Detection probe for detecting pancreatic cancer exosome and application thereof

Abstract

The invention discloses a detection probe for detecting pancreatic cancer exosomes and application of the detection probe, and belongs to the technical field of exosome detection.The detection probe takes silver nanoparticles as a framework and loads Raman reporter molecules, and the Raman reporter molecules are combined with exosome surface protein antibodies; the Raman reporter molecule contains 4-mercaptobenzoic acid; the exosome surface protein antibody comprises a GPC-1 (glypican-1) antibody. The detection probe can complete exosome detection within 20 minutes, pretreatment is not needed, the detection process is convenient and rapid, and the detection probe is suitable for instant detection of clinical samples. Moreover, the lower detection limit is as low as 1 particle / mu L, rapid and sensitive detection of the pancreatic cancer exosome can be realized, and the kit has important significance on clinical diagnosis and research of pancreatic cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of exosome detection, and in particular to a detection probe for detecting pancreatic cancer exosomes and an application thereof. Background Art

[0002] Pancreatic cancer remains one of the most deadly malignant tumors worldwide. According to statistics, the mortality rate of pancreatic cancer patients is as high as over 80%, and the total number of cases is increasing year by year. Due to its relative concealment, lack of specific biomarkers, and low 5-year survival rate, early diagnosis and metastasis monitoring of pancreatic cancer are difficult, clinical diagnosis and treatment are extremely challenging, and the social cost is also quite high. Although traditional biomarkers carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) have improved the diagnostic sensitivity of pancreatic cancer to a certain extent, CA19-9 is also highly expressed in benign pancreatic diseases, and carcinoembryonic antigen is generally expressed in gastrointestinal tumors, and has low specificity in the diagnosis of pancreatic cancer. Conventional testing is difficult to reflect biological properties such as tumor heterogeneity, microenvironment, and activity, and has limited value for the early diagnosis of pancreatic cancer. Therefore, establishing new methods to improve the specificity and sensitivity of pancreatic cancer diagnosis and prognostic evaluation is the key to increasing the early diagnosis rate of pancreatic cancer, prolonging the survival time of pancreatic cancer patients, and improving patient prognosis.

[0003] Existing studies have verified through proteomic analysis that exosomes can identify specific cancer types and distinguish tumor sources. Among them, glypican-1 (GPC-1) is a membrane-anchored protein on pancreatic cancer exosomes. GPC-1 is highly expressed in serum exosomes of pancreatic cancer patients, and its diagnostic specificity and sensitivity can be as high as 100%, and it can distinguish normal people, pancreatic cancer and chronic pancreatitis patients at an early stage. It has obvious value in the survival, prognosis and diagnosis and treatment of pancreatic cancer. The role of exosome surface protein GPC-1 in pancreatic cancer has received widespread attention. Therefore, GPC-1 can be used as a detection target for early diagnosis and treatment of pancreatic cancer and prognosis evaluation, which has important clinical significance and research value.

[0004] Surface-enhanced Raman scattering (SERS) spectroscopy has the advantages of ultra-sensitivity, rapidity and non-destructiveness. It can multiply and amplify the inherent molecular fingerprint information to achieve sensitive and specific detection of biological molecules, pathogens, cancer cells, etc., and has become a rapidly developing field of biological analysis. However, SERS still faces problems such as poor reliability and repeatability of detection results in practical applications. SERS substrate plays a decisive role in SERS enhancement. The selection of substrate materials, preparation methods, and the size, shape, size, uniformity, etc. of the finished product will affect the SERS enhancement effect. Therefore, the reasonable design and preparation of SERS substrates are the key to affecting the reliability and repeatability of detection results. In order to achieve SERS detection with high selectivity, strong specificity and good stability, the preparation of stable and efficient SERS substrate materials and their functionalization have been important research directions in the SERS field for many years.

[0005] The Raman signal cross-sectional area of ​​general material molecules is small and difficult to detect, but the Raman signal cross-sectional area of ​​some material molecules is relatively large and can be more easily detected, so they are mostly selected as signal marker molecules, namely Raman reporter molecules, to improve the sensitivity of detection. Raman reporter molecules are mainly divided into silent zone Raman reporter molecules and non-silent zone Raman reporter molecules according to the distribution of their characteristic peaks. Currently, common Raman reporter molecules include Cy3, ROX, R6G, 4-MBA, etc. At present, the detection objects are generally captured according to methods such as antigen-antibody reaction and nucleic acid aptamers, combined with stable Raman reporter molecules to improve the accuracy of detection. Further improve the selectivity of SERS sensors to manufacture new biosensors. Label-based SERS detection is highly sensitive and repeatable; therefore, different Raman reporter molecules can be used to achieve multi-species detection. Compared with label-free methods, label-based strategies have high sensitivity and repeatability. However, whether pancreatic cancer exosomes can be specifically detected based on SERS sensors, and how to construct SERS sensors that can be efficiently, sensitively and specifically detected, there are few related research reports on existing technologies. Summary of the invention

[0006] The purpose of the present invention is to provide a detection probe for detecting pancreatic cancer exosomes and its application to solve the problems existing in the above-mentioned prior art. The detection probe can realize rapid and sensitive detection of pancreatic cancer exosomes, which is of great significance to the diagnosis and research of clinical pancreatic cancer.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a detection probe for detecting exosomes. The detection probe uses silver nanoparticles as a skeleton and carries Raman reporter molecules. The Raman reporter molecules bind to exosome surface protein antibodies.

[0009] Furthermore, the Raman reporter molecule comprises 4-mercaptobenzoic acid; and the exosome surface protein antibody comprises a GPC-1 antibody.

[0010] Furthermore, the 4-mercaptobenzoic acid is loaded on the silver nanoparticles via the thiol end and covalently bound to the amino group of the GPC-1 antibody via the carboxyl end.

[0011] The present invention also provides a method for preparing the above detection probe, comprising the following steps:

[0012] Sodium citrate, silver nitrate and sodium chloride solution are mixed and stirred to prepare a premixed suspension; the premixed suspension is added to a mixed solution prepared by stirring and heating water and ascorbic acid, and the stirring and heating are continued to obtain a silver nano-colloid solution;

[0013] The Raman reporter molecule is mixed and incubated with the silver nano colloidal solution, and an activator is added to the mixed solution after the mixed incubation to activate the mixture, so as to obtain a silver nano Raman reporter molecule coupling mixture;

[0014] The detection probe is obtained by adding an exosome surface protein antibody to the silver nano-Raman reporter molecule coupling mixture for mixed reaction.

[0015] Furthermore, the volume ratio of the sodium citrate solution, silver nitrate solution and sodium chloride solution is 20:5:4; wherein the concentrations of the sodium citrate solution and the silver nitrate solution are both 1wt%, and the concentration of the sodium chloride solution is 20mM.

[0016] Furthermore, the volume ratio of the ultrapure water to the ascorbic acid is (400-500):1; wherein the concentration of the ascorbic acid is 0.1M.

[0017] Furthermore, the mixing volume ratio of the Raman reporter molecule and the silver nanocolloid solution is 1:10; the activator is a mixed solution prepared by dissolving 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in 2-(N-morpholine)ethanesulfonic acid.

[0018] Furthermore, the added amount of the mixed solution and the activator is 1:1, and the activation condition is incubation at 37° C. for 30 minutes.

[0019] The present invention also provides an application of the above detection probe in preparing a product for detecting pancreatic cancer exosomes.

[0020] The present invention discloses the following technical effects:

[0021] The present invention prepares a detection probe for detecting pancreatic cancer exosomes. The detection probe uses sodium citrate and silver nitrate as raw materials, synthesizes a silver nanocolloid solution by a reduction method, and forms a stable covalent Ag-S bond with a silver nanomaterial by activating the thiol group of the Raman reporter molecule 4-MBA, completing the Raman reporter molecule binding of the nanomaterial, and then covalently binds the activated Raman reporter molecule 4-MBA carboxyl group to the GPC-1 amino group to prepare a rapid and sensitive detection probe for pancreatic cancer exosomes that is coupled with a silver nanomaterial 4-MBA reporter molecule and targets GPC-1. The detection probe completes the detection of exosomes within 20 minutes without pretreatment, and the detection process is convenient and fast, which is suitable for real-time detection of clinical samples. In addition, the detection limit is as low as 1 particle / μL, which can achieve rapid and sensitive detection of pancreatic cancer exosomes.

[0022] In summary, the detection of exosomes by the present invention is significantly superior to conventional detection methods in terms of speed and efficiency, and the operation process is simple. The 4-MBA reporter molecule in the detection probe is combined with the colloidal solution formed by silver nanoparticles to ensure the enhancement of the substrate Raman signal, and the Raman reporter molecule is used to achieve a relatively stable detection of the Raman signal. At the same time, the surface protein GPC-1 used has a high specificity for pancreatic cancer exosomes, and the combination of Raman reporter molecules significantly improves the sensitivity of detection. It can be seen that the detection probe is of great significance for the diagnosis and research of clinical pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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.

[0024] Figure 1 It is a schematic diagram of the detection of the present invention;

[0025] Figure 2 It is the UV absorption peak diagram of Ag and AgNPs-4-MBA;

[0026] Figure 3 The Raman spectra detection results of silver nanoparticles, silver nano-Raman tags, and silver nano-detection probes;

[0027] Figure 4 Electron micrograph of AgNPs-4-MBA-antiGPC-1@exosomes;

[0028] Figure 5 The sensitivity test results of AgNPs-4-MBA-antiGPC-1 detection probe;

[0029] Figure 6 The specific detection results of AgNPs-4-MBA-antiGPC-1 detection probe for pancreatic cancer exosomes;

[0030] Figure 7 The detection results of serum samples from pancreatic cancer patients using AgNPs-4-MBA-antiGPC-1 detection probe. DETAILED DESCRIPTION

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] Main reagent sources:

[0037] Antibody diluent, 2-(N-morpholinoethanesulphonic acid) (MES) buffer, and bovine serum albumin (BSA) were purchased from Shanghai MacLean; phosphate buffer saline (PBS) was purchased from Wuhan Sewell; 1-ethyl-(3-dimethylaminopropyl) carbodiimide (C8H 17 N3, EDC), N-hydroxysuccinimide (C4H5NO3, NHS) were purchased from Thermo Fisher Scientific (China), GPC-1 antibody was purchased from Abcam (Shanghai) Trading Co., Ltd., sodium citrate (Na3C6H5O7), silver nitrate (AgNO3), sodium chloride (NaCl), ascorbic acid (C6H8O6), and 4-mercaptobenzoic acid (4-Mercaptobenzoic acid, 4-MBA) were purchased from Shanghai Aladdin Biochemical Technology.

[0038] The other reagents, unless otherwise indicated, are conventional reagents in the art and can also be purchased commercially.

[0039] The invention develops a rapid and sensitive detection method for pancreatic cancer exosomes by coupling silver nanomaterials with 4-MBA reporter molecules and taking GPC-1 as a target. The method comprises the following steps: firstly, sodium citrate and silver nitrate are used as raw materials, a silver nanocolloid solution is synthesized by a reduction method, silver nanoparticles are coupled with Raman reporter molecules 4-MBA, a Raman label AgNPs-4-MBA is synthesized, and the hydroxyl group of the exosome surface protein GPC-1 antibody is covalently bonded to the synthesized AgNPs-4-MBA carboxyl group to form an AgNPs-4-MBA-antiGPC-1 complex; the AgNPs-4-MBA-antiGPC-1 complex specifically recognizes the pancreatic cancer exosome surface protein GPC-1 through the surface-modified GPC-1 antibody, thereby completing the specific recognition and sensitive detection of pancreatic cancer GPC-1 positive specimens. Figure 1 This is a schematic diagram of the detection principle of the present invention. The specific research is as follows:

[0040] Example 1 Preparation and characterization of silver nanoparticle detection probes

[0041] (1) Preparation of silver nanoparticle (AgNPs) colloidal solution

[0042] 1 mL of 1 wt% sodium citrate, 0.25 mL of 1 wt% silver nitrate and 0.2 mL of 20 mM sodium chloride solution were mixed and stirred on a magnetic stirrer for 5 minutes to form a uniform premixed suspension and then set aside for use. 47.5 mL of ultrapure water and 100 μL of 0.1 M ascorbic acid were heated to boiling under strong stirring, and the premixed suspension was added thereto after 1 minute, and the mixture was stirred and heated in the dark for 1 hour to obtain a light yellow stable silver nanoparticle (AgNPs) colloidal solution.

[0043] (2) Preparation of silver nano-Raman reporter molecule coupling mixture Ag-4-MBA

[0044] Take 100 μL of 1 mM 4-MBA and add it to 1 mL of silver nanocolloid and incubate it overnight at room temperature to activate the thiol end of the Raman reporter molecule 4-MBA, so that the activated thiol group forms a stable covalent Ag-S bond with the silver nanomaterial, completing the binding of the nanomaterial and the Raman reporter molecule. After incubation overnight, the solution can be seen to change from light yellow to gray. Centrifuge at 5000 rpm for 4 minutes to remove excess unbound Raman reporter molecule 4-MBA, and then use 1 mL of 2-(N-morpholinoline)ethanesulfonic acid (MES, pH = 4.7) to dissolve the mixed solution prepared by EDC and NHS (the concentrations of EDC and NHS are both 1 mM), mix the mixed solution with equal volumes of silver nano-Raman reporter molecule coupling mixture, incubate on a 37°C metal bath for 30 minutes to activate the carboxyl end of the Raman reporter molecule, and obtain the silver nano-Raman reporter molecule coupling mixture Ag-4-MBA. The silver nano-Raman reporter molecule coupling mixture was measured using a UV-visible spectrophotometer, and the results are as follows: Figure 2 As shown, Ag-4-MBA has a single absorption peak that shifts to the right (from 423 nm to 467 nm), which means that the coupling effect of Ag-4-MBA is good and stable.

[0045] (3) Preparation of Ag-4-MBA-antiGPC-1 detection probe

[0046] Add excess GPC-1 antibody to Ag-4-MBA and react in a metal bath at 37°C for 2 hours to covalently bind the activated Raman reporter molecule 4-MBA carboxyl group to the GPC-1 amino group to prepare a silver nanoparticle detection probe with enhanced Raman signal. Centrifuge at 5000rpm for 4 minutes to remove excess antibody, and suspend the precipitate in PBS containing 1% BSA to obtain AgNPs-4-MBA-antiGPC-1 detection probe.

[0047] Detection of Raman spectral intensity of silver nanoparticle detection probe:

[0048] The silicon wafer was pre-soaked in acetone for 24 hours to reduce interference, rinsed three times with flowing ultrapure water and dried in nitrogen. Silver nanoparticles, silver nano-Raman labels, and silver nano-detection probes were added to the pre-treated silicon wafer and measured on a Raman spectrometer. The measurement results are shown in Figure 3 , with a Raman shift of 1081 cm -1 The characteristic peak of 4-MBA can be seen, and the results show that the silver nano-Raman label has an obvious Raman signal enhancement effect, and after modifying the GPC-1 antibody, it can still maintain a high spectral intensity.

[0049] Example 2 Sensitivity and specificity analysis of silver nanoparticle detection probes

[0050] The pancreatic cancer exosome samples were serially diluted to 1×10 9 , 1×10 8 , 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 After adding an equal amount of AgNPs-4-MBA-antiGPC-1 detection probe prepared in Example 1, the mixture was incubated in a 37°C constant temperature metal bath for 5-20 min, centrifuged at 5000 rpm to remove unbound exosomes, and the precipitate was resuspended in 50 μL PBS buffer. The precipitate was examined by electron microscopy, and the results were as follows: Figure 4 , the results showed that the AgNPs-4-MBA-antiGPC-1 detection probe specifically recognized GPC-1 positive exosomes. Figure 5 The sensitivity test results of the AgNPs-4-MBA-antiGPC-1 detection probe show that the detection limit of the detection probe is as low as 1×10 3 particles / mL, which can achieve sensitive detection of exosomes.

[0051] Equal amounts of AgNPs-4-MBA-antiGPC-1 detection probes were added to pancreatic cancer cell panc-1 exosome samples and liver cancer cell huh7 exosome samples, respectively, and the results were measured by Raman spectrometer to evaluate the specificity of the detection system for pancreatic cancer exosome detection. Figure 6 ,The results showed that the AgNPs-4-MBA-antiGPC-1 detection probe specifically detected pancreatic cancer exosomes.

[0052] Example 3: Detection of serum samples of pancreatic disease patients using silver nanoparticle detection probes

[0053] Blood samples from pancreatic cancer patients and healthy controls were collected separately, and the serum was transferred to a sterile enzyme-free EP tube with a pipette. 50 μL of the prepared CD63-modified magnetic MOFs beads were added to 200 μL of serum sample, incubated in a 37°C constant temperature metal bath for 5 minutes, and centrifuged at 13,000 rpm to discard the supernatant. After resuspending with PBS buffer, 200 μL AgNPs-4-MBA-antiGPC-1 detection probe was added, and the mixture was incubated in a 37°C constant temperature metal bath for 20 minutes and centrifuged at 5,000 rpm. The precipitate was resuspended with 50 μL PBS buffer and dropped onto the pretreated silicon wafer for measurement on a Raman spectrometer. Detection results Figure 7 The results showed that serum samples from pancreatic cancer patients had a -1 The Raman shift intensity at 1081 cm was significantly increased, with a mean of 14754 ± 3987 (mean ± SEM). -1 The mean Raman shift intensity at 6724±276 (mean±SEM). When the SERS intensity is higher than 7315, the sensitivity of pancreatic cancer diagnosis can reach 100.0% and the specificity can reach 94.44%.

[0054] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A detection probe for detecting pancreatic cancer exosomes, characterized in that: The detection probe uses silver nanoparticles as a skeleton and carries Raman reporter molecules, which are combined with exosome surface protein antibodies.

2. The detection probe according to claim 1, characterized in that The Raman reporter molecule comprises 4-mercaptobenzoic acid; and the exosome surface protein antibody comprises a GPC-1 antibody.

3. The detection probe according to claim 2, characterized in that: The 4-mercaptobenzoic acid is loaded on the silver nanoparticles via the thiol end and covalently combined with the amino group of the GPC-1 antibody via the carboxyl end.

4. A method for preparing a detection probe according to any one of claims 1 to 3, characterized in that: The following steps are involved: Sodium citrate, silver nitrate and sodium chloride solution are mixed and stirred to prepare a premixed suspension; the premixed suspension is added to a mixed solution prepared by stirring and heating water and ascorbic acid, and the stirring and heating are continued to obtain a silver nano-colloid solution; The Raman reporter molecule is mixed and incubated with the silver nano colloidal solution, and an activator is added to the mixed solution after the mixed incubation to activate the mixture, so as to obtain a silver nano Raman reporter molecule coupling mixture; The detection probe is obtained by adding an exosome surface protein antibody to the silver nano-Raman reporter molecule coupling mixture for mixed reaction.

5. The preparation method according to claim 4, characterized in that: The volume ratio of the sodium citrate solution, silver nitrate solution and sodium chloride solution is 20:5:4; wherein the concentrations of the sodium citrate solution and the silver nitrate solution are both 1wt%, and the concentration of the sodium chloride solution is 20mM.

6. The preparation method according to claim 4, characterized in that: The volume ratio of the ultrapure water to the ascorbic acid is (400-500):1; wherein the concentration of the ascorbic acid is 0.1M.

7. The preparation method according to claim 4, characterized in that: The mixing volume ratio of the Raman reporter molecule to the silver nano-colloid solution is 1:10; the activator is a mixed solution prepared by dissolving 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in 2-(N-morpholine)ethanesulfonic acid.

8. The preparation method according to claim 4, characterized in that: The volume ratio of the mixed solution to the activator is 1:1, and the activation condition is incubation at 37° C. for 30 min.

9. Use of the detection probe according to any one of claims 1 to 3 in the preparation of a product for detecting pancreatic cancer exosomes.

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