A method for detecting p53 gene based on SERS tandem strategy of click chemistry reaction

Abstract

This invention discloses a method for detecting the p53 gene using a SERS tandem strategy based on click chemistry, belonging to the field of biodetection technology. This method cleverly introduces a target DNA-triggered HCR polymer to enrich CuONPs for catalyzing the click chemistry reaction, converting the p53 gene count into the CuONP count. After acidification and reduction, the released Cu(I) is used to catalyze the click reaction, leading to the consumption of Raman signal molecules. Unreacted Raman signal molecules in the supernatant are detected by magnetic separation, and the Raman signal of alkynes is then read out. The p53 gene concentration is obtained from the alkyne Raman signal. This method features high sensitivity, low detection limit (0.0174 pM), universality, simple detection process, and accurate quantitative detection. This method demonstrates good performance in detecting the p53 gene in serum samples within a low concentration range and has promising clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biodetection technology and relates to a method for detecting the p53 gene using a SERS tandem strategy based on click chemistry. Specifically, it relates to a method for detecting the p53 gene using SERS based on a click chemistry catalyzed by CuO nanoparticles enriched in long-chain DNA polymers. Background Technology

[0002] Cancer accounts for one-eighth of all deaths worldwide, and most cancers are discovered at an advanced stage, making early diagnosis and prognosis crucial. The p53 gene is a cancer suppressor gene, often referred to as the "guardian of the genome." Mutations in the p53 gene mostly occur in the early stages of cancer, affecting the activity of tumor suppressor factors and contributing to more than half of all human cancers. Therefore, sensitive detection of the p53 gene in biological samples is of great significance for early cancer prevention. To date, analytical methods based on electrochemistry, colorimetry, chemiluminescence, and fluorescence have been used to detect the p53 gene. However, these methods have inherent limitations, such as damaging the sample, susceptibility to photobleaching and photoquenching, and complex equipment operation. Therefore, establishing a rapid, simple, non-toxic, and ultra-sensitive method for detecting the p53 gene is of paramount importance.

[0003] For decades, surface-enhanced Raman scattering (SERS) has received widespread attention as a biotechnology and bioanalytical method with excellent multi-pathway reuse capabilities, high selectivity, and non-destructive nature. Compared with other analytical detection methods, SERS has the following unique advantages: (1) Raman is resistant to photoquenching and photobleaching, ensuring the stability of repeated experiments. (2) The Raman peak of water is extremely weak, making it possible to quickly and conveniently detect solvent water samples. (3) Raman spectra have rich fingerprint information and narrow spectral width, with high resolution, making SERS possible for multiplex analysis. SERS can amplify the original signal by several orders of magnitude through electromagnetic and chemical enhancement, and then amplify the signal again through clever sensor design. Therefore, it is particularly important to cleverly design an analytical detection strategy that combines SERS for trace detection of biological substances.

[0004] In recent years, click chemistry has been widely applied in biosensor systems as a typical reaction. A click chemistry biosensor based on Cu(I)-mediated 1,3-dipolar cycloaddition of azides and alkynes has become a research hotspot in biochemical detection and bioimaging. This is because the alkyne group involved in the reaction has unique chemical and spectroscopic properties, giving it a unique advantage in Raman detection. Chemically, the alkyne group is a small molecule (C≡C has only two atoms), exogenous (C≡C does not exist in organisms), and non-orthogonal (it does not react with other substances in organisms). Spectroscopically, due to the unique vibrational mode of the alkyne group, the Raman signal appears in the Raman silent region (1800–2800 cm⁻¹). -1 This differs from traditional Raman signal molecules. Furthermore, in the Raman quiescent region, alkynes exhibit stronger Raman intensities than other Raman signal molecules in the quiescent region. Therefore, alkyne-modified gold nanoparticles can be used as signal molecules to obtain highly sensitive and selective SERS signals in complex biological matrices.

[0005] Currently, with the development of DNA nanotechnology, combining analytical techniques with nucleic acid amplification strategies has enabled a series of powerful signal amplification and detection methods. Among these strategies, hybridization chain reaction (HCR) amplification exhibits mild reaction conditions, requires no enzymes, and demonstrates high selectivity and sensitivity to targets, showing great potential for signal amplification. Furthermore, HCR has synthesized a nicked polymer, making it possible to construct nanoparticle binding sites on the polymer. Moreover, HCR has proven highly efficient and effective for enzyme-free detection of DNA and miRNA. Summary of the Invention

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide a method for detecting the p53 gene based on a SERS tandem strategy of click chemistry reaction.

[0007] This invention develops a novel click chemistry-based SERS tandem strategy for detecting the p53 gene, exhibiting high selectivity and sensitivity. This strategy cleverly introduces a target DNA-triggered HCR polymer to enrich CuO nanoparticles for catalyzing the click chemistry reaction. The target DNA-triggered HCR captures copper oxide nanoparticles (CuONPs), forming a CuONP-modified DNA polymer, thus converting the number of the target DNA p53 gene into the number of copper oxide nanoparticles. After acid treatment and reduction, the released Cu(I) from the CuONPs is used to catalyze the click reaction, linking Raman signal molecules and azide-modified magnetic nanoparticles. Finally, after magnetic separation, the number of Raman signal molecules in the solution decreases, leading to a reduction in the alkyne SERS signal. Quantification of these steps allows the concentration of the p53 gene to be determined from the alkyne Raman signal.

[0008] The method of the present invention has the characteristics of high sensitivity, universality, simple detection process, and accurate quantitative detection.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] In this invention, we constructed a novel SERS tandem strategy to detect the p53 gene. For example... Figure 1 As shown, a p53 gene-triggered hybridization chain reaction was first constructed to synthesize a long-chain DNA polymer, and then CuONPs were enriched on the DNA polymer. This established a link between the p53 gene signal and the quantity of CuONPs under a non-enzymatic isothermal environment. Then, a "click" chemical reaction catalyzed between azide compounds and alkyne molecules by Cu(I) obtained through acidification and reduction led to the consumption of Raman signal molecules. Finally, unreacted Raman signal molecules in the supernatant were detected using magnetic separation technology, and the Raman signal of the alkyne was subsequently read out.

[0011] A method for detecting the p53 gene using a SERS tandem strategy based on click chemistry includes the following steps:

[0012] (1) Preparation of Raman signal probe

[0013] Using gold nanoparticles as a substrate, a Raman signal probe was synthesized by combining alkyne-containing Raman signal molecules through covalent bonds (Au-N) and electrostatic interactions.

[0014] (2) Preparation of azide-modified magnetic beads

[0015] After activating the carboxyl groups of carboxyl magnetic beads with an activator, azide compounds are modified onto them through amide bonds to obtain azide-modified magnetic beads.

[0016] (3) Synthesis of CuONP-enriched long-chain DNA polymers:

[0017] (A) After activating the carboxyl groups on Fe3O4@SiO2 with an activator, it was incubated with PO at room temperature to obtain PO / Fe3O4@SiO2;

[0018] (B) The p53 gene was incubated with P0 / Fe3O4@SiO2 at room temperature to obtain p53 / P0 / Fe3O4@SiO2;

[0019] (C) After activating the thiol-modified H1 and H2, they were incubated with p53 / P0 / Fe3O4@SiO2 at room temperature to obtain long-chain DNA polymers by completing the hybridization chain reaction;

[0020] (D) CuONPs were mixed with long-chain DNA polymers, incubated at room temperature, and then aged with salt; then magnetic separation was performed to obtain long-chain DNA polymers enriched with CuONPs.

[0021] The specific principle is as follows: (a) A long-chain DNA polymer is formed on Fe3O4@SiO2 microbeads by inducing a hybridization chain reaction using the p53 gene. Simply put, in the presence of a activator, the carboxyl groups on the Fe3O4@SiO2 microbeads are activated into active esters. The active esters on the Fe3O4@SiO2 surface further conjugate with the amino terminus (-NH2) of the DNA chain labeled P0. This modifies the Fe3O4@SiO2 microbeads with the base chain P0. Part of the p53 gene can pair complementaryly with one end of the P0 base, while the other part can open the HCR starting material chains H1 and H2. (b) CuONPs are modified at the gaps in the long-chain DNA polymer, and polymer-conjugated CuONPs are formed between H1 and H2. Then, magnetic separation technology is used to separate the CuONP-enriched long-chain DNA polymer from the supernatant. After acidification and reduction, a certain concentration of Cu(I) is obtained, which is used to catalyze subsequent click chemistry reactions, thereby consuming Raman signal molecules.

[0022] (4) Alkyne signal readout and SERS signal detection

[0023] (a) The long-chain DNA polymer enriched with CuONPs induced by different concentrations of p53 gene was acidified and reduced, and then mixed with the Raman signal probe in step (1) and the azide-modified magnetic beads in step (2) to perform a click chemical reaction to consume the Raman signal molecules. After magnetic separation, the unreacted Raman signal molecules in the supernatant were tested for Raman signal intensity.

[0024] (b) A standard curve was established based on the relationship between the concentration of the p53 gene and the reduction (ΔI) of the Raman signal intensity in the supernatant after magnetic separation relative to the blank control (Control), so as to use the reduction of Raman signal intensity to quantitatively detect the p53 gene.

[0025] The specific principle is as follows: the more p53 gene added, the more Cu(I) is obtained, the more Raman signal molecules participate in the click chemical reaction, the fewer Raman signal molecules remain in the supernatant, the weaker the measured Raman signal intensity, and the greater the reduction (ΔI) in Raman signal intensity relative to the blank control. The triple bond peak (It) in the Raman silent region of alkyne-containing Raman signal molecules (such as PEAN) is used. 1990The concentration of p53 gene and the concentration of Raman signal molecules in the supernatant after magnetic separation were used as characteristic peaks. A standard curve was established based on the correspondence between the concentration of p53 gene and the concentration of Raman signal molecules in the supernatant after magnetic separation, so as to use the Raman signal in the supernatant to quantitatively detect p53 gene.

[0026] In the above method:

[0027] The alkyne-containing Raman signal molecule mentioned in step (1) is PEAN (4-ethynylaniline). The concentration of PEAN used in the experiment is 75-125 μM and the volume is 10-20 μL / 1 mL AuNPs; the preferred concentration and volume are 100 μM and 16 μL / 1 mL AuNPs.

[0028] The average particle size of the gold nanoparticles in step (1) is 40-50 nm, preferably 45 nm; the concentration of the gold nanoparticles used is 0.75-1.25 mmol / L, preferably 1 mmol / L.

[0029] The carboxyl magnetic beads mentioned in step (2) are magnetic nanoparticles with carboxyl groups modified on their surface, specifically Fe3O4 with carboxyl groups (-COOH) modified on its surface. The average particle size of the carboxyl magnetic beads is 200 nm to 300 nm.

[0030] The azide compound mentioned in step (2) is 11-azido-3,6,9-trioxaundecan-1-amine;

[0031] The activators mentioned in step (2) are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS); first, the activator EDC is added and the mixing reaction time is 10 min, and then NHS is added to the above reaction system and the mixing reaction time is 30 min.

[0032] In step (3), to modify the gaps in the long-chain DNA polymer with CuONP particles and induce the formation of conjugated CuONPs between H1 and H2, we processed the two standard DNAs into hairpin structures and labeled them with thiol functional groups at the 3′- or 5′- ends. Secondly, the thiol-labeled H1 and H2 (-SH) needed to be activated before use. The 100 μM thiol-modified H1 and H2 were activated with freshly prepared 10 mM TCEP.

[0033] The activators mentioned in step (3)(A) are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS);

[0034] The sequence of P0 in step (3)(A) is: 5′-ACCTGGACTGAACGAGTCTTCCA-3′;

[0035] The sequence of the p53 gene mentioned in step (3)(B) is: 5′-TCATCACACTGGAAGACTC-3′;

[0036] The sequence of H1 mentioned in step (3)(C) is as follows:

[0037] 5′-ATGACGCCTGACTGGCTAGCCGGCAGCTAGCCAGTCACGGCGTCATAGTCCATTTTTT-SH-3′;

[0038] The sequence of H2 mentioned in step (3)(C) is as follows:

[0039] 5′-SH-TTTTTTTGCCGGCTAGCCAGTCACGGCGTCATTGGACTATGACGCCGTGACTGGCTAGC-3′;

[0040] The Fe3O4@SiO2 mentioned in step (3)(A) is Fe3O4 micro magnetic beads coated with silica with carboxyl groups modified on the surface, and its average particle size is about 4 to 5 μm;

[0041] The average particle size of CuONPs described in step (3)(D) is 45-55 nm, preferably 50 nm;

[0042] In step (3)(C), the activation treatment is performed using TCEP; the activation time is 5 to 7 hours; preferably 6 hours.

[0043] In steps (3) (A), (B), (C), and (D), the room temperature is 25–35°C.

[0044] In step (3)(A), the incubation time is 0.5 to 1.5 hours; preferably 1 hour.

[0045] In step (3)(B), the incubation time is 0.5 to 1.5 hours; preferably 1 hour.

[0046] In step (3)(C), the incubation time is 2 to 4 hours, preferably 3 hours;

[0047] In step (3)(D), the incubation time is 2 to 4 hours, preferably 3 hours;

[0048] In step (3)(D), the salt is NaCl.

[0049] The magnetic separation described in steps (3)(D) and (4)(a) refers to the separation of magnetic compounds from supernatant by using the physical action of a magnetic field. Compared with the traditional step of cleaning the solid substrate to remove residues, this method is more efficient.

[0050] The acidification mentioned in step (4)(a) refers to dissolving CuONPs into Cu(II) with hydrochloric acid; the reduction refers to reducing Cu(II) into Cu(I) with sodium ascorbate, which is used to catalyze the subsequent click chemical reaction between azide and alkyne; wherein the concentration of sodium ascorbate used is 10 mM;

[0051] The Raman signal mentioned in step (4)(a) is obtained by a Raman spectrometer: the Raman spectrometer is a 785nm excited BW&TEK Raman spectrometer, and the obtained Raman spectrum range is 400-3000 cm⁻¹. -1 The laser power is approximately 400mW, and the signal collection time is 1–5s (preferably 1s).

[0052] In step (4)(a), the concentrations of p53 gene at different concentrations range from 0 mol to 10 pM, namely 0 mol, 0.01 pM, 0.05 pM, 0.1 pM, 0.5 pM, 1.0 pM, 5.0 pM, and 10 pM; where 0 mol is the blank control.

[0053] The Raman silence zone mentioned in step (4) refers to 1800–2800 cm. -1 Raman shift; triple bond peak (I 1990 (This refers to the PEAN molecule at 1990 cm⁻¹) -1 The peak intensity at that location. Among them is the characteristic peak (520 cm⁻¹) on a silicon wafer substrate. -1 The strength of the (location) is calibrated;

[0054] In step (4)(a), the Raman signal intensity is preferably I. 1990 / I 520 The Raman signal intensity, where I 1990 It is the PEAN molecule at 1990 cm -1 Peak intensity at I 520 The characteristic peak (520 cm⁻¹) of the silicon substrate used for calibration. -1 The intensity of (location).

[0055] In step (4)(b), a standard curve is established based on the reduction in Raman signal intensity relative to the blank control, with the equation as follows:

[0056] Y = 8.271LgX + 26.873, R 2=0.993, where Y represents the change in relative Raman signal intensity compared to the blank control (Control), and X represents the concentration of the p53 gene (pM) (0 < X ​​≤ 10 pM).

[0057] The limit of detection (LOD) and limit of quantitation (LOQ) of this strategy are 0.0174 pM and 0.0583 pM, respectively. The accuracy of the strategy, expressed as RSD, ranges from 3.14% to 6.21%.

[0058] The present invention has the following advantages and effects compared with the prior art:

[0059] (1) This invention utilizes a multi-stage amplification strategy of HCR. This enzyme-free amplification technology at room temperature is more adaptable to environmental conditions and more selective.

[0060] (2) Using AuNPs as the Raman enhancement material in this experiment has several advantages. As is well known, gold nanoparticles are easy to synthesize and have good stability. This makes the experiment simpler and easier to operate.

[0061] (3) This invention utilizes alkyne compounds as Raman signal molecules. The Raman signal of alkyne compounds is located in the Raman quiescent region (1800–2800 cm⁻¹). -1 It exhibits superior characteristics, unlike other Raman signaling molecules, which avoids interference from the biological background, demonstrating the great potential of this method in clinical trials;

[0062] (4) The click chemical reaction of 1,3-dipolar cycloaddition of azides and alkynes mediated by Cu(I) is introduced in this invention. Unlike other biological enzyme-linked reactions, the chemical reaction is more adaptable to the environment, faster, and more specific.

[0063] (5) This invention has universality. The strategy of altering the original hairpin strand of HCR based on the target DNA sequence can be applied to the detection of other low-concentration DNA and miRNA;

[0064] (6) This invention has good reproducibility. This invention obtains the corresponding results by detecting the Raman spectral signal of the solution. Compared with the traditional solid-phase Raman enhanced substrate method, the Raman spectral signal monitoring data in the solution is more stable, reliable and reproducible.

[0065] (7) The present invention is simple to operate, has good detection stability, high detection sensitivity, and low detection limit (0.0174pM).

[0066] (8) The method of this invention can obtain highly sensitive and selective SERS signals in complex biological matrices. This method utilizes a multi-step amplification strategy, including nanoparticle-modified HCR polymers and a "click" reaction, reducing the detection limit to 0.0174 pM. The results show that the method of this invention has good performance in detecting the p53 gene in serum samples within a low concentration range, and has promising clinical application prospects. Attached Figure Description

[0067] Figure 1 This is a schematic diagram of the method for detecting p53 gene SERS using a click chemical reaction catalyzed by CuONPs enriched in long-chain DNA polymers according to the present invention; wherein, A: schematic diagram of constructing long-chain DNA polymers enriched with CuONPs; B: schematic diagram of Raman signal detection.

[0068] Figure 2 These are characterization images of gold nanoparticles; where A: TEM image of 45nm AuNPs; B: UV-Vis spectrum of 45nm AuNPs.

[0069] Figure 3 This is a flowchart of the preparation process of PEAN-functionalized AuNPs (A) and azide-modified magnetic beads (B).

[0070] Figure 4 The reaction of the p53 gene with H1 and H2 was shown using 4S gelred staining gel electrophoresis: Lanes 2-6: p53 gene (500nM, 200nM, 100nM, 50nM, 20nM) mixed with H1 and H2; Lane 7: H1 and H2 mixed; Lane 1: 25bp marker.

[0071] Figure 5 The images show scanning electron microscope (SEM) images of CuONPs successfully enriched on the Fe3O4@SiO2 surface when the p53 gene is present; the inset in the upper right corner is a SEM image of the Fe3O4@SiO2 surface without p53-triggered HCR.

[0072] Figure 6 Na + K + Ca 2+ Zn 2+ Using metal ions as catalysts, explore Cu 2+ The selectivity of catalytic click chemical reactions; where the ordinate ΔI represents the reduction in Raman signal intensity relative to the blank control (Control). From the image, we can see that only Cu... 2+ Only then can a click chemical reaction be triggered, which in turn causes a change in the Raman signal.

[0073] Figure 7 Studying Cu through "click" chemical reactions 2+ Relationship between concentration and SERS signal; where A: different Cu concentrations 2+ SERS spectra at different concentrations, from top to bottom Cu 2+ The concentrations were 0, 0.01, 0.1, 0.5, 1.0, 2.5, 5.0, and 10 mM, respectively, with 0 mol serving as the blank control. The Raman signal intensity increased with increasing Cu content. 2+ The concentration increases and decreases; B: Different Cu 2+ Concentration and relative to blank control (Control, Cu) 2+ A standard detection curve showing the reduction in Raman signal intensity (ΔI) at a concentration of 0%. The concentrations of the Raman signal molecule and the azide magnetic beads were 10... -4 mol / L. Incubate the reaction for 1 h.

[0074] Figure 8 The images show SERS patterns (A) of different concentrations (0–10 pM) of p53 gene in standard solutions, and standard detection curves (B) showing the reduction (ΔI) of Raman signal intensity of different concentrations of p53 gene relative to the blank control (Control, p53 gene concentration is 0). In A, the concentrations of p53 gene from top to bottom are 0 mol, 0.01 pM, 0.05 pM, 0.1 pM, 0.5 pM, 1.0 pM, 5.0 pM, and 10 pM, where 0 mol is the blank control (Control).

[0075] Figure 9 This is a specificity experiment; where a: blank; b: uncoordinated DNA strand of p53 gene; c: single mismatched DNA strand of p53 gene; d: target DNA of p53 gene; e: target DNA mixed with other DNA; the vertical axis ΔI represents the reduction in Raman signal intensity relative to the blank control (Control) (ΔI).

[0076] Figure 10 This is a repeatability assessment of the strategy; where the vertical axis ΔI represents the reduction in Raman signal intensity relative to the control (ΔI). The strategy accuracy is represented by the RSD of this graph, which ranges from 3.14% to 6.21%. Detailed Implementation

[0077] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0078] In the following examples, experimental methods without specific conditions and environments are generally performed under conventional conditions or conditions recommended by the manufacturer. In this invention, PEAN represents the Raman signal molecule 4-ethynylaniline; Fe3O4@SiO2 represents Fe3O4 micromagnetic beads coated with carboxyl-modified silica, with an average particle size of approximately 4–5 μm (Fe3O4@SiO2 was purchased from Maclean's Ltd., Shanghai, China); TCEP represents tris(2-carboxyethyl)phosphine; PBS represents phosphate buffer; EDC represents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; and NHS represents N-hydroxysuccinimide.

[0079] A schematic diagram of a method for detecting p53 gene SERS based on CuONPs-catalyzed click chemical reactions enriched in long-chain DNA polymers, as shown below. Figure 1 As shown.

[0080] Example 1

[0081] (1) Preparation of gold nanoparticles

[0082] In short, 50 mL of water and 25 mM 600 μL of HAuCl4 aqueous solution were added to a round-bottom flask. The solution was heated to boiling, and then 500 μL of sodium citrate solution was rapidly added with rapid stirring to bring the final concentration of sodium citrate in the reaction system to 1 wt%. The solution turned black after approximately 25 seconds and changed from black to wine-red after approximately 70 seconds. Boiling was continued for 15 minutes to complete the reduction reaction. Then, the solution was cooled to room temperature with continuous stirring. The results showed that the prepared solution was red (wine-red), and the obtained solution was a solution of gold nanoparticles (AuNPs) with a particle size of 45 nm. Figure 2 A is a transmission electron microscope (TEM) image of gold nanoparticles prepared under optimized conditions. Figure 2 B is the UV-Vis absorption spectrum of AuNPs at 45 nm.

[0083] (2) Preparation of PEAN-functionalized AuNPs

[0084] 4-ethynylaniline-functionalized AuNPs were synthesized via covalent (Au-N) functionalization and electrostatic interactions. Under the combined action of these two forces, alkynes were adsorbed onto the AuNP surface. This ensured that the 4-ethynylaniline-modified AuNPs exhibited a relatively good and stable Raman signal. 160 μL of a 100 μM aqueous solution of 4-ethynylaniline was added to 10 mL of a 1 mmol / L AuNPs solution at 45 nm prepared in step (1). The mixture was then gently stirred at room temperature for 20 minutes. Finally, the resulting solution was centrifuged at 8000 rpm for 10 minutes, resuspended, and the unmodified 4-ethynylaniline solution on the gold nanoparticles was removed. A flowchart of the preparation process for PEAN-functionalized AuNPs is shown below. Figure 3 As shown in Figure A.

[0085] (3) Preparation of azide-modified magnetic beads

[0086] 11-Azide-3,6,9-trioxaundecan-1-amine is a long-chain azide compound with an amino group at one end. We coupled the azide compound to magnetic beads using stable covalent bonds. The covalent bond between the modified carboxyl group (-COOH) on the magnetic beads and the (-NH2) group on the azide compound is achieved through EDC and NHS chemical covalent coupling. First, 100 μL of 5 mg mL... -1 Carboxyl-functionalized magnetic beads (i.e., Fe3O4 with carboxyl groups on the surface, purchased from Aladdin Reagent (Shanghai) Co., Ltd.) were washed three times with 1 mL PBS buffer (pH = 7.4, 0.01 M) to remove surface impurities. Then, 5 mg mL of PBS was added sequentially to the above solution. -1 30 μL each of EDC and NHS were added and gently stirred sequentially for 10 min and 30 min at room temperature to obtain activated MPs solutions. Then, 100 μL of 1 mM 11-azido-3,6,9-trioxaundecan-1-amine was added to the activated MPs solution. The mixture was incubated at 25 °C for 1.5 h. 20 μL of BSA (final concentration 2.5%) was added to the solution to ensure that no other exposed activation sites were present on the magnetic bead surface. Finally, the azide-modified magnetic beads were purified by magnetic separation and resuspended in 1 mL of PBS buffer (pH = 7.4, 0.01 M) to remove free reagents. The preparation flow chart of azide-modified magnetic beads is shown below. Figure 3 As shown in B.

[0087] (4) Construction of long-chain DNA polymers enriched with CuONPs triggered by p53 gene

[0088] Before constructing the polymer, Fe3O4@SiO2 should be washed at least three times with PBS buffer to remove surface impurities. Figure 1As shown, firstly, the carboxyl groups on the surface of Fe3O4@SiO2 were activated by EDC and NHS, and then incubated with P0 (P0-NH2) at room temperature for 1 h. Free P0 in the solution was removed by magnetic separation and resuspending in PBS to obtain P0 / Fe3O4@SiO2. Then, the p53 gene was incubated with the P0 / Fe3O4@SiO2 mixture for another 1 h to remove free p53, resulting in p53 / P0 / Fe3O4@SiO2. Secondly, the thiol-modified H1 and H2 (-SH) needed to be activated before use. 100 μM thiol-modified H1 and H2 were activated by freshly prepared 10 mM TCEP for 6 h. Afterwards, 1.0 μM activated H1 and H2 were added to the p53 / P0 / Fe3O4@SiO2 mixture and incubated for 3 h. In this way, a long-chain DNA polymer containing many gaps was synthesized; the electrophoresis results of the long-chain DNA polymer using 4S gelred staining gel electrophoresis are as follows: Figure 4 As shown; from Figure 4 We synthesized a long-chain DNA polymer in the presence of the p53 gene (different concentrations of p53 gene: 500 nM, 200 nM, 100 nM, 50 nM, and 20 nM). We then introduced CuONPs. 1 mg of CuONPs was added to 1 mL of PBS solution, and the CuONPs were dispersed in the PBS solution by sonication for 10 min. Afterward, the CuONPs PBS solution was added to the long-chain DNA polymer and incubated at room temperature for 3 h. Then, 1.0 M NaCl solution was gradually added until the desired NaCl concentration was obtained (final concentration 0.1 M). Magnetic separation and resuspending were performed using PBS buffer (pH 7.4, 0.01 M) to remove free and excess substances and the supernatant. Finally, a long-chain DNA polymer of CuONPs was constructed on the Fe3O4@SiO2 surface. Figure 5 This is a scanning electron microscope image of a long-chain DNA polymer enriched with CuONPs constructed under optimized conditions. From... Figure 5 As can be seen, we successfully enriched CuONPs in Fe3O4@SiO2.

[0089] The sequence of P0 is: 5′-ACCTGGACTGAACGAGTCTTCCA-3′;

[0090] The sequence of the p53 gene is: 5′-TCATCACACTGGAAGACTC-3′;

[0091] The sequence of H1 is: 5′-ATGACGCCGTGACTGGCTAGCCGGCAGCTAGCCAGTCACGGCGTCATAGTCCATTTTTT-SH-3′;

[0092] The sequence of H2 is: 5′-SH-TTTTTTTGCCGGCTAGCCAGTCACGGCGTCATTGGACTATGACGCCGTGACTGGCTAGC-3′;

[0093] (5) CuONPs enriched in long-chain DNA polymers release Cu 2+

[0094] A suitable amount of hydrochloric acid solution was added to long-chain DNA polymers enriched with CuONPs induced by the p53 gene at different concentrations (0.01 pM, 0.05 pM, 0.1 pM, 0.5 pM, 1.0 pM, 5.0 pM, 10 pM, where 0 mol was the blank control). After the reaction system reacted for 10 min, the supernatant was collected. 10 μL of the collected supernatant and 10 μL of 10 mM sodium ascorbate were added to a solution containing PEAN-functionalized AuNPs from step (2) and azide-modified magnetic beads from step (3). The reaction was carried out at room temperature for 1 h. After magnetic separation, the supernatant was used to detect the unreacted Raman signal molecules.

[0095] Although we already know that Cu(I) is a highly selective catalyst for the cycloaddition reactions of azides and alkynes, we also conducted specificity and selectivity experiments to evaluate the interference of other metal ions in human serum on this strategy. Furthermore, we explored the selectivity of the Cu(I)-catalyzed reaction, and the results are as follows: Figure 6 As shown, we can see that only in Cu 2+ The change in Raman signal only occurred in the presence of a reducing agent, demonstrating the specificity of Cu(I) for this reaction.

[0096] We studied Cu through "click" chemical reactions. 2+ The relationship between concentration and SERS signal, using different Cu concentrations. 2+ Concentrations (0, 0.01, 0.1, 0.5, 1.0, 2.5, 5.0, 10 mM, with 0 mol serving as a blank control) were used to explore the Raman signal of PEAN and Cu. 2+ The correlation of concentrations, the results are as follows: Figure 7 As shown, the Raman signal intensity varies with Cu 2+ The concentration decreases with increasing Cu concentration; and the decrease is related to the decrease in Cu concentration. 2 + The increase in concentration, relative to the blank control (Control, Cu) 2+ The decrease in Raman signal intensity (ΔI) at a concentration of 0 gradually increases.

[0097] (6) Measurement of Raman signals and readout of alkynyl Raman signals

[0098] The Raman signals of alkyne signal molecules collected in the supernatant from the above experiments were measured. We used a Raman spectrometer with an excitation source of 785 nm to acquire signals from the Raman signal probe in the supernatant. The laser power reaching the sample was 400 mW, and the signal acquisition time was 1 s. After signal acquisition, baseline processing was performed on the data using Origin software to obtain clear and intuitive SERS spectra (e.g., ...). Figure 8 ).

[0099] from Figure 8 It is evident that as the concentration of the p53 gene to be detected increases, the collected SERS signal gradually decreases, while the reduction in Raman signal intensity (ΔI) relative to the blank control gradually increases. Within the p53 gene concentration range of 0.01–10 pM, the relationship between the two conforms to Y = 8.271LgX + 26.873, R 2 =0.993, where Y represents the relative change in Raman signal intensity compared to the blank control (Control, p53 gene concentration is 0), and X represents the p53 gene concentration (pM); indicating that effective quantitative analysis can be performed within this range. The limit of detection (LOD) of this invention is 0.0174 pM, and the limit of quantitation (LOQ) is 0.0583 pM (LOD = 3σ / s, where [s] represents the calibration slope, and [σ] is the standard deviation of six blank sample determinations; LOQ = 10σ / s, where [s] represents the calibration slope, and [σ] is the standard deviation of six blank sample determinations).

[0100] Example 2

[0101] To illustrate the specificity of this invention, single-mismatch DNA strands and unmatched DNA strands of the p53 gene were designed, with the sequences as follows:

[0102] Single mismatched DNA strand: 5′-TCATCACACTGGAAGA A TC-3′;

[0103] Uncoordinated DNA strand: 5′-ACTGCTAGAGATTTTCCACA-3′;

[0104] The specific implementation steps are as described in Example 1, wherein the concentration of the p53 gene is 10 pM; the change in relative Raman signal intensity compared with the blank control (Control, p53 gene concentration is 0) can be referred to... Figure 9 This demonstrates that the present invention has strong specificity.

[0105] Example 3

[0106] To test the reproducibility of this strategy, we conducted reproducibility experiments, following the steps outlined in Example 1. The p53 gene concentration was 1 pM. For both within-group and between-group experiments (9 groups), the following procedures were followed: Figure 10 As shown, the relative standard deviation of the 9 data within the group was 3.14%, and the relative standard deviation of the 9 data between the groups was 6.21%, indicating that the strategy has good repeatability.

[0107] Example 4

[0108] To verify the application effect of this invention in actual samples, we added the p53 gene to human serum samples to simulate real samples. We added different concentrations (0.05, 0.5, 1, 5 pM) of the p53 gene to the serum samples, then diluted them, and then used these samples for detection and analysis. The specific implementation steps are as described in Example 1. The results are shown in Table 1. As can be seen from Table 1, this method can also be applied well in real serum samples.

[0109] Table 1. Results of determining p53 gene in simulated serum using the nanodetection platform of the present invention.

[0110] 1 0.05 0.048±0.01 96.00 13.97 2 0.5 0.519±0.0 103.80 7.12 3 1 0.917±0.16 91.70 17.44 4 5 5.642±0.23 112.84 4.13

[0111] 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. sequence list <110> South China Normal University <120> A method for detecting the p53 gene using a SERS tandem strategy based on click chemistry. <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> P0 <400> 1 acctggactg aacgagtctt cca 23 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> p53 gene <400> 2 tcatcacact ggaagactc 19 <210> 3 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> H1 <220> <222> (59)..(59) <223> SH modifier <400> 3 atgacgccgt gactggctag ccggcagcta gccagtcacg gcgtcatagt ccatttttt 59 <210> 4 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> H2 <220> <222> (1)..(1) <223> SH modifier <400> 4 tttttttgcc ggctagccag tcacggcgtc attggactat gacgccgtga ctggctagc 59

Claims

1. A method for detecting the p53 gene using a SERS tandem strategy based on click chemistry, characterized in that, Specifically, the steps include the following: (1) Preparation of Raman signal probe Using gold nanoparticles as a substrate, a Raman signal probe was synthesized by combining alkyne-containing Raman signal molecules through covalent bonds and electrostatic interactions; the alkyne-containing Raman signal molecule was 4-ethynylaniline. (2) Preparation of azide-modified magnetic beads After activating the carboxyl groups of selected carboxyl magnetic beads with an activator, an azide compound is modified onto them through amide bonds to obtain azide-modified magnetic beads; the azide compound is 11-azido-3,6,9-trioxaundecan-1-amine. (3) Synthesis of long-chain DNA polymers enriched with CuONPs: (A) After activating the carboxyl groups on Fe3O4@SiO2 with an activator, it was incubated with PO at room temperature to obtain PO / Fe3O4@SiO2; (B) The p53 gene was incubated with P0 / Fe3O4@SiO2 at room temperature to obtain p53 / P0 / Fe3O4@SiO2; (C) After activating the thiol-modified H1 and H2, they were incubated with p53 / P0 / Fe3O4@SiO2 at room temperature to obtain long-chain DNA polymers by completing the hybridization chain reaction; (D) CuONPs were mixed with long-chain DNA polymers, incubated at room temperature, and then aged with salt; then magnetic separation was performed to obtain long-chain DNA polymers enriched with CuONPs. (4) Alkyne signal readout and SERS signal detection (a) The long-chain DNA polymer enriched with CuONPs induced by different concentrations of p53 gene was acidified and reduced, and then mixed with the Raman signal probe in step (1) and the azide-modified magnetic beads in step (2) to perform a click chemical reaction to consume the Raman signal molecules. After magnetic separation, the supernatant was taken and the unreacted Raman signal molecules were tested for Raman signal intensity to obtain the Raman signal intensity. (b) A standard curve was established based on the relationship between the concentration of the p53 gene and the reduction in Raman signal intensity in the supernatant after magnetic separation relative to the blank control, so as to use the reduction in Raman signal intensity to quantitatively detect the p53 gene.

2. The method according to claim 1, characterized in that: The sequence of P0 in step (3) (A) is: 5′-ACCTGGACTGAACGAGTCTTCCA-3′; The sequence of the p53 gene mentioned in step (3)(B) is: 5′-TCATCACACTGGAAGACTC-3′.

3. The method according to claim 1, characterized in that: The sequence of H1 mentioned in step (3)(C) is: 5′-ATGACGCCTGACTGGCTAGCCGGCAGCTAGCCAGTCACGGCGTCATAGTCCATTTTTT-SH-3′; The sequence of H2 mentioned in step (3)(C) is as follows: 5′-SH-TTTTTTTGCCGGCTAGCCAGTCACGGCGTCATTGGACTATGACGCCGTGACTGGCTAGC-3′.

4. The method according to any one of claims 1 to 3, characterized in that: The average particle size of the gold nanoparticles mentioned in step (1) is 40–50 nm; The carboxyl magnetic beads mentioned in step (2) are magnetic nanoparticles with carboxyl groups modified on the surface, and their average particle size is 200 nm to 300 nm. The activator mentioned in step (2) is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide; The activator mentioned in step (3) (A) is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide; The Fe3O4@SiO2 mentioned in step (3) (A) is Fe3O4 micro magnetic beads coated with carboxyl-modified silica, with an average particle size of 4 to 5 μm; The average particle size of CuONPs described in step (3) (D) is 45-55 nm.

5. The method according to any one of claims 1 to 3, characterized in that: In step (3) (C), the activation treatment is performed using TCEP; the activation time is 5-7 hours. In steps (3) (A), (B), (C), and (D), the room temperature is 25–35°C. In step (3) (A), the incubation time is 0.5 to 1.5 hours; In step (3) (B), the incubation time is 0.5 to 1.5 hours; In step (3) (C), the incubation time is 2 to 4 hours; In step (3) (D), the incubation time is 2 to 4 hours.

6. The method according to any one of claims 1 to 3, characterized in that: In step (3) (D), the salt is NaCl; The acidification mentioned in step (4)(a) refers to dissolving CuONPs into Cu(II) with hydrochloric acid; the reduction refers to reducing Cu(II) into Cu(I) with sodium ascorbate.

7. The method according to any one of claims 1 to 3, characterized in that: In step (4)(a), the concentrations of p53 gene at different concentrations range from 0 mol to 10 pM, namely 0 mol, 0.01 pM, 0.05 pM, 0.1 pM, 0.5 pM, 1.0 pM, 5.0 pM, and 10 pM; where 0 mol is the blank control.

8. The method according to any one of claims 1 to 3, characterized in that: The Raman signal in step (4) (a) is measured by a Raman spectrometer with an excitation wavelength of 785 nm, a Raman spectrum range of 400-3000 cm -1 , a laser power of 400 mW, and a signal collection time of 1-5 s.

9. The method according to claim 1, characterized in that: The Raman signal of the alkyne-containing Raman signal molecule is located in the Raman quiescent region, which is defined as 1800–2800 cm⁻¹. -1 The Raman shift is at 1990 cm⁻¹; among them, the characteristic peak intensity of the 4-ethynylaniline molecule is at 1990 cm⁻¹. -1 At this point, the characteristic peak at 520 cm⁻¹ on the silicon wafer substrate was observed. -1 The intensity at that location is calibrated; In step (4)(a), the Raman signal intensity is I. 1990 / I 520 The Raman signal intensity, where I 1990 It is the 4-ethynylaniline molecule at 1990 cm⁻¹ -1 Peak intensity at I 520 The characteristic peak at 520 cm⁻¹ is from the silicon substrate used for calibration. -1 The strength at that location.

Images