Preparation method, product and application of SERS (Surface Enhanced Raman Scattering) sensor for detecting colorectal cancer precancerous lesion biomarker

Abstract

The invention provides a preparation method, a product and application of an SERS (Surface Enhanced Raman Scattering) sensor for detecting a colorectal cancer precancerous lesion biomarker, and belongs to the technical field of biology. The preparation method comprises the following steps: firstly, obtaining a nanoring array by utilizing a liquid-liquid interface self-assembly method, annealing treatment and gold magnetron sputtering treatment, and then coupling and modifying Raman signal molecules and aptamer complementary chains to the surface of a gold nanocube through Au-S bonds so as to prepare two SERS nanoprobes; an aptamer chain is modified on the surface of a nanoring array, an SERS nanoprobe is combined with a complementary basic group in the aptamer chain and is fixed on the nanoring array, and the SERS sensor is constructed. The SERS sensor is embedded into the PDMS chip to obtain the SERS microarray chip, and the SERS microarray chip has the advantages of high sensitivity, high specificity, high detection speed, high flux and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a preparation method, product and application of a SERS sensor for detecting biomarkers of colorectal precancerous lesions. Background Art

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors in the gastrointestinal tract. Due to the insignificance of its early symptoms, CRC is easily overlooked by patients, resulting in most patients being in the late stage of the disease when diagnosed. The high morbidity and mortality of CRC pose a major threat to human life and health. Therefore, early detection and intervention of precancerous lesions are of vital importance to improving the survival rate of CRC patients. At present, although traditional methods such as colonoscopy occupy an important position in the diagnosis of CRC, their invasiveness, operational complexity and possible discomfort of patients limit the large-scale popularization and application of these methods in the general population. In view of this, multi-target fecal DNA detection, as a new non-invasive CRC screening method in recent years, has been widely used due to its advantages of being non-invasive, painless, safe and convenient. However, this method still faces challenges such as high cost and insufficient specificity. In order to overcome these limitations, current research is committed to exploring new CRC biomarkers and diagnostic tools. For example, Lee et al. developed a surface plasmon resonance biosensor based on nuclear heterogeneous ribonucleoprotein A1 (hnRNPA1) and applied it to the analysis of colorectal cancer plasma samples. In addition, Shen's team found that S100 calcium-binding protein P (S100P) was expressed at elevated levels in CRC patient tissues and was closely associated with the invasiveness and metastasis of CRC.

[0003] The detection methods of protein biomarkers have been widely reported, covering enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC) and mass spectrometry. However, these methods have disadvantages such as complex operation and poor accuracy, and are not suitable for large-scale sample analysis. Surface enhanced Raman spectroscopy (SERS) is a vibration-based fingerprint spectroscopy technology with extremely high sensitivity and accuracy. SERS can enhance the originally weak Raman signal by using surface nanostructures and surface plasmon resonance effects, thereby achieving accurate detection of trace compounds. SERS has the advantages of no interference from water, fast response speed, and non-destructive analysis, and has great potential in the field of body fluid detection and analysis. Compared with the traditional "sandwich strategy" that requires two incubation cycles, the SERS technology combined with the adapter recognition-release detection strategy has the advantages of high specificity, strong affinity, low cost, simple and fast operation, and has received widespread attention in the detection of protein biomarkers.

[0004] Although SERS has shown great potential in the field of protein biomarker detection, its clinical application is greatly limited due to its shortcomings such as large sample consumption and poor reproducibility of the detection process. How to prepare a SERS substrate with stable structure, dense hot spots and uniform distribution is a difficult problem that needs to be solved urgently. Au / SnO2 nanoring arrays (Au / SnO2 NRAs), characterized by their uniform size, regular arrangement, and close contact ring structure, have shown significant advantages in high intensity and high reproducibility in SERS enhancement effect due to their rough surface and narrow nanogaps between particles. As an integrated chip platform, microarray chips have the advantages of high throughput, low sample consumption, high safety, and compact and portable structure, making it possible for simple and rapid sample detection. In recent years, the combination of SERS technology and chips has attracted more and more attention from researchers, injecting new vitality into fields such as biological detection and medical diagnosis. Summary of the invention

[0005] Based on the above content, the purpose of the present invention is to provide a preparation method, product and application of a SERS sensor for detecting biomarkers of colorectal precancerous lesions.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] One of the technical solutions of the present invention is a method for preparing a SERS sensor for detecting biomarkers of colorectal precancerous lesions, comprising the following steps:

[0008] After mixing the SnCl4 solution containing PS microspheres, n-hexane and anhydrous ethanol, a PS sphere monolayer film was obtained by liquid-liquid interface self-assembly method;

[0009] The PS ball monolayer film is sequentially subjected to annealing treatment and gold magnetron sputtering treatment to obtain a nanoring array;

[0010] The Raman signal molecule 4-MBA and the aptamer complementary chain cDNA1 were modified to the surface of the gold nanocube through Au-S bond coupling to obtain SERS nanoprobe 1; the Raman signal molecule DTNB and the aptamer complementary chain cDNA2 were modified to the surface of the gold nanocube through Au-S bond coupling to obtain SERS nanoprobe 2;

[0011] The aptamer chain H is modified on the surface of the nanoring array, and the SERS nanoprobe 1 and the SERS nanoprobe 2 are combined with the complementary bases in the aptamer chain H and fixed on the nanoring array to obtain the SERS sensor;

[0012] The adaptor chain H is H1 and H2; the sequence of H1 is shown in SEQ ID NO.1; the sequence of H2 is shown in SEQ ID NO.2; the sequence of cDNA1 is shown in SEQ ID NO.3; and the sequence of cDNA2 is shown in SEQ ID NO.4.

[0013] The second technical solution of the present invention is a SERS sensor prepared by the above preparation method for simultaneously detecting hnRNPA1 and S100P, biomarkers of colorectal precancerous lesions.

[0014] The third technical solution of the present invention is a SERS microarray chip, which is obtained by embedding the above-mentioned SERS sensor into a PDMS chip.

[0015] A fourth technical solution of the present invention is an application of the above-mentioned SERS microarray chip in the simultaneous detection of colorectal precancerous lesion biomarkers heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) and recombinant human S100 calcium-binding protein P (S100P) for non-disease diagnosis and treatment purposes.

[0016] The present invention discloses the following technical effects:

[0017] The nano-ring array (Au / SnO2 NRAs array) prepared by the present invention has the advantages of stable structure, regular arrangement, dense hot spots and uniform distribution, and can be prepared on a large scale.

[0018] The SERS microarray chip prepared by the present invention has the advantages of high sensitivity, strong specificity, simple operation, fast detection speed and high throughput.

[0019] The SERS microarray chip prepared by the present invention has excellent stability, outstanding specificity and good reproducibility, and provides strong technical support for the early detection, timely intervention and treatment of CRC. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The present invention is a schematic diagram of the preparation process of the SERS microarray chip and the detection of the colorectal precancerous lesion biomarkers hnRNP A1 and S100P.

[0022] Figure 2SEM photo A, TEM photo B, high-resolution TEM photo C, local magnification photo and SAED diffraction photo D, UV-vis-NIR spectrum E, and Raman spectrum F of AuNCs labeled with 4-MBA and 4-MBA prepared in Example 1.

[0023] Figure 3 Figure 2 is the SEM image of the PS microsphere monolayer template in Example 2 (A); the SEM image of the SnO2 NBAs array (B); the SEM image of the Au / SnO2 NRAs array at low magnification (C); the SEM image of the Au / SnO2 NRAs array at high magnification (D); the electromagnetic field simulation image of the Au / SnO2 NRAs array (E); the Raman spectra of the Au / SnO2 NRAs array labeled with 4-MBA and 4-MBA (F); the SERS spectra of 10 points randomly selected from the surface of the Au / SnO2 NRAs array labeled with 4-MBA (G); the SERS spectra of 10 points randomly selected from the surface of the Au / SnO2 NRAs array labeled with 4-MBA (1078 cm -1 The corresponding SERS intensity histogram at H; 1078 cm after the 4-MBA labeled Au / SnO2 NRAs array was stored at room temperature for different days -1 The corresponding SERS intensity line graph I.

[0024] Figure 4 These are the optimization of cDNA concentration (A), incubation temperature (B), and incubation time (C) in the effect verification example.

[0025] Figure 5 Figure A is the SERS spectra of the SERS microarray chip detecting hnRNP A1 and S100P, only hnRNP A1, only S100P, AFP, CEA and blank control in the effect verification example; Figure B is the SERS spectra of the SERS microarray chip detecting hnRNP A1 and S100P, only hnRNP A1, only S100P, AFP, CEA and blank control at 1073 cm -1 and 1330cm -1 SERS intensity histogram at 1073 cm-1; reproducibility of SERS microarray chip; SERS microarray chips prepared from different batches to detect hnRNP A1 and S100P -1 and 1330cm -1 SERS intensity histogram at D.

[0026] Figure 6 Figure A shows the SERS spectra of the SERS microarray chip used to detect different concentrations of hnRNP A1 and S100P dispersed in serum. -1The linear relationship curve B between the characteristic peak intensity at 1330 cm and the logarithmic value of hnRNP A1 concentration; S100P at different concentrations at 1330 cm -1 The linear relationship curve C between the characteristic peak intensity and the logarithmic value of S100P concentration.

[0027] Figure 7 A, B, and C are colonoscopy, CT, and pathological HE staining images of healthy people, respectively; D, E, and F are colonoscopy, CT, and pathological HE staining images of patients with colorectal precancerous lesions, respectively; G, H, and I are colonoscopy, CT, and pathological HE staining images of patients with colorectal cancer, respectively.

[0028] Figure 8 Figure A shows the actual SERS microarray chip in the effect verification example; Figure B shows the average SERS spectra of the serum of healthy people, patients with colorectal precancerous lesions, and colorectal cancer patients; Figure C shows the average SERS spectra of the serum of healthy people, patients with colorectal precancerous lesions, and colorectal cancer patients at 1073 cm -1 and 1330cm -1 SERS intensity histogram at C. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] The present invention firstly uses liquid-liquid interface self-assembly method, annealing treatment, and small magnetron sputtering to prepare closely contacted and orderly arranged nanoring arrays (Au / SnO2 NRAs), and uses seed growth method to prepare gold nanocubes (AuNCs). Raman signal molecules (4-MBA and DTNB) and aptamer complementary chains (cDNA1 and cDNA2) are modified to the surface of AuNCs through Au-S bond coupling to prepare two SERS nanoprobes. The surface of Au / SnO2 NRAs array is modified with aptamer chains (Apt-hnRNPA1 and Apt-S100P), and the SERS nanoprobes are combined with complementary bases in the aptamer chains and fixed on the Au / SnO2 NRAs array to construct a new SERS sensor. During the detection process, due to the strong affinity and high specificity between the aptamer and the target protein, the SERS nanoprobes originally bound to the array surface will be competitively replaced, and the SERS nanoprobes carrying the Raman signal molecules will fall off the SERS sensor, resulting in a decrease in the SERS signal intensity. The results showed that the SERS microarray chip has excellent detection performance and can complete rapid detection within 15 minutes. The detection limit (LOD) of hnRNPA1 is as low as 6.20pg / mL, and the detection limit of S100P is as low as 6.51pg / mL. The present invention combines SERS technology with microarray chips and realizes the detection of hnRNPA1 and S100P in serum samples of patients with colorectal precancerous lesions based on the aptamer recognition-release strategy, providing strong support for the early detection, risk assessment and treatment monitoring of colorectal precancerous lesions, and has broad application prospects.

[0035] Specifically, the present invention provides a method for preparing a SERS sensor for simultaneously detecting hnRNPA1 and S100P, a biomarker of colorectal precancerous lesions, comprising the following steps:

[0036] After mixing the SnCl4 solution containing PS microspheres, n-hexane and anhydrous ethanol, a PS sphere monolayer film was obtained by liquid-liquid interface self-assembly method;

[0037] The PS ball monolayer film is sequentially subjected to annealing treatment and gold magnetron sputtering treatment to obtain a nanoring array (denoted as Au / SnO2 NRAs array);

[0038] The Raman signal molecule 4-MBA and the aptamer complementary chain cDNA1 were modified to the surface of the gold nanocube through Au-S bond coupling to obtain SERS nanoprobe 1; the Raman signal molecule DTNB and the aptamer complementary chain cDNA2 were modified to the surface of the gold nanocube through Au-S bond coupling to obtain SERS nanoprobe 2;

[0039] The aptamer chain H is modified on the surface of the nanoring array, and the SERS nanoprobe 1 and the SERS nanoprobe 2 are combined with the complementary bases in the aptamer chain and fixed on the nanoring array to obtain the SERS sensor;

[0040] The adaptor chain H is H1 and H2; the sequence of H1 is shown in SEQ ID NO.1; the sequence of H2 is shown in SEQ ID NO.2; the sequence of cDNA1 is shown in SEQ ID NO.3; and the sequence of cDNA2 is shown in SEQ ID NO.4.

[0041] In some embodiments of the present invention, the volume ratio of the SnCl4 solution containing PS microspheres, n-hexane and anhydrous ethanol is 6:2:2; the content of PS microspheres in the SnCl4 solution containing PS microspheres is 1 mg / mL.

[0042] In some embodiments of the present invention, the concentration of the SnCl4 solution is 0.05M.

[0043] In some embodiments of the present invention, after obtaining the PS sphere monolayer film, the step of transferring the PS sphere monolayer film using a hydrophilized silicon wafer is further included. The present invention does not specifically limit the method for hydrophilizing the silicon wafer, and a technical solution familiar to those skilled in the art can be selected.

[0044] In some embodiments of the present invention, the annealing treatment is performed at a temperature of 400° C. for 2 hours; the gold magnetron sputtering treatment is performed for a sputtering time of 8 minutes, a current of 30 mA, and an equivalent deposition thickness of 180 nm.

[0045] In some embodiments of the present invention, the gold nanocubes (AuNCs) are prepared by a seed growth method; the specific steps of the seed growth method are:

[0046] After the HAuCl4 solution and the CTAC solution were mixed evenly, freshly prepared icy NaBH4 solution was quickly added while continuously stirring, and the mixture was stirred vigorously for 2 minutes, then stopped stirring, and allowed to stand at room temperature to obtain a seed solution;

[0047] The seed solution is added to the growth solution, stirred evenly, and allowed to stand to obtain a gold nanocube solution; the growth solution is obtained by mixing a CTAC solution, a HAuCl4 solution, an ascorbic acid (AA) solution and water.

[0048] In some embodiments of the present invention, the concentration of the HAuCl4 solution is 0.01M; the concentration of the CTAC solution is 0.1M; the concentration of the NaBH4 solution is 10mM; and the volume ratio of the HAuCl4 solution, CTAC solution and NaBH4 solution is 0.25:7.5:0.6.

[0049] In some embodiments of the present invention, the concentration of the CTAC solution in the growth solution is 0.1 M, the concentration of the HAuCl4 solution is 0.01 M, and the concentration of AA is 0.1 M; the volume ratio of the CTAC solution, HAuCl4 solution, AA solution and water in the growth solution is 3:0.5:2:15.

[0050] In some embodiments of the present invention, the standing time is 1 day.

[0051] In some embodiments of the present invention, the Raman signal molecule 4-MBA and the aptamer complementary chain cDNA1 are coupled and modified to the surface of the gold nanocube through Au-S bonds to obtain the SERS nanoprobe 1 in the following specific steps:

[0052] The gold nanocube solution and the Raman signal molecule 4-MBA solution were mixed and stirred for reaction, and then the resulting reaction solution was added to the activated aptamer complementary chain cDNA1 solution for incubation;

[0053] After the incubation is completed, a BSA solution is added to the reaction solution obtained by the incubation to react, thereby obtaining the SERS nanoprobe 1;

[0054] The volume ratio of the gold nanocube solution to the Raman signal molecule 4-MBA solution is 8 mL:200 μL; the concentration of the Raman signal molecule 4-MBA solution is 5 mM;

[0055] The stirring reaction was carried out at room temperature for 1 hour; the incubation was carried out at 37° C., with a humidity of 80% for 2 hours; and the reaction was carried out at room temperature for 2 hours.

[0056] In some embodiments of the present invention, the Raman signal molecule DTNB and the aptamer complementary chain cDNA2 are coupled and modified to the surface of the gold nanocube through Au-S bonds to obtain the SERS nanoprobe 2 in the following specific steps:

[0057] The gold nanocube solution and the Raman signal molecule DTNB solution were mixed and stirred for reaction, and then the resulting reaction solution was added to the activated aptamer complementary chain cDNA2 solution for incubation;

[0058] After the incubation is completed, a BSA solution is added to the reaction solution obtained by the incubation to react, thereby obtaining the SERS nanoprobe 2;

[0059] The volume ratio of the gold nanocube solution to the Raman signal molecule DTNB solution is 8 mL:200 μL; the concentration of the Raman signal molecule DTNB solution is 5 mM;

[0060] The stirring reaction was carried out at room temperature for 1 hour; the incubation was carried out at 37° C., with a humidity of 80% for 2 hours; and the reaction was carried out at room temperature for 2 hours.

[0061] In some embodiments of the present invention, activating the aptamer complementary chain cDNA1 or the aptamer complementary chain cDNA2 specifically comprises: using freshly prepared TCEP buffer (50mM) to activate the aptamer complementary chain cDNA1 or the aptamer complementary chain cDNA2 to obtain an activated aptamer complementary chain cDNA1 solution or an activated aptamer complementary chain cDNA2 solution.

[0062] In some embodiments of the present invention, the concentration of the BSA solution is 1 wt %; after the reaction I is completed, the steps of centrifugal purification and constant volume are also included.

[0063] In some embodiments of the present invention, the aptamer chain H is modified on the surface of the nanoring array, and the SERS nanoprobe 1 and the SERS nanoprobe 2 are combined with the complementary bases in the aptamer chain H and fixed on the nanoring array. The specific steps of obtaining the SERS sensor are:

[0064] The activated aptamer chain H solution is added dropwise onto the surface of the nanoring array, followed by incubation to obtain an aptamer chain-coupled nanoring array;

[0065] The SERS nanoprobe 1 and the SERS nanoprobe 2 are dropped onto the surface of the aptamer chain-coupled nanoring array, followed by incubation to obtain the SERS sensor.

[0066] In some embodiments of the present invention, the activation of the aptamer chain H is specifically: using a freshly prepared TCEP buffer to activate the aptamer chain H to obtain an activated aptamer chain solution.

[0067] In some embodiments of the present invention, the activated aptamer chain solution is dripped onto the surface of the nanoring array, followed by incubation at a temperature of 37° C., a humidity of 80%, and a time of 2 h.

[0068] In some embodiments of the present invention, after obtaining the Au / SnO2 NRAs array coupled with aptamer chains, a step of washing away unconnected aptamer chains using PBS buffer is also included.

[0069] In some embodiments of the present invention, the SERS nanoprobe is added dropwise to the surface of the nanoring array coupled with the aptamer chain, and then incubated at a temperature of 37°C, a humidity of 80%, and a time of 1.5 hours. After the incubation, the SERS nanoprobe is partially complementary to the aptamer chain base pairing, so that the SERS nanoprobe is anchored on the Au / SnO2 NRAs array, and the SERS sensor is successfully prepared.

[0070] The present invention also provides a SERS sensor prepared by the above preparation method for simultaneously detecting hnRNPA1 and S100P, biomarkers of colorectal precancerous lesions.

[0071] The present invention also provides a SERS microarray chip, which is obtained by embedding the above SERS sensor into a PDMS chip.

[0072] In some embodiments of the present invention, the method for preparing the SERS microarray chip is specifically as follows:

[0073] 1) Use AutoCAD software to design the size, shape and distribution of the microarray chip to ensure that the chip can meet the experimental requirements. Use photolithography technology to build a microarray chip mold, spin-coat SU-8 on the cleaned silicon wafer, and obtain the microarray mold after curing, exposure and development;

[0074] 2) Place a container containing a PDMS mixture (the PDMS mixture is a mixture of PDMS and a crosslinker at a mass ratio of 10:1, and the crosslinker is a crosslinker commonly used for crosslinking PDMS, such as a platinum catalyst) into a vacuum chamber. Start the vacuum pump, start to evacuate the vacuum chamber, and gradually reduce the air pressure in the chamber. During this process, the bubbles in the PDMS solution will expand and rise to the liquid surface as the air pressure decreases, and eventually break or escape the solution. Continue for ten minutes until no more bubbles are observed in the solution. Pour the vacuum-treated mixture into the microarray mold, and after the vacuum treatment again, place it in an 80°C oven for baking and curing for 3 hours to obtain a PDMS chip. After cooling to room temperature, the PDMS chip is punched, cleaned, dried, and surface-modified (using a plasma machine for treatment, plasma treatment can change the chemical structure of the PDMS surface, thereby increasing the hydrophilicity of the PDMS chip) according to the design drawing, and then the SERS sensor is embedded in the prepared PDMS chip to obtain a designed SERS microarray chip.

[0075] The present invention combines the respective advantages of SERS technology and microarray chips to construct a new high-throughput SERS microarray chip based on aptamer recognition-release strategy, which can achieve highly sensitive recognition and rapid detection of hnRNP A1 and S100P. Prior to this, the present invention first constructed an Au / SnO2 NRAs array with a close contact ring structure, which is composed of a highly ordered, precisely dimensioned rough ring structure, showing an excellent SERS enhancement effect. SnO2 has good biocompatibility and is easy to chemically modify. Subsequently, gold nanocube particles (AuNCs) were synthesized and modified with Raman signal molecules (4-MBA and DTNB) and aptamer complementary chains (cDNA1 and cDNA2) to prepare two SERS nanoprobes. The SERS nanoprobes were combined with the complementary bases in the aptamer chain and fixed on the Au / SnO2NRAs array, and a new SERS sensor was successfully constructed. When hnRNP A1 and S100P are present in the analyte, the SERS probe with Raman signal molecules will competitively fall off, resulting in a significant decrease in the SERS signal intensity of the characteristic peak, because the affinity between the aptamer and the target protein is much higher than that of the complementary chain (cDNA1 and cDNA2). Finally, the detection results of clinical samples were compared and analyzed using the SERS chip with the ELISA test results. The SERS chip can accurately meet the detection requirements of hnRNP A1 and S100P in clinical serum samples. In summary, the SERS chip using this new aptamer recognition-release strategy, as an advanced SERS analysis platform, not only opens up a new perspective for clinical detection of biomarkers, but also provides a new idea for screening CRC.

[0076] The present invention also provides the use of the SERS microarray chip in the simultaneous detection of colorectal precancerous lesion biomarkers hnRNPA1 and S100P for non-disease diagnosis and treatment purposes.

[0077] In order to better understand the present invention, the content of the present invention is further explained in conjunction with the following examples, but the content of the present invention is not limited to the following examples. The preparation process of the SERS microarray chip of the present invention and the schematic diagram of detecting the colorectal precancerous lesion biomarkers hnRNPA1 and S100P are shown in FIG. Figure 1 shown.

[0078] Unless otherwise specified, the raw materials and reagents used in the examples can be obtained through commercial channels.

[0079] The instruments and test conditions used in the present invention are as follows:

[0080] The scanning electron microscope (SEM) images were obtained using a S-4800II field emission scanning electron microscope produced by Hitachi, Japan.

[0081] The transmission electron microscopy (TEM) images were obtained using a Tecnai G2 F30 field emission transmission electron microscope from FEI Company, USA.

[0082] Raman spectra were measured by the Invia Reflex laser microscope Raman spectrometer produced by Renishaw, UK. The laser wavelength was 785 nm, the exposure time was 10 s, the laser intensity was 50 mW, the objective lens was 50×, and the detection range was 600-1800 cm -1 .

[0083] The method for using the SERS microarray chip to detect hnRNPA1 and S100P in a sample to be detected comprises the following steps:

[0084] 1) Dissolve hnRNPA1 and S100P standards in serum and prepare 10 -12 -10 -6 g / mL concentration gradient mixture. 15 μL of the mixture was added to the reaction area of ​​the SERS microarray chip for simultaneous detection of colorectal precancerous lesion biomarkers hnRNPA1 and S100P for incubation (incubation time was 15 minutes). After PBS washing, SERS detection was performed to capture the characteristic signals of 4-MBA and DTNB, and based on 1073 cm -1 With 1330cm -1 The wave number was used to construct the concentration logarithm-signal intensity working curve.

[0085] 2) Serum samples from healthy people, patients with colorectal precancerous lesions, and patients with colorectal cancer were collected and added to the reaction area of ​​the SERS microarray chip for simultaneous detection of colorectal precancerous lesion biomarkers hnRNPA1 and S100P, and reacted at room temperature. After PBS washing, SERS detection was used to obtain 4-MBA and DTNB signals.

[0086] 3) Substituting the signal intensity in step 2) into the working curve in step 1) to determine the concentrations of hnRNPA1 and S100P in the sample to be tested.

[0087] Example 1

[0088] The preparation steps of SERS nanoprobe are as follows:

[0089] 1) Take a beaker with a capacity of 10 mL, add 0.25 mL HAuCl4 solution (0.01 M) and 7.5 mL CTAC solution (0.1 M) in sequence, and mix well. While stirring continuously, quickly add 0.6 mL of freshly prepared icy NaBH4 solution (10 mM), stir vigorously for 2 minutes, stop stirring, and stand at room temperature for two hours to obtain a seed solution, which is diluted 10 times for later use.

[0090] 2) The growth solution was prepared by mixing 3 mL of CTAC solution (0.1 M), 0.5 mL of HAuCl4 solution (0.01 M), 2 mL of AA solution (0.1 M) and 15 mL of deionized water. 10 μL of the diluted seed solution was added to the growth solution, stirred gently and allowed to stand at room temperature for 1 day to obtain the AuNCs solution.

[0091] 3) Add 8 mL of the AuNCs solution obtained in step 2) and 200 μL of the Raman signal molecule 4-MBA solution (5 mM) into a 25 mL beaker, stir vigorously for 1 hour to obtain AuNCs@4-MBA; the same experimental process is used to obtain AuNCs@DTNB.

[0092] 4) Take 5 μL of freshly prepared TCEP buffer (50 mM) to activate the aptamer complementary chain (cDNA1), then add the AuNCs@4-MBA from step 3) to the activated cDNA1 solution and incubate in a constant temperature incubator at 37°C and 80% humidity for 2 hours.

[0093] 5) Add 15 μL of BSA solution (1% wt) to the incubated reaction solution and react for 2 hours. After two rounds of centrifugation and purification, the solution was fixed to volume in PBS solution to obtain the SERS nanoprobe AuNCs@4-MBA@cDNA1. The same experimental process can be used to obtain the SERS nanoprobe AuNCs@DTNB@cDNA2.

[0094] Characterization of AuNCs morphology and SERS effect:

[0095] The morphology and structure of AuNCs were detected by SEM, TEM, HRTEM and SAED imaging diffraction patterns.

[0096] Renishaw InVia laser Raman spectrometer was used for SERS detection, and the parameters were set as follows: objective lens 50×, laser wavelength 785nm, power 5mW, data acquisition time 10s. To ensure the validity of the data, the spectrum of each sample was averaged at 5 random points during the reaction process, and pre-processed using Origin 2021 software to correct the baseline and smooth the spectrum for subsequent analysis.

[0097] Figure 2 As shown in Figure A, AuNCs have a regular hexahedral structure with consistent size and morphology, and an average edge length of 68 nm. Figure 2 Medium B and Figure 2 As shown in Figure C, AuNCs have sharp corners and a high specific surface area. This makes AuNCs have excellent localized surface plasmon resonance (LSPR) effect and easy to carry out various chemical modifications. Figure 2As shown in Figure 5D, the lattice spacing of AuNCs is 0.236 nm, which is consistent with the theoretical value of the {111} lattice plane of the gold crystal structure. Figure 2 Figure E shows that the AuNCs samples all have a sharp LSPR peak near 532nm. In addition, according to the enhancement factor (EF) formula, we also used 4-MBA at 1078cm -1 The SERS activity of AuNCs was evaluated by the characteristic peak intensity at Figure 2 F):EF=(I SERS / C SERS ) / (I Raman / C Raman ), when C Raman =10 -1 M, C SERS =10 -5 When M, the EF value is 1.52×10 5 . AuNCs have excellent SERS enhancement effect.

[0098] Example 2

[0099] The preparation steps of Au / SnO2 NRAs array are as follows:

[0100] 1) Immerse 10 mg of PS microspheres in 10 mL of 0.05 M SnCl4 solution for later use.

[0101] 2) Using the liquid-liquid interface self-assembly method, 6 mL of SnCl4 solution containing PS microspheres, 2 mL of n-hexane, and 2 mL of anhydrous ethanol were added to a 25 mL empty beaker in sequence, and the solution was rapidly self-assembled on the liquid surface to obtain a PS sphere monolayer film, which was then transferred using a hydrophilic silicon wafer to obtain a PS microsphere monolayer template.

[0102] 3) The PS microsphere monolayer template obtained in step 2) was annealed at 400° C. for 2 hours to obtain a flat bowl-shaped tin oxide structure array (SnO2 NBAs).

[0103] 4) The SnO2 NBAs array obtained in step 3) is placed in a small magnetron sputtering instrument, the current is set to 30 mA to sputter gold nanoparticles, the sputtering time is 8 minutes, and the equivalent deposition thickness is 180 nm, thereby obtaining a closely contacted and orderly arranged Au / SnO2NRAs array.

[0104] Characterization of the morphology and SERS effect of Au / SnO2 NRAs array:

[0105] The morphological changes during the preparation of Au / SnO2 NRAs arrays were characterized. PS microspheres (diameter 500 nm) immersed in low concentration SnCl4 solution self-assembled on the hydrophilic treated silicon wafer ( Figure 3 A), which is highly ordered, laying a solid experimental foundation for the subsequent preparation of size-controlled Au / SnO2NRAs arrays. After annealing, the PS microsphere array presents a flat bowl-shaped tin oxide structure array. Figure 3 Figure B is the SEM image of the intermediate SnO2 NBAs array. Figure 3 Middle C and Figure 3 D in the middle shows the SEM images of the Au / SnO2 NRAs array at different magnifications. After gold deposition, the flat bowl-shaped SnO2 NBAs array is transformed into a regularly arranged ring-shaped Au / SnO2 NRAs array with a diameter of about 500nm. A three-dimensional model of the Au / SnO2 NRAs array was established and simulated to understand the electric field intensity distribution of the Au / SnO2 NRAs array ( Figure 3 In order to evaluate the SERS activity of the Au / SnO2 NRAs array, the Au / SnO2 NRAs array was labeled with 4-MBA and SERS spectroscopy was performed ( Figure 3 F) When C Raman =10 -1 M, C SERS =10 -8 When M, the EF value is 2.53×10 8 . SERS spectroscopy analysis was performed on 10 random points on the surface of the 4-MBA labeled Au / SnO2 NRAs array ( Figure 3 Middle G), 1078cm -1 The relative standard deviation (RSD) of the Raman characteristic peak intensity at Figure 3 Finally, the stability of the Au / SnO2 NRAs array was evaluated by SERS detection of the Au / SnO2 NRAs array stored at room temperature for different days (1 day, 5 days, 10 days and 15 days), and the 1078 cm -1 The RSD of the Raman characteristic peak intensity at is equal to 5.49% ( Figure 3 In (I), Au / SnO2 NRAs have excellent storage stability.

[0106] Example 3

[0107] The preparation steps of SERS sensor are as follows:

[0108] 1) Activate the aptamer chains (Apt-hnRNPA1 and Apt-S100P) with freshly prepared TCEP buffer, drop 200 μL of the activated Apt-hnRNPA1 and Apt-S100P solution onto the Au / SnO2 NRAs array prepared in Example 2, and incubate in a constant temperature incubator at 37°C and 80% humidity for 2 h to obtain the Au / SnO2 NRAs array coupled with the aptamer chains. Then rinse with PBS buffer to wash away the unconnected aptamer chains.

[0109] 2) Take 60 μL of the SERS nanoprobe prepared in Example 1 and add it dropwise to the surface of the Au / SnO2NRAs array coupled with the aptamer chain obtained in step 1. After incubation in a constant temperature incubator at 37°C for 1.5 hours, rinse with PBS buffer. The SERS nanoprobe is partially complementary to the bases of the aptamer chain, so that the SERS nanoprobe is anchored on the Au / SnO2NRAs array, and the SERS sensor is successfully prepared.

[0110] Example 4

[0111] The preparation steps of SERS microarray chip are as follows:

[0112] 1) Use AutoCAD software to design the size, shape and distribution of the microarray chip to ensure that the chip can meet the experimental requirements. Use photolithography technology to build a microarray chip mold, spin-coat SU-8 on the cleaned silicon wafer, and obtain the microarray mold after curing, exposure and development;

[0113] 2) Place a container containing a PDMS mixture (the PDMS mixture is a mixture of PDMS and a platinum catalyst in a mass ratio of 10:1) into a vacuum chamber. Start the vacuum pump, start to evacuate the vacuum chamber, and gradually reduce the air pressure in the chamber. During this process, the bubbles in the PDMS solution will expand and rise to the liquid surface as the air pressure decreases, and eventually break or escape the solution. Continue for ten minutes until no more bubbles are observed in the solution. Pour the vacuum-treated mixture into the microarray mold, and after vacuum treatment again, place it in an 80°C oven for baking and curing for 3 hours to obtain a PDMS chip. After cooling to room temperature, the PDMS chip is punched, cleaned, dried, and surface-modified (using a plasma machine for treatment, plasma treatment can change the chemical structure of the PDMS surface, thereby increasing the hydrophilicity of the PDMS chip) according to the design drawing, and then the SERS sensor prepared in Example 3 is embedded in the prepared PDMS chip to obtain a designed SERS microarray chip.

[0114] Effect verification example:

[0115] 1. Screening of parameter optimization

[0116] 1.1 A systematic screening of cDNA concentrations was conducted, and the experimental design covered multiple cDNA concentration gradients (1nM, 10nM, 100nM, 1μM, 10μM, 100μM), which were used to modify the surface of AuNCs and subsequently fixed on the Au / SnO2 NRAs array to prepare SERS sensors for detecting the SERS spectra of specific biomarkers (hnRNPA1 and S100P). Figure 4 As shown in A, 1073cm -1 and 1330cm -1 The SERS intensity at 1 μM increases with the increase of cDNA concentration. When the cDNA concentration reaches 1 μM, the growth trend of SERS intensity tends to be stable, which can be explained as the coupled cDNA is close to saturation. Therefore, the optimal concentration of cDNA is 1 μM to prepare the subsequent SERS sensor.

[0117] 1.2 Incubation temperature is the main factor affecting detection performance. The experimental design covers multiple incubation temperature gradients (34°C, 35°C, 36°C, 37°C, 38°C, 39°C) to detect the SERS spectra of the mixed solution of hnRNPA1 and S100P. Figure 4 As shown in B, 1073cm -1 and 1330cm -1 The SERS intensity at 37°C increases with the increase of temperature, but when the temperature reaches the critical point of 37°C and continues to increase, the SERS intensity of the characteristic peak decreases. Therefore, 37°C is selected as the optimal incubation temperature.

[0118] 1.3 Drop the hnRNPA1 and S100P mixture onto the reaction area of ​​the SERS microarray chip and incubate at 37°C. Take it out every 5 minutes, wash the reaction area with PBS, and then perform SERS detection to determine the optimal hybridization reaction time (i.e., incubation time). Figure 4 As shown in C, with the extension of incubation time, 1073 cm -1 and 1330cm -1 The SERS intensity at the position shows a downward trend. When the incubation time reaches 15 minutes, the SERS intensity gradually stabilizes. This is because the affinity and specificity between the aptamer and the target protein are strong, and the SERS nanoprobes originally bound to the surface of the Au / SnO2 NRAs array are competitively replaced, and the SERS nanoprobes carrying the Raman signal molecules fall off, resulting in a decrease in the intensity of the SERS signal. Therefore, the optimal incubation time is 15 minutes.

[0119] 2. Specificity and reproducibility of SERS microarray chips

[0120] The blank was used as the control group, CEA and AFP were used as interferences, and hnRNPA1 alone, S100P alone, and hnRNP A1 and S100P mixed solution were used as the target group (all with a concentration of 100 ng / mL). The nucleotide sequences used in the experiment are shown in Table 1. The test solutions were incubated with the reaction area of ​​the SERS microarray chip, hybridized in a 37°C incubator for 15 minutes, and the reaction area was washed with PBS. The SERS detection was then performed to evaluate the specificity of the SERS microarray chip. The SERS spectra of the hnRNPA1 and S100P mixed solution (concentration of 1 pg / mL) were detected using chips prepared from five different batches. Five points were randomly selected in the reaction area of ​​each batch of chips for SERS measurement, and then the average spectrum was calculated. The reproducibility of the SERS microarray chip was evaluated.

[0121] Table 1 Nucleotide sequences used in the experiments

[0122]

[0123] like Figure 5 As shown in A, the SERS spectrum of the control group did not change significantly. Figure 5 In B, it can be observed more intuitively that when the target exists, the 1073cm -1 and 1330cm -1 The SERS intensity of the characteristic peak at the position is significantly reduced. Therefore, the SERS microarray chip as an analysis platform can accurately distinguish the target and other interferents with excellent specificity. Repeatability is another important parameter that affects the actual detection results of the chip. Figure 5 As shown in Figure 5C and Figure 5D, five batches of microarray chips were prepared at different times and subjected to SERS detection. There was no significant difference in the SERS spectra at 1073 cm -1 and 1330cm -1 The RSD values ​​of the characteristic peak intensities at 400 nm and 800 nm were 5.79% and 5.96% respectively. The SERS microarray chip has good reproducibility.

[0124] 3. Quantitative SERS detection of hnRNPA1 and S100P

[0125] hnRNP A1 and S100P (hnRNP A1:S100P=1:1) were dispersed into serum and prepared into different concentrations (10 -12 -10 -6 g / mL) serum sample solution, and the SERS microarray chip prepared in Example 4 was used to quantitatively detect hnRNPA1 and S100P simultaneously.

[0126] like Figure 6 As shown in A, with the increase of hnRNPA1 and S100P concentrations, the 1073 cm-1 and 1330cm -1 The SERS intensity at the position shows a clear downward trend. This is because the aptamer has a high degree of specificity and affinity with the target, which causes the SERS nanoprobe carrying the signal molecule to detach and the SERS intensity to decrease. Figure 6 As shown in Figure 6B and Figure 6C, the logarithm of the concentration of hnRNPA1 and S100P showed a good linear relationship with the SERS intensity, and the standard curve equations were y = -2688.67x -14642.47, R 2 = 0.9843 and y = -2980.61x - 16112.55, R 2 =0.9836. The LOD values ​​of hnRNPA1 and S100P were calculated to be 6.20 pg / mL and 6.51 pg / mL. Compared with other detection methods (Table 2), the SERS analysis platform of the integrated microarray chip has a wider detection range, higher sensitivity, and a very short detection time, and the detection results can be quickly obtained in just 15 minutes. The SERS microarray chip provided by the present invention has great application potential in the diagnosis and screening of cancer.

[0127] Table 2. Comparison of SERS microarray chips with other methods in biomarker detection

[0128]

[0129] 4. SERS microarray chip analysis of clinical samples ( Figure 8 (A in the middle is a real picture of the SERS microarray chip)

[0130] CRC diagnosis requires a rigorous and systematic process of comprehensive consideration to ensure the accuracy and comprehensiveness of the diagnostic results. Comprehensive diagnosis of patients is performed through colonoscopy, CT and pathological HE staining. Figure 7 As shown in Figures AC, colonoscopy shows that the intestinal mucosa of a healthy person is smooth and has clear vascular texture. Intestinal wall thickness imaging is normal, with no thickening or masses. Pathological analysis shows that the intestinal mucosal tissue structure is intact, with cells arranged neatly and without anisotropy. Figure 7 As shown in the middle DF, raised lesions can be seen in the rectum, and the surface mucosa is uneven and nodular. The boundary between the intestine and surrounding structures (such as the liver and pancreas) is unclear. The pathological diagnosis is the presence of villous tubular adenoma with high-grade intraepithelial neoplasia in the rectal mucosa. Figure 7 As shown in the middle GI, there are inflammatory raised lesions in the colorectum. The depth of infiltration is deep, and the pathological diagnosis is confirmed as colorectal cancer.

[0131] Serum clinical samples were collected from 30 healthy people, 30 patients diagnosed with colorectal precancerous lesions by pathological examination, and 30 patients with colorectal precancerous lesions. The SERS microarray chip was used to perform SERS detection on the 90 serum clinical samples collected, and the average spectrum of healthy people and patients was obtained ( Figure 8 (middle B). Figure 8 Middle C shows 1073cm -1 and 1330cm -1 The SERS intensity corresponding to the characteristic peak at 1073 cm -1 and 1330cm -1 Substitute the SERS intensity corresponding to the characteristic peak into the corresponding linear regression equation ( Figure 6 Medium B and Figure 6 C), the expression levels of hnRNPA1 and S100P in the serum of healthy people and patients can be calculated. The test results were compared with the ELISA test results. Compared with healthy people, the expression levels of hnRNPA1 and S100P in the serum of patients with CRC precancerous lesions and CRC patients gradually increased. The test results of SERS microarray were highly consistent with the results of ELISA (Table 3). Therefore, this chip can accurately meet the detection needs of hnRNPA1 and S100P in clinical serum samples, which is helpful for the diagnosis and detection of colorectal precancerous lesions.

[0132] Table 3 SERS and qRT-PCR results of clinical samples

[0133]

[0134] 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a SERS sensor for detecting biomarkers of colorectal precancerous lesions, characterized in that: The following steps are involved: After mixing the SnCl4 solution containing PS microspheres, n-hexane and anhydrous ethanol, a PS sphere monolayer film was obtained by liquid-liquid interface self-assembly method; The PS ball monolayer film is sequentially subjected to annealing treatment and gold magnetron sputtering treatment to obtain a nanoring array; The Raman signal molecule 4-MBA and the aptamer complementary chain cDNA1 were modified to the surface of the gold nanocube through Au-S bond coupling to obtain SERS nanoprobe 1; the Raman signal molecule DTNB and the aptamer complementary chain cDNA2 were modified to the surface of the gold nanocube through Au-S bond coupling to obtain SERS nanoprobe 2; The aptamer chain H is modified on the surface of the nanoring array, and the SERS nanoprobe 1 and the SERS nanoprobe 2 are combined with the complementary bases in the aptamer chain H and fixed on the nanoring array to obtain the SERS sensor; The adaptor chain H is H1 and H2; the sequence of H1 is shown in SEQ ID NO.1; the sequence of H2 is shown in SEQ ID NO.2; the sequence of cDNA1 is shown in SEQ ID NO.3; and the sequence of cDNA2 is shown in SEQ ID NO.

4.

2. The method for preparing a SERS sensor for detecting biomarkers of colorectal precancerous lesions according to claim 1, characterized in that: The volume ratio of the SnCl4 solution containing PS microspheres, n-hexane and anhydrous ethanol is 6:2:2; the content of PS microspheres in the SnCl4 solution containing PS microspheres is 1 mg / mL.

3. The method for preparing a SERS sensor for detecting biomarkers of colorectal precancerous lesions according to claim 1, characterized in that: After obtaining the PS ball single-layer film, the method further includes a step of transferring the PS ball single-layer film using a hydrophilic silicon wafer.

4. The method for preparing a SERS sensor for detecting biomarkers of colorectal precancerous lesions according to claim 1, characterized in that: The temperature of the annealing treatment is 400° C. and the time is 2 hours; the sputtering time of the gold magnetron sputtering treatment is 8 minutes, the current is 30 mA, and the equivalent deposition thickness is 180 nm.

5. The method for preparing a SERS sensor for detecting biomarkers of colorectal precancerous lesions according to claim 1, characterized in that: The gold nanocubes are prepared by a seed growth method; the specific steps of the seed growth method are: After the HAuCl4 solution and the CTAC solution are evenly mixed, the freshly prepared icy NaBH4 solution is quickly added during continuous stirring. After the vigorous stirring is completed, the solution is allowed to stand at room temperature to obtain a seed solution. The seed solution is added to the growth solution, stirred evenly, and then allowed to stand to obtain a gold nanocube solution; the growth solution is obtained by mixing a CTAC solution, a HAuCl4 solution, an ascorbic acid solution and water.

6. The method for preparing a SERS sensor for detecting biomarkers of colorectal precancerous lesions according to claim 1, characterized in that: The specific steps of coupling and modifying the Raman signal molecule 4-MBA and the aptamer complementary chain cDNA1 to the surface of the gold nanocube through Au-S bond to obtain the SERS nanoprobe 1 are as follows: The gold nanocube solution and the Raman signal molecule 4-MBA solution were mixed and stirred for reaction, and then the resulting reaction solution was added to the activated aptamer complementary chain cDNA1 solution for incubation; After the incubation is completed, a BSA solution is added to the reaction solution obtained by the incubation to react, thereby obtaining the SERS nanoprobe 1; The specific steps of coupling and modifying the Raman signal molecule DTNB and the aptamer complementary chain cDNA2 to the surface of the gold nanocube through Au-S bond to obtain the SERS nanoprobe 2 are as follows: The gold nanocube solution and the Raman signal molecule DTNB solution were mixed and stirred for reaction, and then the resulting reaction solution was added to the activated aptamer complementary chain cDNA2 solution for incubation; After the incubation is completed, a BSA solution is added to the reaction solution obtained by the incubation to react and obtain the SERS nanoprobe 2.

7. The method for preparing a SERS sensor for detecting biomarkers of colorectal precancerous lesions according to claim 1, characterized in that: The specific steps of modifying the aptamer chain H on the surface of the nanoring array, combining the SERS nanoprobe 1 and the SERS nanoprobe 2 with the complementary bases in the aptamer chain H and fixing them on the nanoring array to obtain the SERS sensor are as follows: The activated aptamer chain H solution is added dropwise onto the surface of the nanoring array, followed by incubation to obtain an aptamer chain coupled nanoring array; The SERS nanoprobe 1 and the SERS nanoprobe 2 are dropped onto the surface of the aptamer chain-coupled nanoring array, followed by incubation to obtain the SERS sensor.

8. A SERS sensor for detecting biomarkers of colorectal precancerous lesions prepared by the preparation method according to any one of claims 1 to 7.

9. A SERS microarray chip, characterized in that: The SERS sensor according to claim 8 is embedded in a PDMS chip.

10. Use of the SERS microarray chip according to claim 9 in the simultaneous detection of hnRNPA1 and S100P, biomarkers of colorectal precancerous lesions, for purposes other than disease diagnosis and treatment.

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