SERS probe, characterized in that it includes magnetic bead double-stranded DNA probe and SERS probe; The magnetic bead double-stranded DNA probe comprises a magnetic bead, a protein aptamer DNA strand (CEA-aptamer-12) connected to the magnetic bead, and an aptamer-protective DNA strand (CEA-aptamer-12-half) that partially hybridizes with the CEA-aptamer-12. The CEA-aptamer-12 is a DNA aptamer of the target protein, and its sequence includes a recognition portion for recognizing and binding the target protein and a capture portion for subsequent capture. The CEA-aptamer-12-half is complementary to the recognition and capture portions of the CEA-aptamer-12 to form a double-stranded structure. The SERS probe comprises noble metal nanoparticles, Raman signal molecules modified on their surface, and a capture DNA strand (CEA-cDNA-12) complementary to the capture portion of the CEA-aptamer-12. The target protein is carcinoembryonic antigen (CEA). The CEA-aptamer-12 is connected to the surface of the magnetic beads via a biotin-avidin system, specifically by modifying the 5' end of the first DNA strand with biotin and coating the surface of the magnetic beads with avidin. The noble metal nanoparticles are gold nanoparticles (AuNPs), and the Raman signal molecules are TAMRA dyes.

[0024] The CEA-cDNA-12 and the Raman signaling molecule TAMRA are linked by Au-S bonds formed between thiol groups and the surface of gold nanoparticles. The aptamer CEA-aptamer-12 has the sequence (Biotin-AAA AAA AAA AAT ACC AGC TTATTC AAT TTT GGT GTG TTG G); the aptamer protective DNA strand (CEA-aptamer-12-half) has the sequence (CCAACA CAC CAA AAT TGA ATA AGC TGG TA); the capture DNA (CEA-cDNA-12) sequence on the SERS probe has the sequence (SH - CCA TCC AAC ACA CCA A); and the Raman tag molecule sequence (TAMRA-DNA) sequence on the SERS probe has the sequence (TAMRA - TTT TTT CCT AGC GAC - SH).

[0025] Example 2 A method for preparing a carcinoembryonic antigen detection kit based on magnetic bead-DNA self-assembly and rapid SERS probe response includes the following steps: (1) Magnetic bead double-stranded DNA probe: The probe includes a magnetic bead, a protein aptamer DNA strand (CEA-aptamer-12) connected to the magnetic bead, and an aptamer protective DNA strand (CEA-aptamer-12-half) that hybridizes with the CEA-aptamer-12; the CEA-aptamer-12 is a DNA aptamer of the target protein, and its sequence includes a recognition portion for recognizing and binding the target protein and a capture portion for subsequent capture; the CEA-aptamer-12-half is complementary to the recognition portion and the capture portion of the CEA-aptamer-12 to form a double-stranded structure; (2) SERS probe: The SERS probe includes noble metal nanoparticles, Raman signal molecules modified on their surface, and a capture DNA strand (CEA-cDNA-12) complementary to the capture portion of the CEA-aptamer-12. (3) The sample to be tested is mixed with magnetic bead double-stranded DNA probe and SERS probe and incubated: When the target protein is present in the sample, the target protein binds to the recognition part of CEA-aptamer-12, causing CEA-aptamer-12-half to dissociate from CEA-aptamer-12, thereby exposing the capture part of CEA-aptamer-12; the incubation conditions in step (3) are 30-40 ℃ and the incubation time is 30-60 minutes; (4) The exposed capture portion hybridizes with CEA-cDNA-12 on the SERS probe to form a complex containing magnetic beads, target protein and SERS probe; (5) Magnetic separation and enrichment: Detect the Raman signal intensity of the magnetic bead complex and determine the concentration of the target protein in the sample based on the Raman signal intensity.

[0026] Example 3 1. Construction of magnetic bead double-stranded DNA probes: (1) Select a DNA aptamer (referred to as CEA-aptamer-12 in this paper) that has high affinity and specificity for the target protein (CEA). Modify the 5' end of CEA-aptamer-12 with biotin.

[0027] (2) The magnetic beads coated with streptavidin are mixed with the biotin-modified CEA-aptamer-12 described above. The first DNA strand is firmly fixed on the surface of the magnetic beads through the high affinity interaction between biotin and streptavidin.

[0028] (3) Design and synthesize a short DNA strand partially complementary to CEA-aptamer-12 as a protective DNA strand for the aptamer (referred to as CEA-aptamer-12-half in this paper).

[0029] (4) CEA-aptamer-12-half is mixed with magnetic beads modified with CEA-aptamer-12 and incubated to allow them to hybridize and form a stable DNA double-stranded structure. At this time, the capture region of the first DNA strand is "closed", forming a magnetic bead double-stranded DNA probe in a "closed" state, which can effectively prevent non-specific adsorption.

[0030] 2. Preparation of SERS probes: (1) Synthesize gold nanoparticles (AuNPs) as SERS-enhancing substrates.

[0031] (2) Design and synthesize two functional DNA strands: one is a DNA strand with Raman reporter molecule (TAMRA) and thiol (-SH) modified at the end (referred to as TAMRA-DNA in this paper); the other is a capture DNA strand with a sequence complementary to the capture region of CEA-aptamer-12 and modified with thiol at the end (referred to as CEA-cDNA-12 in this paper).

[0032] (3) The two DNA strands mentioned above were mixed with the gold nanoparticle solution and covalently linked to the surface of the gold nanoparticles through gold-thiol (Au-S) bonds to prepare a SERS probe that simultaneously carries a Raman reporter molecule and a DNA strand.

[0033] 3. Detection principle and procedure: (1) Incubation and identification: Mix the sample to be tested (with or without the target protein CEA) buffer with the magnetic bead double-stranded DNA probe and SERS probe prepared above and incubate.

[0034] (2) “Activation” and Dissociation: If the target protein CEA is present in the sample, CEA will specifically bind to the recognition region of CEA-aptamer-12 on the magnetic bead. This binding causes a significant change in the aptamer conformation, thereby disrupting the double-stranded structure between CEA-aptamer-12 and CEA-aptamer-12-half, causing CEA-aptamer-12-half to dissociate from the surface of the magnetic bead. This process achieves the “activation” of the CEA-aptamer-12 capture region.

[0035] (3) Capture and assembly: The capture region of CEA-aptamer-12 exposed after "opening" will specifically hybridize with the capture DNA strand (CEA-cDNA-12) on the SERS probe in solution. Thus, the SERS probe is "captured" onto the surface of the magnetic bead, forming a "magnetic bead-aptamer-SERS probe" sandwich structure complex.

[0036] (4) Separation and detection: The above mixture is magnetically separated using an external magnetic field. Free SERS probes that are not bound to the magnetic beads are washed away, while the resulting complexes are enriched.

[0037] (5) Signal readout: Raman spectroscopy was performed on the enriched complex. Since the SERS probes were enriched on the magnetic beads, their Raman signals were concentrated and detected. The measured Raman signal intensity was proportional to the number of captured SERS probes, which in turn was positively correlated with the concentration of the target protein CEA in the sample. By establishing a standard curve of Raman signal intensity versus CEA concentration, accurate quantification of CEA in the sample could be achieved.

[0038] Example 4 CEA detection based on magnetic bead-DNA self-assembly and SERS probes This embodiment details the entire process of preparing a sensing platform and detecting carcinoembryonic antigen (CEA) using the method of the present invention, such as... Figure 1 and 2 As shown.

[0039] Table 1 Nucleic acid sequences used in this invention The nucleic acid sequences are shown in Table 1.

[0040] 1. Preparation of gold nanoparticles (AuNPs) Add 100 mL of 1 mM / L chloroauric acid tetrahydrate solution to a 250 mL three-necked flask and heat to boiling in an oil bath. Quickly add 2.6 mL of 1% trisodium citrate solution, continue heating and observe the color change. The solution color changes from colorless to bluish-black, and finally stabilizes at a wine-red color. Stop heating and allow to cool naturally to room temperature. Analyze the result using a UV-Vis spectrophotometer. Figure 3 a) and transmission electron microscopy (TEM) Figure 3 b) The prepared AuNPs were characterized, confirming that their characteristic absorption peak was around 520 nm, the particle size was approximately 15 nm, and the distribution was uniform. Figure 3 c).

[0041] 2. Preparation of SERS probes Take 3 µL of 100 µM TAMRA-DNA solution and 3 µ L concentration is 10 µ M's CEA-cDNA-12 solution was added to a solution at 14°C. µ In a 1 mM TCEP solution, incubate by rotation at room temperature for 30-45 minutes to reduce the disulfide bonds at the DNA strand ends. Then, add 80 μL of the solution to the above mixture. µ The gold nanoparticle solution prepared in step 1 of step L was shaken to mix thoroughly. 900 mL of [unspecified liquid] was quickly added. µ L-butanol was added and the mixture was shaken vigorously to promote the binding of DNA strands to gold nanoparticles. 20 μL of butanol was added to the system. µ Dissolve L of 0.5×TBE buffer (containing 45 mM Tris-HCl, 1 mM EDTA, 45 mM B(OH)3, pH 8.0), stir rapidly, and centrifuge at 9000 rpm for 10-15 minutes. Discard the supernatant and collect the lower precipitate. Use 80 mL of 0.5×TBE buffer. µ The precipitate was washed three times with L of PBS buffer (containing 10 mM Na₂HPO₄, 2 mM KH₂PO₄, 137 mM NaCl, 2.7 mM KCl, pH ~7.4). Finally, the obtained SERS probe precipitate was washed with 40 mL of PBS buffer. µ Resuspend in L PBS buffer and store at 4 °C for later use. UV-Vis spectroscopy ( Figure 4 The results showed that the prepared SERS probe (curve d) exhibited an absorption peak of 565 nm contributed by TAMRA dye and a 260 nm absorption peak contributed by DNA, in addition to the 520 nm absorption peak of AuNPs, proving that the SERS probe was successfully prepared.

[0042] 3. Preparation of magnetic bead double-stranded DNA probes (1) Preparation of CEA-aptamer-12 modified magnetic beads: Take 5 µ L concentration is 100 µ The CEA-aptamer-12 solution of M was diluted to 150 μL using Buffer I solution (containing 10 mM Tris-HCl, 1 mM EDTA, 1 M NaCl, 0.01%–0.1% Tween-20, pH ~7.5). µ L, used as the DNA adsorption solution. Take 30 µ L-streptavidin magnetic bead suspension, after magnetic separation, the supernatant was discarded, and 300... µ Wash once with L Buffer I. Then wash the above 150... µDNA adsorption buffer (L) was added to the treated magnetic beads, and the mixture was thoroughly resuspended and incubated at room temperature on a rotary mixer for 30-45 minutes. After incubation, magnetic separation was performed, and the supernatant was discarded. The magnetic beads were washed three times with Buffer I, and then twice with PBS buffer. Finally, the beads were washed with 200 μL of PBS buffer. µ The magnetic beads were resuspended in L of PBS buffer to obtain a CEA-aptamer-12 modified magnetic bead solution.

[0043] (2) Formation of DNA double helix: Take the above 200 µ L of CEA-aptamer-12 modified magnetic bead solution was magnetically separated, and the supernatant was discarded. 5 µ L concentration is 100 µ M CEA-aptamer-12-half solution was prepared and PBS buffer was added as a hybridization buffer. The mixture was gently incubated at 42-45 °C with gentle rotation for 3-3.5 hours to allow for complete hybridization. After incubation, the mixture was cooled to room temperature and magnetically separated. The supernatant was discarded to remove unbound CEA-aptamer-12-half. The magnetic beads were washed three times with hybridization buffer to obtain the magnetic bead double-stranded DNA probe in the "off" state.

[0044] 4. CEA testing Aliquot the magnetic bead double-stranded DNA probes prepared in step 3 into multiple centrifuge tubes. Add PBS buffer containing different concentrations of CEA (0, 1, 2, 5, 10 ng / mL) to each tube and incubate gently at room temperature for 2 hours. After incubation, add an equal volume of the SERS probe solution prepared in step 2 to each tube and continue incubating at room temperature for 30 minutes. After incubation, magnetically separate each tube and carefully aspirate the supernatant. Then, centrifuge the tubes at 10,000 rpm and aspirate the remaining PBS buffer using a pipette. Collect the precipitate at the bottom of the tube and drop it onto a clean gold plate, allowing it to air dry at room temperature.

[0045] Finally, Raman spectra of the samples on the gold sheet were acquired using a laser confocal micro Raman spectrometer. The intensity of the characteristic peak of the TAMRA molecule (1358 cm⁻¹) was selected as the quantitative signal.

[0046] Feasibility verification: such as Figure 5As shown, the control group without CEA (curve a) exhibited a very weak Raman signal, indicating that the SERS probe was not attached to the magnetic carrier in the absence of a target, and the double-stranded DNA magnetic bead system was in a "closed" state. In contrast, the experimental group with added CEA (curve b) showed a very strong TAMRA Raman characteristic peak, proving that the addition of CEA successfully triggered the release of the SERS probe, achieving the signal transition from "closed" to "open," thus verifying the feasibility of this method.

[0047] Sensitivity and quantitative analysis: such as Figure 6 As shown in Figure a, a series of Raman spectra were obtained by detecting samples with different CEA concentrations. The results show that the intensity of the Raman characteristic peak at 1358 cm⁻¹ gradually increases with increasing CEA concentration. Linear fitting between the Raman signal intensity corresponding to different concentrations and the CEA concentration revealed a good linear relationship within the concentration range of 1 ng / mL to 10 ng / mL. The linear regression equation was ΔI = 507.72 + 1785.90CCEA, with a correlation coefficient R² of 0.9948. This result demonstrates that the method disclosed in this embodiment can achieve highly sensitive and accurate quantitative detection of CEA protein.

[0048] Example 5 An application of a carcinoembryonic antigen (CEA) detection kit based on magnetic bead-DNA self-assembly and rapid SERS probe in the preparation of products for detecting CEA; the Raman signal intensity of the magnetic bead complex is detected, and the concentration of the target protein in the sample is determined based on the Raman signal intensity.

[0049] This invention relates to a carcinoembryonic antigen (CEA) detection kit based on magnetic bead-DNA self-assembly and a rapid response SERS probe, along with its preparation method and applications, belonging to the field of biodetection technology. The kit is prepared by constructing a magnetic bead double-stranded DNA probe and a surface-enhanced Raman scattering (SERS) probe. The target protein binds to the aptamer, causing the protective DNA strand to dissociate, thus "opening" the aptamer conformation and exposing the capture site. This site hybridizes with the capture DNA strand on the SERS probe, thereby attaching the SERS probe to the magnetic bead. After enriching the complex through magnetic separation, the Raman signal intensity is detected, thereby achieving precise quantification of the target protein. This invention, through signal conversion and enrichment amplification, has the advantages of high sensitivity, high specificity, and rapid response.

[0050] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A carcinoembryonic antigen (CEA) detection kit based on magnetic bead-DNA self-assembly and rapid SERS probe response, characterized in that, Including magnetic bead double-stranded DNA probes and SERS probes; The magnetic bead double-stranded DNA probe includes a magnetic bead, a protein aptamer DNA strand connected to the magnetic bead, namely CEA-aptamer-12, and an aptamer protective DNA strand, namely CEA-aptamer-12-half, which partially hybridizes with the CEA-aptamer-12; the CEA-aptamer-12 is a DNA aptamer of the target protein, and its sequence includes a recognition portion for recognizing and binding the target protein and a capture portion for subsequent capture; The CEA-aptamer-12-half is complementary to the recognition and capture portions of the CEA-aptamer-12, forming a double-chain structure; The SERS probe comprises noble metal nanoparticles, Raman signal molecules modified on their surface, and a capture DNA strand, namely CEA-cDNA-12, that is complementary to the capture portion of the CEA-aptamer-12.

2. The carcinoembryonic antigen detection kit based on magnetic bead-DNA self-assembly and rapid SERS probe response according to claim 1, characterized in that, The target protein is carcinoembryonic antigen (CEA).

3. The carcinoembryonic antigen detection kit based on magnetic bead-DNA self-assembly and rapid SERS probe response according to claim 1, characterized in that, The CEA-aptamer-12 is connected to the surface of the magnetic beads via a biotin-avidin system, specifically by modifying the 5' end of the first DNA strand with biotin and coating the surface of the magnetic beads with avidin.

4. The carcinoembryonic antigen detection kit based on magnetic bead-DNA self-assembly and rapid SERS probe response according to claim 1, characterized in that, The noble metal nanoparticles are gold nanoparticles (AuNPs), and the Raman signal molecule is TAMRA dye.

5. The carcinoembryonic antigen detection kit based on magnetic bead-DNA self-assembly and rapid SERS probe response according to claim 1, characterized in that, The CEA-cDNA-12 and the Raman signaling molecule TAMRA are linked by Au-S bonds formed between thiol groups and the surface of gold nanoparticles.

6. The carcinoembryonic antigen detection kit based on magnetic bead-DNA self-assembly and rapid SERS probe response according to claim 1, characterized in that, The aptamer CEA-aptamer-12 has the sequence Biotin-AAA AAA AAA AAT ACCAGC TTA TTC AAT TTT GGT GTG TTG G; the aptamer-protected DNA strand, CEA-aptamer-12-half, has the sequence CCA ACA CAC CAA AAT TGA ATA AGC TGG TA; the capture DNA on the SERS probe, CEA-cDNA-12, has the sequence SH-CCA TCC AAC ACA CCA A; and the Raman tag molecule sequence on the SERS probe, TAMRA-DNA, has the sequence TAMRA-TT TTT CCT AGC GAC-SH.

7. The method for preparing the carcinoembryonic antigen detection kit based on magnetic bead-DNA self-assembly and rapid SERS probe response according to any one of claims 1-6, characterized in that, Includes the following steps: (1) Magnetic bead double-stranded DNA probe: The probe includes a magnetic bead, a protein aptamer DNA strand connected to the magnetic bead, namely CEA-aptamer-12, and an aptamer protective DNA strand, namely CEA-aptamer-12-half, which partially hybridizes with the CEA-aptamer-12; the CEA-aptamer-12 is a DNA aptamer of the target protein, and its sequence includes a recognition portion for recognizing and binding the target protein and a capture portion for subsequent capture; The CEA-aptamer-12-half is complementary to the recognition and capture portions of the CEA-aptamer-12, forming a double-chain structure; (2) SERS probe: The SERS probe includes noble metal nanoparticles, Raman signal molecules modified on their surface, and a capture DNA strand, namely CEA-cDNA-12, which is complementary to the capture portion of the CEA-aptamer-12. (3) The sample to be tested is mixed with magnetic bead double-stranded DNA probe and SERS probe and incubated: When the target protein is present in the sample, the target protein binds to the recognition part of CEA-aptamer-12, causing CEA-aptamer-12-half to dissociate from CEA-aptamer-12, thereby exposing the capture part of CEA-aptamer-12. (4) The exposed capture portion hybridizes with CEA-cDNA-12 on the SERS probe to form a complex containing magnetic beads, target protein and SERS probe.

8. The method for preparing the carcinoembryonic antigen detection kit based on magnetic bead-DNA self-assembly and rapid SERS probe response according to claim 7, characterized in that, The incubation conditions in step (3) are 30-40 ℃ and the incubation time is 30-60 minutes.

9. The application of the carcinoembryonic antigen detection kit based on magnetic bead-DNA self-assembly and SERS probe rapid response as described in any one of claims 1-6 in the preparation of products for detecting carcinoembryonic antigen (CEA).

10. The application according to claim 9, characterized in that: The Raman signal intensity of the magnetic bead complex was detected, and the concentration of the target protein in the sample was determined based on the Raman signal intensity.

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