Lung cancer marker protein biosensor for regulating and controlling SERS (Surface Enhanced Raman Scattering) probe array based on hydrophilic and hydrophobic effects as well as preparation method and application thereof

Abstract

The invention discloses a lung cancer marker protein biosensor for regulating and controlling an SERS (Surface Enhanced Raman Scattering) probe array based on hydrophilic and hydrophobic effects as well as a preparation method and application of the lung cancer marker protein biosensor, the biosensor is fixed with magnetic beads of DNA (Deoxyribose Nucleic Acid) aptamers, and when the biosensor is specifically combined with a target protein, a section of triggering DNA is competitively released. The trigger DNA is used as a bridge and is connected with an SERS probe with a Raman reporter molecule and a DNA probe modified with a cholesterol hydrophobic group to form a sandwich compound. The compound is dropwise added to a substrate subjected to hydrophobization treatment, and the compound is subjected to self-assembly aggregation under the driving of hydrophilic and hydrophobic interaction, so that SERS probes are close to each other to form a hot spot, and the exponential amplification of a Raman signal is realized. The concentration of the target protein is ingeniously converted into a Raman signal capable of being detected at high sensitivity, accurate quantitative analysis of the target protein is achieved, and the method has important application value in the clinical detection field of early diagnosis of cancers and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical detection, and particularly relates to a lung cancer marker protein biosensor based on hydrophilic-hydrophobic interaction regulated SERS probe array and a preparation method and application thereof. BACKGROUND

[0002] Malignant tumor is one of the major diseases that seriously threaten human life and health at present. Realizing early diagnosis and precise treatment of cancer is the key to improving the survival rate of patients and improving the prognosis. Protein markers are functional proteins produced or abnormally expressed by cells during carcinogenesis, and the concentration changes thereof in blood or body fluid are closely related to the occurrence and development of diseases. Therefore, developing a high-sensitivity and high-specificity protein marker detection method has crucial clinical value for early screening, disease monitoring and efficacy evaluation of cancer. For example, carcinoembryonic antigen (CEA) as a broad-spectrum tumor marker, the abnormal increase of the level thereof is closely related to the occurrence of lung cancer, colorectal cancer and many other cancers.

[0003] Surface-enhanced Raman scattering (SERS) technology has great application potential in the field of biomolecule detection due to its fingerprint identification characteristics, high sensitivity, resistance to photobleaching and small interference of water molecules. The signal enhancement effect of SERS mainly comes from the "hot spots" formed in the gap when noble metal nanoparticles (such as gold and silver nanoparticles) are aggregated. The Raman signal of molecules in the "hot spot" region can be amplified by several orders of magnitude.

[0004] However, in the traditional SERS detection strategy, SERS probes are usually randomly and disorderly dispersed on the detection substrate, and it is difficult to effectively and controllably form high-density "hot spot" regions. The disordered distribution of such probes leads to insufficient signal amplification efficiency and poor reproducibility, which greatly limits the practical application of SERS technology in trace biomolecule detection, especially in the accurate quantitative analysis of low-abundance protein markers. Its sensitivity often cannot meet the needs of early clinical diagnosis.

[0005] Chinese invention patent application CN202111110426.9 proposes a fluorescence aptamer sensor based on magnetic graphene, which is used for detecting CEA. It simplifies the operation by using magnetic separation, realizes rapid and convenient detection, and the anti-pollution peptide coating on the surface effectively reduces the interference in the biological sample and improves the sensitivity and specificity of the detection. However, the preparation process of the sensor involves multiple functionalization steps, and the process is complex, which poses a challenge to ensuring the consistency between batches. In addition, the long-term stability of the magnetic graphene material in practical application is also a factor that needs to be considered.

[0006] Chinese invention patent application CN202211629902.2 proposes a CEA detection method based on DNA nanomachines and CRISPR-Cas12a. It cleverly combines the two technologies to achieve "double cascaded amplification" of the signal, resulting in extremely high sensitivity suitable for early screening. Simultaneously, magnetic separation simplifies the operation steps and shortens the detection time. However, due to the extremely complex system structure involving multiple customized biological reagents and cumbersome preparation processes, the cost is high and batch-to-batch stability control is difficult, posing a challenge to commercial production.

[0007] Therefore, there is an urgent need in this field to develop a novel detection strategy that can achieve controllable and efficient aggregation of SERS probes to stably generate strong SERS signals, thereby solving the problems of insufficient sensitivity and quantitative accuracy in existing technologies. Summary of the Invention

[0008] This invention discloses a lung cancer biosensor protein biosensor based on hydrophilic-hydrophobic interaction-regulated SERS probe array, its preparation method, and its application. This invention aims to overcome the shortcomings of existing technologies and mainly solves the technical problems in existing protein biosensor detection methods based on surface-enhanced Raman scattering (SERS), such as low "hot spot" formation efficiency, unstable signal amplification, and insufficient detection sensitivity and quantitative accuracy due to the random distribution of SERS probes on the substrate.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A biosensor for lung cancer biomarker proteins based on hydrophilic-hydrophobic interaction-regulated SERS probe array, comprising double-stranded DNA magnetic bead probes, SERS probes, hydrophobic DNA probes, and a hydrophobic substrate; The double-stranded DNA magnetic bead probe consists of a 5'-terminal biotin-modified DNA aptamer (FTD-CEA-aptamer) that specifically binds to the target protein marker and is attached to the magnetic bead surface via biotin-avidin interaction; and a trigger DNA (FTD-CEA-armDNA) whose end is partially complementary to the FTD-CEA-aptamer and is attached to the magnetic bead surface via base interaction. The SERS probe comprises noble metal nanoparticles, Raman signaling molecules modified on its surface (FTD-CEA-ROX), and a capture DNA strand (FTD-CEA-cDNA) partially complementary to the FTD-CEA-armDNA. The hydrophobic group of the hydrophobic DNA probe is a cholesterol molecule.

[0010] Furthermore, the hydrophobic substrate is a glass or silicon wafer surface treated with perfluoroalkylsilane or long-chain alkylsilane.

[0011] Further, the noble metal nanoparticles are gold nanoparticles (AuNPs), and the Raman signal molecules are ROX dyes.

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

[0013] Further, the sequence of the aptamer FTD-CEA-aptamer is (Biotin - AAA AAA AAA AAT ACC AGC TTA TTC AAT TTA TCA AT); the sequence of the trigger DNA chain (FTD-CEA-armDNA) is (GGA TTA TTG TTA AAT TGT AAA TTG); the sequence of the capture DNA (FTD-CEA-cDNA) on the SERS probe is (ATT TAA CAA TTA TCC AAAAAAAAAA - SH); the sequence of the Raman label molecule (FTD-CEA-ROX) on the SERS probe is (ROX - TTT TTT CCT AGC GAC - SH); and the sequence of the hydrophobic DNA chain (FTD-CEA-Chol) is (Cholesterol - CAA TTT ATC AAT).

[0014] A preparation method of a lung cancer marker protein biosensor based on hydrophilic-hydrophobic interaction regulation of a SERS probe array, comprising the following steps: (1) Preparation of a double-stranded DNA magnetic bead probe: a 5'-end biotin-modified DNA aptamer (FTD-CEA-aptamer) capable of specifically binding to a target protein marker is connected to the surface of a magnetic bead through biotin-avidin interaction; a trigger DNA (FTD-CEA-armDNA) having a terminal end complementary to the FTD-CEA-aptamer is connected to the surface of the magnetic bead through base interaction, thereby preparing a double-stranded DNA magnetic bead probe; (2) Preparation of a SERS probe: the SERS probe comprises noble metal nanoparticles, Raman signal molecules (FTD-CEA-ROX) modified on the surface of the noble metal nanoparticles, and a capture DNA chain (FTD-CEA-cDNA) complementary to the FTD-CEA-armDNA; (3) Incubation of a sample buffer to be tested with the double-stranded DNA magnetic bead probe; when the target protein marker exists in the sample, it binds to the FTD-CEA-aptamer, causing the FTD-CEA-armDNA to dissociate from the magnetic bead probe and be released into the solution; (4) The solution containing the released FTD-CEA-arm DNA is mixed with the SERS probe and the hydrophobic DNA probe (FTD-CEA-Chol) to form a sandwich complex; the FTD-CEA-arm DNA serves as a connecting bridge, and the sequences at both ends thereof are complementary to the FTD-CEA-cDNA on the SERS probe and the FTD-CEA-Chol base portion, respectively; (5) The sandwich complex formed in step (4) is added dropwise to a hydrophobic substrate surface, and the complex is aggregated on the substrate surface by hydrophilic-hydrophobic interaction, so that the SERS probes are close to each other to form Raman signal "hot spots"; (6) The Raman signal intensity of the hydrophobic substrate surface is detected, and the concentration of the target protein marker in the sample to be tested is determined according to the signal intensity.

[0015] Further, the incubation condition in step (3) is 30-40 ℃, and the incubation time is 30-60 minutes.

[0016] The application of a lung cancer marker protein biosensor based on hydrophilic-hydrophobic interaction regulation of a SERS probe array in the preparation of a product for detecting lung cancer marker proteins, detecting the Raman signal intensity of the hydrophobic substrate surface, and determining the concentration of the target protein marker in the sample to be tested according to the signal intensity.

[0017] Compared with the prior art, the present application has the following beneficial effects: The present application ingeniously converts the target protein concentration into a Raman signal that can be detected with high sensitivity, realizes accurate quantitative analysis of the target protein, and has important application value in the field of clinical detection such as early diagnosis of cancer. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a flowchart of the present application; Figure 2 is a schematic diagram of the present application for detecting lung cancer marker proteins based on hydrophilic-hydrophobic interaction regulation of a SERS probe array; Figure 3 (a) is the ultraviolet absorption curve of the gold nanosol, Figure 3 (b) is the TEM image of the gold nanoparticles, Figure 3 (c) is the particle size distribution graph of the gold nanoparticles; Figure 4 is the ultraviolet spectrum: (a) AuNPs-cDNA; (b) AuNPs-ROX; (c) AuNPs; (d) SERS-Probe; Figure 5 is the water contact angle test, wherein: (a) the water contact angle of glass before hydrophobic chain modification; (b) the water contact angle of glass after hydrophobic chain modification; Figure 6 (a) are the spectral graphs under different concentration conditions, Figure 6 (b) the linear relationship between Raman intensity and different CEA protein concentrations. DETAILED DESCRIPTION

[0019] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0020] Embodiment 1 A lung cancer marker protein biosensor based on hydrophilic-hydrophobic interaction regulation of SERS probe array, comprising a double-stranded DNA magnetic bead probe, a SERS probe, a hydrophobic DNA probe and a hydrophobic substrate. The double-stranded DNA magnetic bead probe: a DNA aptamer (FTD-CEA-aptamer) capable of specific binding to the target protein marker and modified with biotin at the 5' end, connected to the surface of the magnetic bead through biotin-avidin interaction; a trigger DNA (FTD-CEA-armDNA) complementary to the FTD-CEA-aptamer at the end, connected to the surface of the magnetic bead through base interaction; The SERS probe comprises a noble metal nanoparticle, a Raman signal molecule (FTD-CEA-ROX) modified on the surface of the noble metal nanoparticle, and a capture DNA strand (FTD-CEA-cDNA) complementary to the FTD-CEA-armDNA; The hydrophobic group of the hydrophobic DNA probe is a cholesterol molecule; The hydrophobic substrate is the surface of a glass or silicon wafer treated with perfluoroalkylsilane or long-chain alkylsilane; The noble metal nanoparticle is a gold nanoparticle (AuNPs), and the Raman signal molecule is a ROX dye; The target protein marker is carcinoembryonic antigen (CEA).

[0021] The sequence of the aptamer FTD-CEA-aptamer is (Biotin - AAA AAA AAA AAT ACC AGC TTATTC AAT TTA TCAAT); the sequence of the trigger DNA chain (FTD-CEA-armDNA) is (GGA TTA TTG TTA AAT ATTGAT AAA TTG); the sequence of the capture DNA (FTD-CEA-cDNA) on the SERS probe is (ATT TAA CAA TAA TCCAAAAAAAAAA - SH); the sequence of the Raman label molecule (FTD-CEA-ROX) on the SERS probe is (ROX -TTT TTTCCT AGC GAC - SH); and the sequence of the hydrophobic DNA chain (FTD-CEA-Chol) is (Cholesterol - CAA TTT ATCAAT).

[0022] Example 2 A preparation method of a lung cancer marker protein biosensor based on hydrophobic interaction regulation of a SERS probe array, comprising the following steps: (1) Preparation of a double-stranded DNA magnetic bead probe: a 5'-end biotin-modified DNA aptamer (FTD-CEA-aptamer) capable of specifically binding to a target protein marker is connected to the surface of a magnetic bead through biotin-avidin interaction; a trigger DNA (FTD-CEA-armDNA) having a terminal end complementary to the FTD-CEA-aptamer is connected to the surface of the magnetic bead through base interaction, thereby preparing a double-stranded DNA magnetic bead probe; (2) Preparation of a SERS probe: the SERS probe comprises a noble metal nanoparticle, a Raman signal molecule (FTD-CEA-ROX) modified on the surface of the noble metal nanoparticle, and a capture DNA chain (FTD-CEA-cDNA) complementary to the FTD-CEA-armDNA; (3) Incubating a sample buffer to be tested with the double-stranded DNA magnetic bead probe; when the target protein marker exists in the sample, it binds to the FTD-CEA-aptamer, causing the FTD-CEA-armDNA to dissociate from the magnetic bead probe and be released into the solution; the incubation condition is 30-40 ℃, and the incubation time is 30-60 minutes.

[0023] (4) The solution containing the released FTD-CEA-arm DNA is mixed with the SERS probe and the hydrophobic DNA probe (FTD-CEA-Chol) to form a sandwich complex; the FTD-CEA-arm DNA serves as a connecting bridge, and the sequences at both ends thereof are complementary to the FTD-CEA-cDNA on the SERS probe and the FTD-CEA-Chol base portion, respectively; (5) The sandwich complex formed in step (4) is added dropwise to a hydrophobic substrate surface, and the complex is aggregated on the substrate surface by hydrophobic interaction, so that the SERS probes are close to each other to form a Raman signal "hot spot"; (6) The Raman signal intensity of the hydrophobic substrate surface is detected, and the concentration of the target protein marker in the sample to be tested is determined according to the signal intensity.

[0024] Example 3 1. Construction of a protein-responsive trigger DNA release system: A magnetic bead probe is prepared, which uses a magnetic bead as a carrier and fixes a DNA aptamer capable of specifically binding to a target protein marker (such as carcinoembryonic antigen CEA) on the surface thereof by a high-efficiency connection mode such as biotin-avidin. At the same time, a piece of trigger DNA (arm DNA) complementary to the sequence of the DNA aptamer is hybridized with the aptamer to form a stable double-stranded structure, thereby anchoring the trigger DNA on the surface of the magnetic bead.

[0025] 2. Construction of a signal amplification assembly: (1) Preparation of a SERS probe: a Raman reporter molecule (such as ROX) and a piece of capture DNA (cDNA) are covalently modified to the surface of gold nanoparticles (AuNPs) by Au-S bond or the like. The sequence of the capture DNA is complementary to one end of the trigger DNA.

[0026] (2) Preparation of a hydrophobic DNA probe: a piece of DNA sequence is synthesized, one end of which is modified with a hydrophobic group (such as a cholesterol molecule), and the other end of which is complementary to the other end of the trigger DNA.

[0027] 3. Protein detection and signal amplification: (1) Protein recognition and signal conversion: the sample to be tested is mixed with the magnetic bead probe prepared in step 1 and incubated. If the target protein exists in the sample, its affinity to the DNA aptamer is higher than the binding force between the aptamer and the trigger DNA, the target protein will specifically bind to the aptamer, causing the trigger DNA to competitively dissociate from the surface of the magnetic bead and release into the solution. The number of released trigger DNA is positively correlated with the concentration of the target protein in the sample, thereby converting the protein concentration signal into a DNA concentration signal.

[0028] (2) Formation of sandwich complex: The supernatant containing trigger DNA is separated and mixed with the SERS probe and hydrophobic DNA probe prepared in the above step. The released trigger DNA serves as a bridging linker, with its two ends complementary to the capture DNA on the SERS probe and the DNA sequence on the hydrophobic DNA probe, respectively, to self-assemble into a "SERS probe-trigger DNA-hydrophobic DNA probe" sandwich structure complex.

[0029] (3) Hydrophobicity-driven aggregation and signal amplification: The solution containing the sandwich complex is added dropwise to a substrate (such as a hydrophobic glass sheet) that has been pre-treated with hydrophobicity. Under the driving force of hydrophobic interaction, the hydrophobic groups (cholesterol) in the complex spontaneously adsorb to the hydrophobic substrate, thereby driving the entire sandwich complex to undergo efficient and controllable self-assembly aggregation on the substrate surface. This aggregation brings a large number of SERS probes into close proximity at the nanoscale, forming a high-density SERS "hot spot" and achieving exponential amplification of the Raman signal.

[0030] (4) Quantitative detection: The Raman signal intensity on the substrate surface is detected using a Raman spectrometer, and according to the corresponding relationship between the signal intensity and the target protein concentration, accurate quantitative analysis of the target protein marker is achieved.

[0031] Example 4 SERS probe array detection of CEA based on hydrophobicity regulation This Example 4 takes the detection of lung cancer marker carcinoembryonic antigen (CEA) as an example to illustrate the detection method of the present application, and the specific process is shown in Figure 1 and 2 The nucleic acid sequences are shown in Table 1.

[0032] Table 1 Nucleic acid sequences used in the present application 1. Preparation of gold nanoparticles (AuNPs) Add 100 mL of 1 mM / L tetrachloroauric acid 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 and continue heating while observing the color change. When the solution color changes from colorless to blue-black and finally stabilizes to wine red, stop heating and cool naturally to room temperature. Characterize the prepared AuNPs by ultraviolet-visible spectrophotometry ( Figure 3 a) and transmission electron microscopy (TEM) Figure 3 b) to confirm that the characteristic absorption peak is near 520 nm, the particle size is about 15 nm, and the distribution is uniform Figure 3 c).

[0033] 2. Preparation of SERS probe Take 3 µ L FTD-CEA-ROX solution at a concentration of 100 µM and 3 µ L FTD-CEA-cDNA solution at a concentration of 10 µ M was added to 14 µ L TCEP solution at a concentration of 1 mM and incubated at room temperature for 30-45 minutes to reduce the disulfide bonds at the ends of the DNA strands. Subsequently, 80 µ L gold nanoparticle solution prepared in Step 1 was added and shaken to mix. 900 µ L n-butanol was quickly added and shaken vigorously to promote the binding of the DNA strands to the gold nanoparticles. 20 µ L 0.5x TBE buffer (containing 45 mM Tris-HCl, 1 mM EDTA, 45 mM B(OH)3, pH 8.0) was added, and after rapid stirring, centrifuged at 9000 rpm for 10-15 minutes. The supernatant was discarded, and the lower precipitate was collected. The precipitate was washed with 80 µ L PBS buffer (containing 10 mM Na2HPO4, 2 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH ~ 7.4) three times. Finally, the obtained SERS probe precipitate was resuspended with 40 µ L PBS buffer and stored in a 4 °C refrigerator for standby use. The ultraviolet-visible spectrum ( Figure 4 ) shows that the prepared SERS probe (curve d) has an absorption peak at 580 nm contributed by the ROX dye and an absorption peak at 260 nm contributed by the DNA on the basis of the 520 nm absorption peak of the AuNPs, proving that the SERS probe was successfully prepared.

[0034] 3. Preparation of magnetic bead double-stranded DNA probe First, 30 µ L streptavidin magnetic beads were washed twice with Buffer I (10 mM Tris-HCl, 1 mM EDTA, 1 M NaCl, 0.01% Tween-20, pH 7.5). Then, 5 µ L 5'-biotin-modified DNA aptamer (FTD-CEA-aptamer) at a concentration of 100 µ M was diluted in 150 µL Buffer I, mixed with the washed magnetic beads, and incubated at room temperature for 30 minutes to allow the aptamer to be immobilized on the surface of the magnetic beads by biotin-avidin interaction. After incubation, the magnetic beads were separated by a magnetic separator and washed several times with Buffer I and PBS buffer to remove unbound aptamer, obtaining aptamer-modified magnetic beads.

[0035] The aptamer-modified magnetic beads were resuspended in 200 µ L PBS buffer, 5 µ L of trigger DNA (FTD-CEA-arm DNA) with a concentration of 100 µ M was added, and incubated at 45 °C for 3 hours with gentle rotation to allow it to hybridize with the aptamer on the magnetic beads to form a double-stranded structure. After cooling to room temperature, the magnetic beads were separated by a magnetic separator and washed three times with hybridization buffer to remove unbound trigger DNA. Finally, magnetic bead probes that can be used for protein triggering were obtained.

[0036] 4. Preparation of hydrophobic detection substrate A clean glass slide was cleaned by ultrasonic washing with deionized water and anhydrous ethanol, and then dried at 100 °C for 5 minutes. The dried glass slide was placed in an ultraviolet-ozone cleaner for 10-15 minutes. The cleaned glass slide was placed in a solution containing 10 mL of toluene and 95 µ L of perfluorodecyltriethoxysilane, and 0.25 mL of a 1,4-dioxane solution of hydrogen chloride was added as a catalyst. The reaction was carried out at room temperature for 1.5-3 hours. After the reaction was completed, the glass slide was washed with toluene, anhydrous ethanol, 50% aqueous ethanol, and deionized water, and dried at 100 °C, and allowed to cool. The treated glass surface was characterized by water contact angle testing, as shown in Figure 5 The water contact angle of the treated glass surface increased significantly from 39.0° to 119.3°, indicating that the surface was successfully hydrophobized.

[0037] 5. Preparation of hydrophobic DNA probes A DNA sequence (FTD-CEA-Chol) modified with a cholesterol molecule at the 5' end was synthesized, and its sequence was designed to be complementary to the other end of the trigger DNA (FTD-CEA-arm DNA).

[0038] 6. Detection process of CEA protein (1) Protein recognition and trigger DNA release: A series of CEA protein standard buffers with different concentrations (e.g., 0, 1, 2, 6, 10 ng / mL) were prepared. 5 µ L of each concentration of CEA standard was added to 5 µThe prepared magnetic bead probe mixture was incubated gently at room temperature for 2 hours. During this period, the CEA protein binds to the aptamer on the magnetic beads, competitively displaces and releases the trigger DNA.

[0039] (2) Sandwich complex formation: After the incubation, the magnetic beads were separated using a magnetic separator for 3 minutes, and the supernatant was carefully aspirated. The supernatant was taken and added to a buffer containing 5 μL of SERS probe and 5 μL of hydrophobic DNA probe (FTD-CEA-Chol) with a concentration of 1 μM. Incubate at room temperature for 30 minutes to allow the released trigger DNA to act as a bridge to connect the SERS probe and the hydrophobic DNA probe, forming a sandwich complex.

[0040] (3) Signal detection and analysis: The complex solution formed in step (b) was dropped onto a pre-prepared hydrophobic glass slide and naturally dried at room temperature. During the drying process, the complex spontaneously aggregated on the surface of the glass slide driven by hydrophilic-hydrophobic interaction. Laser confocal Raman spectroscopy (laser wavelength 633 nm) was used to collect spectra from the aggregated areas on the glass slide.

[0041] Feasibility verification, sensitivity and quantitative analysis: As shown in Fig. Figure 6 (a), as the concentration of CEA protein increases, the Raman characteristic peak intensity at 1502 cm⁻¹ significantly increases. When there is no CEA (Blank group), the signal is very low.

[0042] As shown in Fig. Figure 6 (b), the Raman signal intensity at different CEA concentrations was quantitatively analyzed. The results show that in the concentration range of 1 ng / mL to 10 ng / mL, the Raman signal intensity (ΔI) and the CEA concentration (C CEA ) show a good linear relationship, the linear regression equation is ΔI = 9207.24 + 1408.73 C CEA , and the correlation coefficient R² is as high as 0.9991.

[0043] The results of Example 4 fully demonstrate that the detection method proposed in the present application can effectively convert the concentration signal of the target protein into a SERS signal that can be detected with high sensitivity, and through the aggregation of probes driven by hydrophilic-hydrophobic interaction, signal amplification is achieved, ultimately achieving excellent detection sensitivity and good quantitative analysis performance.

[0044] Example 5 The application of a lung cancer marker protein biosensor based on hydrophilic-hydrophobic interaction regulated SERS probe array in the preparation of products for detecting lung cancer marker proteins, detecting the Raman signal intensity on the hydrophobic substrate surface, and determining the concentration of the target protein marker in the sample to be tested according to the signal intensity.

[0045] The magnetic beads with DNA aptamer fixed in the present application, when specifically combined with the target protein, will competitively release a trigger DNA. The trigger DNA serves as a bridge to connect a SERS probe with a Raman reporter molecule and a DNA probe modified with a cholesterol hydrophobic group to form a sandwich complex. The complex is added dropwise to a hydrophobically treated substrate, and under the driving force of hydrophilic-hydrophobic interaction, the complex self-assembles and aggregates, causing the SERS probes to approach each other to form 'hot spots', thereby achieving exponential amplification of the Raman signal. The present application ingeniously converts the concentration of the target protein into a Raman signal that can be detected with high sensitivity, achieving accurate quantitative analysis of the target protein and having important application value in the field of clinical detection such as early diagnosis of cancer.

[0046] The above only describes the preferred embodiments of the present application, and it should be pointed out that for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. A lung cancer marker protein biosensor based on hydrophilic-hydrophobic interaction regulated SERS probe array, characterized in that, The double-stranded DNA magnetic bead probe, the SERS probe, the hydrophobic DNA probe, and the hydrophobic substrate; The double-stranded DNA magnetic bead probe comprises a 5'-end biotin-modified DNA aptamer FTD-CEA-aptamer capable of specifically binding to a target protein marker, and a trigger DNA FTD-CEA-armDNA having an end complementary to the FTD-CEA-aptamer and connected to the surface of the magnetic bead through base interaction by biotin-avidin interaction; The SERS probe comprises noble metal nanoparticles, a Raman signal molecule FTD-CEA-ROX modified on the surface of the noble metal nanoparticles, and a capture DNA chain FTD-CEA-cDNA complementary to the FTD-CEA-armDNA; The hydrophobic group of the hydrophobic DNA probe is a cholesterol molecule. 2.The lung cancer marker protein biosensor based on hydrophilic-hydrophobic interaction regulated SERS probe array according to claim 1, wherein, The hydrophobic substrate is a glass or silicon wafer surface treated with perfluoroalkylsilane or long-chain alkylsilane. 3.The lung cancer marker protein biosensor based on hydrophilic-hydrophobic interaction regulated SERS probe array according to claim 1, wherein, The noble metal nanoparticles are gold nanoparticles AuNPs, and the Raman signal molecule is ROX dye. 4.The lung cancer marker protein biosensor based on hydrophilic-hydrophobic interaction regulated SERS probe array according to claim 1, wherein, The target protein marker is carcinoembryonic antigen CEA. 5.The lung cancer marker protein biosensor based on hydrophilic-hydrophobic interaction regulated SERS probe array according to claim 1, wherein, The sequence of the aptamer FTD-CEA-aptamer is Biotin-AAA AAA AAA AAT ACC AGC TTA TTC AAT TTA TCA AT, the sequence of the trigger DNA chain FTD-CEA-armDNA is GGA TTA TTG TTA AAT ATT GAT AAA TTG, the sequence of the capture DNA on the SERS probe FTD-CEA-cDNA is ATT TAA CAA TAA TCC AAAAAAAAAAA-SH, and the sequence of the Raman label molecule on the SERS probe FTD-CEA-ROX is (ROX-TTT TTCCT AGC GAC-SH).

6. The method for preparing lung cancer marker protein biosensor based on hydrophilic-hydrophobic interaction regulated SERS probe array according to any one of claims 1-5, characterized in that, The method comprises the following steps: (1) Preparation of the double-stranded DNA magnetic bead probe: a 5'-end biotin-modified DNA aptamer FTD-CEA-aptamer capable of specifically binding to a target protein marker is connected to the surface of a magnetic bead through biotin-avidin interaction, and a trigger DNA FTD-CEA-armDNA having an end complementary to the FTD-CEA-aptamer is connected to the surface of the magnetic bead through base interaction, thereby obtaining the double-stranded DNA magnetic bead probe; (2) Preparation of the SERS probe: the SERS probe comprises noble metal nanoparticles, a Raman signal molecule FTD-CEA-ROX modified on the surface of the noble metal nanoparticles, and a capture DNA chain FTD-CEA-cDNA complementary to the FTD-CEA-armDNA; (3) Incubate the sample buffer with the double-stranded DNA magnetic bead probe. When the target protein marker is present in the sample, it binds to the FTD-CEA-aptamer, causing the FTD-CEA-arm DNA to dissociate from the magnetic bead probe and release into the solution; (4) Mix the solution containing the released FTD-CEA-arm DNA with the SERS probe and the hydrophobic DNA probe, i.e. FTD-CEA-Chol, to form a sandwich complex; the FTD-CEA-arm DNA acts as a connecting bridge, with its sequence at both ends complementary to the FTD-CEA-cDNA on the SERS probe and the FTD-CEA-Chol base portion, respectively; (5) Drop the sandwich complex formed in step (4) onto the hydrophobic substrate surface, and use hydrophobic and hydrophilic interactions to make the complex aggregate on the substrate surface, thereby bringing the SERS probes close to each other to form Raman signal "hot spots". 7.The method for preparing a lung cancer marker protein biosensor based on hydrophilic-hydrophobic interaction regulated SERS probe array according to claim 6, wherein, The incubation conditions in step (3) are 30-40 ℃, and the incubation time is 30-60 minutes.

8. Use of the lung cancer marker protein biosensor based on hydrophobic and hydrophilic interaction regulation of SERS probe array according to any one of claims 1-5 in the preparation of a product for detecting lung cancer marker proteins.

9. Use according to claim 8, characterized in that: Detect the Raman signal intensity on the surface of the hydrophobic substrate, and determine the concentration of the target protein marker in the sample to be tested according to the signal intensity.

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