A nanoporous gold biosensor for detecting tumor cell-derived exosomes in body fluids, and its preparation method and application

Abstract

The present invention relates to a nanoporous gold biosensor for detecting tumor cell-derived exosomes in body fluids, a preparation method thereof, and an application thereof. The nanoporous gold biosensor comprises: nanoporous gold, an 11-mercaptoundecanoate linked to the nanoporous gold, a nickel ion linked to the carboxyl terminus of the 11-mercaptoundecanoate, and a histidine-tagged CP05 polypeptide linked via the nickel ion.

Description

Technical Field

[0001] The present invention relates to the field of biosensor design and tumor diagnosis technology, and in particular to a surface modification method of a nanoporous gold biosensor for detecting tumor cell-derived exosomes in body fluids. Background Art

[0002] Cancer, a global public health problem, poses a serious threat to human health. Early detection of tumor cells is crucial for cancer treatment. Detecting and treating malignant tumors at an early stage ensures optimal outcomes. Currently, mainstream tumor tissue testing methods include imaging, pathology, tumor marker testing, and genetic testing. However, most of these methods require large sample sizes or complex procedures, hindering early diagnosis.

[0003] As an emerging tumor marker, exosomes are widely present in the body fluids of organisms. Exosomes originate from microvesicles formed by the invagination of lysosomal granules in cells. They carry chemical information reflecting cellular characteristics, including nucleic acids, proteins, lipids, amino acids and metabolites, and can be used for early diagnosis and postoperative detection of cancer. However, the composition of patients' body fluids is complex and the concentration of exosomes is low. It is still very challenging to obtain reliable information through exosomes. Traditional detection methods, such as enzyme-linked immunosorbent assay (ELISA) and western blot analysis, require extensive post-labeling processes and a large number of samples for testing. Given the limitations of these methods, such as cumbersome operations or limited accuracy, traditional exosome analysis methods are often impractical in clinical diagnosis.

[0004] Surface-enhanced Raman spectroscopy (SERS) is a highly sensitive spectroscopic analysis method that can nondestructively detect and identify the chemical structure of characteristic molecules. It has the ability to detect single biological cells. Its ultra-high sensitivity and ability to rapidly identify biomarkers make it an ideal method for early tumor diagnosis. A key foundation for SERS detection is the preparation of highly sensitive substrate materials. Gold and silver nanoparticles produce efficient electromagnetic enhancement under plasmon resonance, making them ideal materials for highly sensitive SERS substrates. However, gold and silver nanoparticles must be dispersed in solutions or colloids, which poses significant storage and handling challenges. Nanoporous gold structures are suitable for capturing exosomes, and their significant surface plasmon resonance effect can significantly enhance Raman spectral signals, making them ideal sensors for exosome detection. However, human blood contains a large number of other biomacromolecules such as proteins and lipids, as well as exosomes derived from normal tissues. Surface-enhanced Raman spectroscopy signals are subject to interference from numerous impurities, making it difficult to accurately identify the source of the signal in blood. Without the ability to specifically isolate tumor-derived exosomes from blood, accurate tumor cell detection using SERS alone is clearly inadequate. Therefore, by linking specific proteins on the nanoporous gold surface to precisely capture exosomes in body fluids, the enrichment of exosomes and signal purification on the nanoporous gold sensor surface can be achieved. Summary of the Invention

[0005] In response to the above problems, the purpose of the present invention is to provide a reliable surface modification technology to produce a high SERS sensitivity biochip that is easy to store and use and has the ability to specifically capture exosomes in body fluids for rapid identification of human tumors.

[0006] The present invention is achieved by the following technical solution: first, nanoporous gold (100-300 nm) with nanopore size matching that of exosomes is selected, and then 11-mercaptoundecanoic acid (MUA), nickel ions (Ni 2+ ) and a histidine-tagged CP05 peptide (His-CP05), ultimately resulting in a nanoporous gold SERS sensor with the specific ability to capture exosomes. After adsorbing exosomes in human body fluids, the sensor generates a corresponding Raman spectrum through Raman analysis. This spectral information can be used to identify the presence of tumor-derived exosomes, thereby determining whether the patient is ill.

[0007] Specifically, in the first aspect, the present invention provides a nanoporous gold biosensor, comprising: nanoporous gold, 11-mercaptoundecanoic acid linked to the nanoporous gold, a nickel ion connected to the carboxyl terminus of the 11-mercaptoundecanoic acid, and a histidine-tagged CP05 polypeptide linked via the nickel ion.

[0008] Preferably, the pore size of the nanoporous gold is 100-300 nm.

[0009] In a second aspect, the present invention provides a method for preparing the above-mentioned nanoporous gold biosensor, comprising the following steps:

[0010] (1) Soaking the nanoporous gold in a 11-mercaptoundecanoic acid solution for 10 to 24 hours to obtain nanoporous gold with 11-mercaptoundecanoic acid linked to the surface;

[0011] (2) immersing the nanoporous gold having 11-mercaptoundecanoic acid linked to its surface obtained in step (1) in a NaOH solution for 3 to 8 minutes for proton exchange to obtain nanoporous gold having sodium 11-mercaptoundecanoate linked to its surface;

[0012] (3) immersing the nanoporous gold having sodium 11-mercaptoundecanoate linked to its surface obtained in step (2) in a nickel salt solution for 1 to 3 hours for proton exchange to obtain nanoporous gold having nickel 11-mercaptoundecanoate linked to its surface;

[0013] (4) adding a histidine-labeled CP05 polypeptide to the nanoporous gold obtained in step (3) whose surface is linked to nickel 11-mercaptoundecanoate, and linking the histidine-labeled CP05 polypeptide to the nanoporous gold through the binding of nickel ions and histidine; and obtaining the nanoporous gold biosensor after drying and washing.

[0014] Preferably, in step (1), the concentration of the 11-mercaptoundecanoic acid solution is 10 to 100 mmol / L, and the solvent is alcohol.

[0015] Preferably, in step (2), the concentration of the NaOH solution is 0.5 to 2 mmol / L.

[0016] Preferably, in step (3), the nickel ion concentration in the nickel salt solution is 0.1 to 1 mol / L.

[0017] Preferably, in step (4), the concentration of the histidine-tagged CP05 polypeptide is 0.01-3 mg / mL.

[0018] In a third aspect, the present invention further provides an application of the above-mentioned nanoporous gold biosensor, wherein the nanoporous gold biosensor is used as a SERS substrate to detect tumor cell-derived exosomes in body fluids by a SERS method.

[0019] A weakly alkaline environment is required when linking the histidine-tagged CP05 peptide to the nanoporous gold surface. The modified nanoporous gold sensor is rinsed with phosphate-buffered saline (PBS) to remove excess peptide. The modified nanoporous gold sensor is used to capture exosomes in blood. The porous gold adsorbs in body fluids for 5 to 15 minutes. After adsorption, the sensor is rinsed with PBS to remove impurities.

[0020] Beneficial effects

[0021] (1) The surface modification method of the present invention is stable and reliable, and the functional groups are firmly bonded to the porous gold and are not easily detached;

[0022] (2) The nanoporous gold sensor prepared by the present invention can accurately capture exosomes in body fluids, achieve exosome enrichment, and eliminate the interference of impurity signals in body fluids;

[0023] (3) The nanoporous gold sensor prepared by the present invention can directly detect exosomes in body fluids and analyze the source of exosomes by Raman spectroscopy without the need for any indirect markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a scanning electron microscope image of the selected nanoporous gold surface;

[0025] Figure 2 This is the infrared spectrum of MUA modified with nanoporous gold sheets;

[0026] Figure 3 This is the Raman spectrum of the CP05 peptide modified with nanoporous gold sheets;

[0027] Figure 4 The Raman spectra of the modified nanoporous gold sensor after adsorption of A549 exosomes, HCT-116 exosomes, and exosomes in normal blood;

[0028] Figure 5 The Raman spectra of the modified nanoporous gold sensor after adsorbing HCT-116 exosomes at different concentrations;

[0029] Figure 6 The Raman spectra of the modified nanoporous gold sensor after adsorption in the mixed body fluid of A549 exosomes, HCT-116 exosomes and normal blood;

[0030] Figure 7Figure a is the Raman spectrum of the CP05 polypeptide modified by the nanoporous gold sheet in Example 2; b is the Raman spectrum of the CP05 polypeptide modified by the nanoporous gold sheet in Example 3; c is the Raman spectrum of the CP05 polypeptide modified by the nanoporous gold sheet in Example 5; d is the Raman spectrum of the CP05 polypeptide modified by the nanoporous gold sheet in Example 6. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to specific embodiments, but the protection scope of the present invention is not limited to the contents described.

[0032] The present invention provides a nanoporous gold biosensor for detecting tumor cell-derived exosomes in body fluids, comprising nanoporous gold, 11-mercaptoundecanoate linked to the nanoporous gold, nickel ions linked to the carboxyl terminus of the 11-mercaptoundecanoate, and a histidine-tagged CP05 polypeptide linked via the nickel ions. The pore size of the nanoporous gold is preferably 100-300 nm, which matches the size of exosomes (100-200 nm), and can well confine the exosomes within the nanopores.

[0033] The specific preparation method mainly includes the following steps: selecting nanoporous gold with pores that match the size of exosomes (preferably pore diameters between 100 and 300 nm) to allow exosomes to enter; then, immersing the nanoporous gold in 2 ml of a 10-100 mmol / L MUA alcohol solution for 10-24 hours. This allows the terminal thiol (-SH) groups of 11-mercaptoundecanoic acid to fully adsorb and bond with the porous gold, resulting in a nanoporous gold sheet with MUA attached to its surface. The concentration of the MUA alcohol solution must be controlled within an appropriate range. Too low a concentration will result in insufficient 11-mercaptoundecanoic acid adsorption on the porous gold surface, while too high a concentration will not significantly increase the adsorption capacity.

[0034] The nanoporous gold sheet with MUA attached to its surface is then immersed in 5-10 ml of a 0.5-2 mmol / L NaOH solution for 3-8 minutes to attach sodium ions to the carboxyl termini of the MUA and create an alkaline environment. The sodium ions in the NaOH solution attach to the carboxyl termini, while the alkaline environment of the NaOH solution renders histidine negatively charged in subsequent steps. Too low a NaOH concentration will result in insufficient alkalinity, hindering subsequent histidine adsorption. Too high a concentration will result in excessive alkalinity, leading to protein denaturation.

[0035] Next, the nanoporous gold with sodium ions attached to the carboxyl termini of the MUA was immersed in 0.5–1 ml of a 0.1–1 mol / L NiSO₄ solution for 1–3 hours to perform proton exchange and attach nickel ions to the carboxyl termini of the MUA. A low NiSO₄ concentration would result in insufficient nickel ion adsorption, while a high concentration would cause excessive nickel ion precipitation on the gold surface, affecting the subsequent Raman signal.

[0036] Finally, 10 μL of His-CP05 at a concentration of 0.01 to 3 mg / mL was added to the porous gold surface. Under alkaline conditions, nickel ions and histidine are positively charged and negatively charged, respectively. The nickel ions bind to the histidine, thereby attaching the CP05 peptide to the porous gold surface. A low concentration of the histidine-tagged CP05 peptide will result in insufficient adsorption of the peptide on the porous gold surface, while a high concentration will not significantly increase adsorption.

[0037] The porous gold sheet was dried at room temperature to 55°C and then rinsed with PBS buffer to obtain the nanoporous gold biosensor. The nanoporous gold sheet sensor was ultrasonically adsorbed in human body fluids, dried, rinsed with PBS buffer, and then dried again. The source of the exosomes in the body fluids was determined by Raman spectroscopy of the porous gold sheet.

[0038] The ultrasonic adsorption time is 5 to 15 minutes, and the drying temperature is room temperature to 55°C.

[0039] A weakly alkaline environment is required when attaching the histidine-tagged CP05 peptide to the porous gold surface. The modified nanoporous gold sensor is rinsed with PBS buffer to remove excess peptide. The modified nanoporous gold sensor is then used to capture exosomes in blood. The porous gold adsorbs in body fluids for 5 to 15 minutes. After adsorption of exosomes, the sensor is rinsed with PBS buffer to remove impurities.

[0040] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.

[0041] Example 1

[0042] A nanoporous gold sheet with an average pore size of 200 nm was selected as the substrate ( Figure 1 , nanopore size is 100~300nm); the nanoporous gold was immersed in 2ml MUA alcohol solution with a concentration of 80mmol / L for 16h. Figure 2 As shown, the infrared spectra of the porous gold sheet in step (1) and the porous gold sheet in alcohol were tested, and the main characteristic band of MUA was located at Figure 2 The vertical boxes correspond to the vibration of carboxyl and alkyl chains respectively. -1 and 1411cm -1 The absorption bands at the positions are respectively attributed to the stretching vibration of C=O of the carboxyl group and the symmetric stretching vibration of -CO2-, indicating that MUA is successfully connected to the gold sheet; the porous gold sheet in step (1) is immersed in a NaOH solution with a volume of 8 ml and a concentration of 1 mmol / L for 5 min to connect sodium ions to the carboxyl end of MUA and create an alkaline environment; the porous gold sheet in step (2) is immersed in a NiSO4 solution with a volume of 0.8 ml and a concentration of 500 mmol / L for 2 h to perform proton exchange, thereby connecting nickel ions to the end of MUA; in step (4), His-CP05 with a volume of 10 μl and a concentration of 1 mg / ml is added to the surface of the porous gold sheet, and the CP05 polypeptide is connected to the porous gold through the combination of nickel ions and histidine. The porous gold is dried at 50°C and then rinsed with PBS, as shown in FIG. Figure 3 As shown in the figure, the Raman spectra of the nanoporous gold sensor before and after rinsing were tested. The signals of CP05 were clear and stable before and after rinsing, indicating that the CP05 polypeptide was successfully connected to the surface of the porous gold sheet. The obtained sensor surface was adsorbed with exosomes from human lung cancer (A549), exosomes from intestinal cancer (HCT-116) and exosomes from normal human blood, respectively. Ultrasonic adsorption was performed for 10 minutes, and then rinsed with PBS and dried at 50°C. Finally, Raman testing was performed to obtain the Raman spectrum. Figure 4 As shown, the three types of exosomes can be clearly distinguished by Raman spectroscopy.

[0043] In the above operating conditions, the concentration of exosomes was 10 8 particles / mL, which is lower than the actual exosome concentration in body fluids (10 9 particles / ml), indicating that the prepared SERS sensor is sensitive enough; after reducing the concentration of exosomes by one order of magnitude (10 7 particles / ml), and the HCT-116 exosomes were tested according to the same procedure. Figure 5 As shown, the Raman signal intensity remains basically unchanged, indicating that the surface-modified functional groups can quickly and accurately capture exosomes in body fluids and achieve exosome enrichment.

[0044] Furthermore, A549 exosomes and HCT-116 exosomes were mixed in normal human blood, and the nanoporous gold sensor was ultrasonically adsorbed in the mixed fluid for 10 minutes, then rinsed with PBS and dried at 50°C. Finally, Raman testing was performed to obtain 170 points of Raman spectrum scanning with a scanning area of 120×120μm. 2 .like Figure 6As shown, the spectra can be found to be divided into three categories, indicating that the sensor can distinguish exosome signals from different sources in complex body fluids.

[0045] Example 2

[0046] In this embodiment, the other steps are the same as those in Example 1. The concentration of the MUA alcohol solution in step (1) is changed to 10 mmol / L, and the adsorption time is changed to 10 h; the volume of the sodium hydroxide solution in step (2) is changed to 5 ml, the concentration is changed to 0.5 mmol / L, and the adsorption time is changed to 3 min; the volume of the NiSO4 solution in step (3) is changed to 0.5 ml, the concentration is changed to 100 mmol / L, and the adsorption time is changed to 1 h; the concentration of His-CP05 in step (4) is changed to 0.01 mg / ml; and the ultrasonic adsorption time is changed to 5 min. Figure 7 As shown in Figure a, the Raman spectrum shows that the CP05 polypeptide is successfully attached to the porous gold surface, and the intensity is slightly lower than that of Example 1. The exosome capture and detection results are the same as those of Example 1.

[0047] Example 3

[0048] In this embodiment, the other steps are the same as those in Example 1. The concentration of the MUA alcohol solution in step (1) is changed to 30 mmol / L, and the adsorption time is changed to 12 h; the volume of the sodium hydroxide solution in step (2) is changed to 5 ml, the concentration is changed to 0.8 mmol / L, and the adsorption time is changed to 3 min; the volume of the NiSO4 solution in step (3) is changed to 0.5 ml, the concentration is changed to 200 mmol / L, and the adsorption time is changed to 1.5 h; the concentration of His-CP05 in step (4) is changed to 0.05 mg / ml; and the ultrasonic adsorption time is changed to 5 min. Figure 7 As shown in Figure b, the Raman spectrum shows that the CP05 polypeptide is successfully attached to the porous gold surface, and the intensity is slightly lower than that of Example 1. The exosome capture and detection results are the same as those of Example 1.

[0049] Example 4

[0050] In this example, the other steps were the same as in Example 3. The concentration of the MUA alcohol solution was changed to 50 mmol / L, the concentration of the sodium hydroxide solution was changed to 1 mmol / L, and the concentration of the NiSO4 solution was changed to 300 mmol / L. The results of CP05 polypeptide modification on the nanoporous gold surface and the exosome capture and detection results were the same as in Example 3.

[0051] Example 5

[0052] In this example, the other steps are the same as those in Example 1. The concentration of His-CP05 in step (4) is changed to 2 mg / ml; all drying temperatures are changed to 55°C. Figure 7As shown in Figure c, the results of CP05 polypeptide modification on the nanoporous gold surface are the same as those in Example 1, with a slightly improved intensity. The exosome capture and detection results are also the same as those in Example 1.

[0053] Example 6

[0054] In this embodiment, the other steps are the same as those in Example 1. The concentration of the MUA alcohol solution in step (1) is changed to 100 mmol / L, and the adsorption time is changed to 24 h; the volume of the sodium hydroxide solution in step (2) is changed to 10 ml, the concentration is changed to 2 mmol / L, and the adsorption time is changed to 8 min; the volume of the NiSO4 solution in step (3) is changed to 1 ml, the concentration is changed to 1 mol / L, and the adsorption time is changed to 3 h; the concentration of His-CP05 in step (4) is changed to 3 mg / ml; and the ultrasonic adsorption time is changed to 15 min. Figure 7 As shown in Figure d, the Raman spectrum shows that the CP05 polypeptide is successfully attached to the porous gold surface, and the intensity is improved compared with Example 1. The exosome capture and detection results are the same as in Example 1.

Claims

1. A nanoporous gold biosensor, characterized in that: The nanoporous gold biosensor comprises nanoporous gold, 11-mercaptoundecanoic acid linked to the nanoporous gold, nickel ions connected to the carboxyl end of the 11-mercaptoundecanoic acid, and a histidine-tagged CP05 polypeptide linked via the nickel ions.

2. The nanoporous gold biosensor according to claim 1, characterized in that The pore size of the nanoporous gold is 100-300 nm.

3. A method for preparing the nanoporous gold biosensor according to claim 1 or 2, characterized in that: The following steps are involved: (1) Soaking the nanoporous gold in a 11-mercaptoundecanoic acid solution for 10 to 24 hours to obtain nanoporous gold with 11-mercaptoundecanoic acid linked to the surface; (2) immersing the nanoporous gold having 11-mercaptoundecanoic acid linked to its surface obtained in step (1) in a NaOH solution for 3 to 8 minutes for proton exchange to obtain nanoporous gold having sodium 11-mercaptoundecanoate linked to its surface; (3) immersing the nanoporous gold having sodium 11-mercaptoundecanoate linked to its surface obtained in step (2) in a nickel salt solution for 1 to 3 hours for proton exchange to obtain nanoporous gold having nickel 11-mercaptoundecanoate linked to its surface; (4) Adding the histidine-labeled CP05 polypeptide to the nanoporous gold obtained in step (3) whose surface is linked to nickel 11-mercaptoundecanoate, and linking the histidine-labeled CP05 polypeptide to the nanoporous gold through the binding of nickel ions and histidine; and obtaining the nanoporous gold biosensor after drying and washing.

4. The preparation method according to claim 3, characterized in that In the step (1), the concentration of the 11-mercaptoundecanoic acid solution is 10 to 100 mmol / L, and the solvent is alcohol.

5. The preparation method according to claim 3 or 4, characterized in that In step (2), the concentration of the NaOH solution is 0.5 to 2 mmol / L.

6. The preparation method according to any one of claims 3 to 5, characterized in that In the step (3), the nickel ion concentration in the nickel salt solution is 0.1 to 1 mol / L.

7. The preparation method according to any one of claims 3 to 6, characterized in that In step (4), the concentration of the histidine-tagged CP05 polypeptide is 0.01 to 3 mg / mL.

8. Use of the nanoporous gold biosensor according to claim 1 or 2, characterized in that: The nanoporous gold biosensor is used as a surface enhanced Raman spectroscopy (SERS) substrate to detect tumor cell-derived exosomes in body fluids using the SERS method.

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