Black phosphorus / manganese ion composite nanosensor and its preparation method and application

Abstract

A black phosphorus / manganese ion composite nanosensor is prepared by ultrasonically exfoliating a 100nm-5μm black phosphorus nanosheet suspension with an ultrasonic cell crusher in an ice bath to obtain a 50-100nm black phosphorus nanosheet dispersion; and sequentially adding a divalent manganese ion solution, a black phosphorus nanosheet dispersion, and a fluorescent group-labeled nucleic acid aptamer solution or a nucleic acid probe solution into a biological buffer solution, followed by mixing to fully disperse the black phosphorus nanosheets in a liquid medium comprising the biological buffer solution, the divalent manganese ion solution, the fluorescent group-labeled nucleic acid aptamer solution, and / or the fluorescent group-labeled nucleic acid probe solution. The prepared liquid nanosensor system is a black phosphorus / manganese ion composite nanosensor, which can be applied to a kit and a detection system for detecting cancer cell markers, and can exert an effect of quantitatively detecting colorectal cancer exosomes.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a black phosphorus / manganese ion composite nanosensor and a preparation method and application thereof. Background Art

[0002] Colorectal cancer is a common digestive tract malignancy that occurs in the colon and rectum. It has a high incidence and mortality rate worldwide, and seriously threatens human life and health. The difficulty in preventing and treating colorectal cancer lies in the lack of specific clinical symptoms in its early stages. Most patients are already in the middle or late stages when they seek medical treatment. The recurrence rate after treatment is high and the prognosis is poor. [1] Therefore, it is particularly important to develop new technologies for effective, accurate and sensitive colorectal cancer screening and diagnosis.

[0003] Although colonoscopy is still the "gold standard" for colorectal cancer screening and diagnosis [2] However, colonoscopy is an invasive examination method, which will cause great pain to patients and cannot obtain an accurate diagnosis before the formation of tumors visible to the naked eye in the early stage. At present, the conventional immunological detection methods used for colorectal cancer screening and diagnosis are limited in clinical application due to their low specificity and sensitivity. Therefore, the development of an effective and reliable new technology for the clinical diagnosis of colorectal cancer has been a long-term goal of researchers.

[0004] At present, a large number of studies and practices have shown that circulating microRNA (miRNA) in serum is closely related to the occurrence, staging, metastasis and recurrence of various tumors, and can be used as a biomarker for tumor diagnosis. [3,4] Circulating miRNAs in serum are stably present by binding to lipoproteins or being enriched in extracellular vesicles (EVs) such as exosomes. [5,6] Compared with lipoprotein binding, miRNA enriched in exosomes has stronger biological activity [7,8] Because the nucleic acid substances in serum come from a wide range of sources, and the miRNA contained in exosomes can be confirmed to be secreted by living cells in the tissue and has special functionality. The latest research shows that there are specific miRNAs expressed in the serum exosomes of colorectal cancer patients, which can be used for colorectal cancer screening, early diagnosis, staging, typing, monitoring recurrence and prognosis prediction [9-11] .

[0005] miR-21 is a miRNA with an autonomous transcription unit located in the FRA17B fragile region of chromosome 17q23.2

[12] Through traditional Northern blot, microarray and RT-PCR and other conventional detection methods, it was found that miR-21 was upregulated in a variety of solid tumors such as colorectal cancer, lung cancer, pancreatic cancer, prostate cancer, gastric cancer and breast cancer. This suggests that miR-21 plays an important role in the occurrence and development of a variety of tumors and can be used for the diagnosis, treatment and prognosis of tumors, which has important clinical significance; but at the same time, miR-21, as a proto-oncogene, is closely related to the occurrence and development of a variety of tumors. However, due to its lack of specificity, it is difficult to accurately and effectively diagnose colorectal cancer at an early stage by simply measuring the miR-21 indicator.

[0006] Hui Wang

[13] et al. used graphene oxide-DNA aptamers and DNase I enzyme to assist in fluorescence signal amplification to detect colorectal cancer exosomes. Protein CD63 is a signature protein present on all exosomes, and protein EpCAM is a unique protein on exosomes derived from colorectal cancer. CD63 and EpCAM aptamers were labeled with different fluorescent groups and adsorbed to the surface of graphene oxide through π-π bonds. Due to fluorescence resonance energy transfer, fluorescence quenching, in the presence of colon cancer exosomes, CD63 and EpCAM aptamers on the graphene oxide surface can fold into a special spatial structure to recognize the corresponding proteins of colon cancer exosomes, form a complex and detach from the graphene oxide surface, and the quenched fluorescence is restored. At the same time, the DNA aptamer bound to the exosome is hydrolyzed with DNase I enzyme, so that the exosome can bind to more DNA aptamers, achieving the purpose of fluorescence signal amplification, and can detect trace clinical samples.

[0007] However, this method has the following drawbacks:

[0008] (1) DNA aptamers are adsorbed to the graphene oxide surface through π-π bonds. This binding mode is not strong and is easily interfered by nonspecific proteins in complex biological samples, resulting in high background signals and false positives.

[0009] (2) This technology only uses the indicator that the levels of EpCAM and CD63 on exosomes derived from colon cancer serum are higher than those in the healthy control group. It is difficult to accurately diagnose colorectal cancer and the technical indicators are single.

[0010] References:

[0011] [1] Xing Jie, Li Peng, Zhang Shutian. Current status of colorectal cancer prevention and treatment in China[J]. Current status of colorectal cancer prevention and treatment in China, 2013, 2(6): 288–291.

[0012] [2] Chen X, Ba Y, Ma L, Cai X, Yin Y, Wang K, Guo J, Zhang Y, Chen J,Guo X, Li Q, Li X, Wang W, Zhang Y, Wang J, Jiang X, Xiang Y, Xu C, Zheng P,Zhang J, Li R, Zhang H, Shang X, Gong T, Ning G, Wang J, Zen K, Zhang J, Zhang CY. Characterization of microRNAs in serum: a novel class ofbiomarkers for diagnosis of cancer and other diseases[J]. Cell Res., 2008, 18(10):997–1006.

[0013] [3] Lee YS, Dutta A. MicroRNAs in cancer[J]. Annu. Rev. Fr. Pathol.,2009, 4:199–227.

[0014] [4] Arroyo JD, Chevillet JR, Kroh EM, Ruf IK, Pritchard CC,Gibson DF, Mitchell PS, Bennett CF, Pogosova-Agadjanyan EL, Stirewalt DL, Tait JF, Tewari M. Argonaute2 complexes carry a population of circulatingmicroRNAs independent of vesicles in human plasma[J]. PNAS, 2011, 108(12):5003–5008.

[0015] [5] Ortiz-Quintero B. Cell-free microRNAs in blood and other body fluids, as cancer biomarkers[J]. Cell Proli., 2016, 49(3):281–303.

[0016] [6] Cheng L, Sharples R A, Scicluna B J, Hill A F. Exosomes provide aprotective and enriched source of miRNA for biomarker profiling compared tointracellular and cell-free blood[J]. J. Extracell. Vesicles, 2014, 3: 1–14。

[0017] [7]Matsumura T, Sugimachi K, Iinuma H, et al. Exosomal microRNA inserum is a novel biomarker of recurrence in human colorectal cancer[J]. Br.J. Cancer., 2015, 113(2): 275–281。

[0018] [8] Tovar-Camargo O A, Toden S, Goel A. Exosomal microRNA Biomarkers:Emerging Frontiers in Colorectal and Other Human Cancers[J]. Expert Rev. Mol.Diagn., 2016, 16(5): 553–567。

[0019] [9] Wang J, Yan F, Zhao Q, Zhan F, Wang R, Wang L, Zhang Y, Huang X.Circulating exosomal miR-125a-3p as a novel biomarker for early-stage coloncancer[J]. Sci. Rep., 2017, 7(1): 4150。

[0020]

[10] Matsumura T, Sugimachi K, Iinuma H, et al. Exosomal microRNA inserum is a novel biomarker of recurrence in human colorectal cancer[J]. Br.J. Cancer., 2015, 113(2): 275–281。

[0021]

[11] Tovar-Camargo O A, Toden S, Goel A. Exosomal microRNABiomarkers: Emerging Frontiers in Colorectal and Other Human Cancers[J].Expert Rev. Mol. Diagn., 2016, 16(5): 553–567。

[0022]

[12] Fujita S,Ito T,Mizutani T,et al. miR_21 Gene exp ressiontriggered by AP_1is sustained through a double_negative feedback mechanism[J].J Mol Biol,2008,378(3):492-504。

[0023]

[13] Hui W, Hui C, Zhipeng H, Tengda Li, Anmei D, Jilie K. DNase Ienzyme-aided fluorescence signal amplification based on grapheneoxide-DNAaptamer interactions for colorectal cancer exosome detection[J].Talanta,2018, 184:219-226。 Summary of the Invention

[0024] The purpose of the present invention is to provide a black phosphorus / manganese ion composite nanosensor, which can strongly bind to nucleic acids and effectively increase the stability of black phosphorus nanosheets after modifying black phosphorus nanosheets with manganese ions; and when the black phosphorus / manganese ion composite nanosensor is prepared with miR-21 recognition probe as nucleic acid probe and CD63 aptamer and EpCAM aptamer as nucleic acid aptamer, it can play an effect of quantitative detection of colorectal cancer exosomes, and increase the specificity of colorectal cancer diagnosis by dual detection of colorectal cancer exosome surface proteins and internal miRNA.

[0025] To achieve the above purpose, the present invention is implemented by the following technical means:

[0026] A black phosphorus / manganese ion composite nanosensor is a liquid nanosensor system prepared by fully dispersing black phosphorus nanosheets with an average sheet diameter of 50-100 nm in a biological buffer, a divalent manganese ion solution, a nucleic acid aptamer solution labeled with a fluorescent group and / or a nucleic acid probe solution as a liquid medium.

[0027] Furthermore, in the black phosphorus / manganese ion composite nanosensor, at a non-first operating temperature, the base of the nucleic acid probe labeled with a fluorescent group is bound to the black phosphorus surface through the coordination of the divalent manganese ion, so that the fluorescence is quenched;

[0028] After being incubated with the exosomes to be tested at a first working temperature, the nucleic acid probe binds to the tumor-related miRNA inside the exosomes to be tested, so that the nucleic acid probe is detached from the black phosphorus surface and the quenched fluorescence is restored;

[0029] At a temperature other than the second operating temperature, the base of the nucleic acid aptamer labeled with the fluorescent group is bound to the black phosphorus surface through the coordination of the divalent manganese ion, so that the fluorescence is quenched;

[0030] After being incubated with the exosomes to be tested at the second working temperature, the nucleic acid aptamer binds to the specific protein on the surface of the exosomes to be tested, so that the nucleic acid aptamer is detached from the black phosphorus surface and the quenched fluorescence is restored.

[0031] Furthermore, the biological buffer can be selected from one of 4-hydroxyethylpiperazineethanesulfonic acid buffer (HEPES buffer) or tris(hydroxymethylaminomethane) hydrochloride buffer solution (Tris-HCL buffer); the divalent manganese ion solution can be selected from any one of manganese chloride solution, manganese phosphate solution, and manganese carbonate solution; the black phosphorus nanosheets are added in the form of a black phosphorus nanosheet dispersion and dispersed in the liquid medium.

[0032] Furthermore, the biological buffer is 4-hydroxyethylpiperazineethanesulfonic acid buffer, the divalent manganese ion solution is manganese chloride solution, and the mass ratio of 4-hydroxyethylpiperazineethanesulfonic acid, black phosphorus nanosheets, and manganese chloride is (10-50):1:(20-50).

[0033] The black phosphorus / manganese ion composite nanosensor provided in this scheme modifies naked black phosphorus by divalent manganese ion coordination. After the modification of black phosphorus, it can strongly bind to the base of nucleic acid aptamer or nucleic acid probe through metal cation-π bond, which increases the stability of black phosphorus on the one hand, and increases the specificity of the nanosensor constructed based on black phosphorus on the other hand, and can resist the interference of nonspecific substrates.

[0034] In addition, this scheme can control the working temperature suitable for nucleic acid aptamers and / or nucleic acid probes, so that the nucleic acid aptamers and / or nucleic acid probes labeled with fluorescent groups are detached from the black phosphorus surface, bind to specific proteins on the surface of exosomes and / or tumor-related miRNAs inside, and restore the quenched fluorescence. The difference in the quenching and recovery process of fluorescence is statistically significant, which proves that the black phosphorus / manganese ion composite nanosensor has good specificity and is suitable as a tool for specific detection of cancer cells. It can be applied to kits and detection systems for diagnosing and detecting cancer cell markers to achieve exosome-specific protein detection and internal tumor-related miRNA detection.

[0035] To achieve the above-mentioned purpose, further, the nucleic acid probe labeled with a fluorescent group is a miR-21 recognition probe labeled with a first fluorescent group, the tumor-related miRNA is miR-21, and the first working temperature is 37°C; the nucleic acid aptamer labeled with a fluorescent group is a CD63 aptamer labeled with a second fluorescent group and / or an EpCAM aptamer labeled with a third fluorescent group, the specific protein corresponds to protein CD63 and / or protein EpCAM, and the second working temperature is 4°C.

[0036] Furthermore, the mass ratio of the miR-21 recognition probe labeled with the first fluorescent group to the black phosphorus nanosheet is 1: (8-18); the mass ratio of the CD63 aptamer labeled with the second fluorescent group to the black phosphorus nanosheet is 1: (5-15); and the mass ratio of the EpCAM aptamer labeled with the third fluorescent group to the black phosphorus nanosheet is 1: (2-12).

[0037] Different fluorescent groups were used to label the CD63 aptamer, EpCAM aptamer and miR-21 recognition probe respectively. The modified black phosphorus nanosheets after divalent manganese ion coordination modification could strongly adsorb and recognize the CD63 aptamer, EpCAM aptamer and miR-21 recognition probe. Under non-first and second working temperatures, such as room temperature (20-25°C), the fluorescence was quenched by black phosphorus due to the transfer of fluorescence resonance ability, forming a black phosphorus / manganese ion composite nanosensor. When colorectal cancer exosomes are present, as colorectal cancer surface proteins CD63 and EpCAM, when incubated at the second working temperature of 4°C, the CD63 aptamer and EpCAM aptamer adsorbed by the black phosphorus / manganese ion composite nanosensor will fold into a special spatial structure, specifically recognize the proteins CD63 and EpCAM on the surface of the exosomes and combine to form a complex, the CD63 aptamer and EpCAM aptamer will detach from the black phosphorus surface, and the quenched fluorescence will be restored. According to the change in fluorescence value, the exosome content can be determined; when incubated at the first working temperature of 37°C, the black phosphorus / manganese ion composite nanosensor mainly penetrates the exosome membrane and enters the interior of the exosome. The miR-21 recognition probe adsorbed by the black phosphorus / manganese ion composite nanosensor can specifically combine with the miR-21 inside the exosome to form a complementary double chain, the miR-21 recognition probe will detach from the black phosphorus surface, and the quenched fluorescence will be restored. According to the change in fluorescence value of the system, the quantitative detection of miR-21 can be performed.

[0038] The black phosphorus / manganese ion composite nanosensor provided in this scheme can be applied to the kit and detection system for quantitative detection of colorectal cancer exosomes. The content of colorectal cancer exosomes is determined by detecting two types of indicators, namely, colorectal cancer exosome protein CD63 and protein EpCAM as well as its internal miR-21. Compared with the existing technology that only uses the levels of EpCAM and CD63 on exosomes as diagnostic indicators for colorectal cancer, this can further increase the accuracy of colon cancer diagnosis.

[0039] The present invention also provides a method for preparing the above-mentioned black phosphorus / manganese ion composite nanosensor, which is characterized by the following steps:

[0040] S1: In an ice bath, a suspension of black phosphorus nanosheets with an average sheet diameter of 100 nm-5 μm is ultrasonically exfoliated by an ultrasonic cell crusher to obtain a dispersion of black phosphorus nanosheets with an average sheet diameter of 50-100 nm and fully dispersed;

[0041] S2: Adding divalent manganese ion solution, black phosphorus nanosheet dispersion, fluorescent group labeled nucleic acid aptamer solution and / or fluorescent group labeled nucleic acid probe solution to the biological buffer in sequence and mixing them well, the black phosphorus / manganese ion composite nanosensor is obtained.

[0042] Furthermore, the method can be carried out by the following steps:

[0043] S1: In an ice bath, 0.2 mg / mL black phosphorus nanosheet suspension was sonicated intermittently for 30-45 min using an ultrasonic cell crusher at a power of 200 W in a mode of sonicating for 3-5 s and pausing for 3-5 s to obtain a dispersion of black phosphorus nanosheets with an average sheet diameter of 50-100 nm and fully dispersed.

[0044] S2: The mixing step is as follows: after sufficient shaking at the first working temperature and / or the second working temperature, centrifuging at 10000-14000 rpm for 30-60 min at room temperature, removing the supernatant after centrifugation, and then adding an equal amount of biological buffer solution to the supernatant removed.

[0045] The black phosphorus / manganese ion composite nanosensor prepared by the technical solution of the present invention has the characteristics of simple method, quick operation, high detection accuracy and strong specificity, and does not require complicated processing of samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Comparison of ultraviolet absorption of black phosphorus, black phosphorus / manganese ion group and black phosphorus / manganese ion / nucleic acid group on the 1st, 7th, 14th, 21st and 28th day;

[0047] Figure 2 It is the fluorescence value of the system after adding different concentrations of EDTA solution, phosphate solution and urea solution to the black phosphorus / manganese ion / nucleic acid group;

[0048] Figure 3 After adding 200 nM t1, M1, M2, and M3 nucleic acid solutions to the nanosensor, the change in the fluorescence value of the system ( -1) (λex / λem=490 / 570 nm) (***P<0.001);

[0049] Figure 4 The fluorescence spectra of the black phosphorus / manganese ion / CD63 nanosensor after adding different concentrations of exosome suspensions derived from the plasma of colorectal cancer patients (A) and the linear relationship between the concentration of exosomes derived from the plasma of colorectal cancer patients and the fluorescence intensity (B);

[0050] Figure 5 After adding different concentrations of exosome suspensions derived from plasma of patients with colon cancer, the fluorescence spectra of the black phosphorus / manganese ion / EpCAM nanosensor (A) and the linear relationship between the concentration of exosomes derived from plasma of patients with colon cancer and the fluorescence intensity (B);

[0051] Figure 6Confocal images of SW480 exosomes and NCM460 exosomes treated with black phosphorus / manganese ion / CD63 nanosensor and black phosphorus / manganese ion / miR-21 nanosensor under different conditions (scale bar is 10 μm);

[0052] Figure 7 The fluorescence spectra after adding different concentrations of colon cancer plasma exosomes to the black phosphorus / manganese ion / miR-21 nanosensor (A) and the standard curve of colon cancer plasma exosome concentration and fluorescence intensity (B). DETAILED DESCRIPTION

[0053] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0054] Example 1 Stability test of bare black phosphorus nanosheets modified by divalent manganese ions

[0055] In this embodiment, a black phosphorus nanosheet dispersion is prepared by treating a black phosphorus nanosheet suspension with an average sheet diameter of 100 nm-5 μm (purchased from Jiangsu Xianfeng Nanomaterial Technology Co., Ltd., product model XF207) as a raw material. The preparation is specifically carried out using the following steps:

[0056] S1.0 Preparation of black phosphorus nanosheet dispersion: Under ice bath, 0.2 mg / mL of black phosphorus nanosheet suspension with an average flake size of 100 nm-5 μm was ultrasonically sonicated for 30 min at a power of 200 W in a mode of 3 s on and 4 s off to obtain a fully dispersed black phosphorus nanosheet dispersion with an average flake size of 50-100 nm.

[0057] This example is based on the black phosphorus nanosheet dispersion prepared in step S1.0, and a black phosphorus group, a black phosphorus / manganese ion group, and a black phosphorus / manganese ion / nucleic acid group are established to determine the stability difference of the black phosphorus nanosheet modified with divalent manganese ions relative to the bare black phosphorus nanosheet, as follows:

[0058] S1.1 Preparation of black phosphorus group test samples: add 1 mL of the black phosphorus nanosheet dispersion prepared in the manner of Example 1 to 4 mL of 10 mM 4-hydroxyethylpiperazineethanesulfonic acid buffer (HEPES), mix well, and prepare black phosphorus group test samples;

[0059] S1.2 Preparation of black phosphorus / manganese ion group test samples: add 2.5 mL of 10 mM manganese chloride solution and 1.5 mL of black phosphorus nanosheet dispersion prepared in the manner of Example 1 to 1.5 mL of 10 mM HEPES, mix well, and prepare black phosphorus / manganese ion group test samples;

[0060] S1.3 Preparation of black phosphorus / manganese ion / nucleic acid group test samples: Add 2.5 mL of 10 mM manganese chloride solution, 0.5 mL of 1 μM Alexa Fluor 555 fluorescent group-labeled A1 probe nucleic acid solution, and 1 mL of black phosphorus nanosheet dispersion prepared in the manner of Example 1 to 1 mL of 10 mM HEPES, mix well, and prepare black phosphorus / manganese ion / nucleic acid group test samples.

[0061] The samples of the black phosphorus group, black phosphorus / manganese ion group, and black phosphorus / manganese ion / nucleic acid group were exposed to air at room temperature without being shielded from light. The absorbance of the three groups was tested using an ultraviolet spectrophotometer every day for 30 days. The test results were as follows: Figure 1 ; The nucleic acid sequence of A1 probe labeled with Alexa Fluor 555 fluorescent group is shown in Table 1.

[0062] like Figure 1 As shown, the black phosphorus group, black phosphorus / manganese ion group and black phosphorus / manganese ion / nucleic acid group of test samples were placed at room temperature and exposed to the air without being shielded from light. The absorbance of the black phosphorus group decreased rapidly. This is because the phosphorus atoms on its surface are oxidized to form phosphate anions, causing the naked black phosphorus to be oxidized in the air. Therefore, without any modification of the naked black phosphorus, the sensor constructed based on this has the defect of instability.

[0063] Compared with the black phosphorus group, the absorbance of the two tested samples, the black phosphorus / manganese ion group and the black phosphorus / manganese ion / nucleic acid group, changed less with time, proving that manganese ions improved the stability of black phosphorus. This is because divalent manganese ions attached to the black phosphorus surface, passivated the lone pair of electrons on the black phosphorus surface, and hindered the binding of the lone pair of electrons with oxygen atoms. In addition, the adsorption of nucleic acid polyanions led to an increase in the negative charge on the surface of the black phosphorus / manganese ion / nucleic acid group, making the system more stable. Therefore, the stability of the black phosphorus / manganese ion group and the black phosphorus / manganese ion / nucleic acid group is greater than that of unmodified black phosphorus.

[0064] Example 2 Exploring the adsorption principle and adsorption site of nucleic acids

[0065] In order to explore the adsorption principle and adsorption site of nucleic acids, EDTA solution, phosphate solution (PBS), and urea solution of different concentrations were added to the black phosphorus / manganese ion composite nanosensor sample to be tested, and the fluorescence value was detected to explore and verify the adsorption site of nucleic acids in the black phosphorus / manganese ion composite nanosensor, as follows:

[0066] S2.1: Add 250 μL of 10 mM manganese chloride solution, 50 μL of the black phosphorus nanosheet dispersion prepared in step S1.0 of Example 1, and 100 μL of 1 μM Alexa Fluor 555-labeled A1 probe nucleic acid solution to 100 μL of 10 mM HEPES buffer, mix them uniformly, and then shake them at a first working temperature of 37° C. for 3 h to obtain a black phosphorus / manganese ion test sample;

[0067] S2.2: The above system was centrifuged at room temperature (25°C) and 14000 rpm for 30 min. After centrifugation, 450 μL of the supernatant was removed, and 450 μL of 10 mM HEPES solution was added and mixed to prepare the black phosphorus / manganese ion composite nanosensor sample to be tested;

[0068] S2.3: Add the black phosphorus / manganese ion composite nanosensor sample to be tested to the 96-well plate, 100 μL per well, and incubate at the first working temperature of 37°C for 30 min;

[0069] S2.4: Add the following into the 96-well plate: Figure 2 Different concentrations of EDTA solution, urea solution, and phosphate solution were added to three replicate wells in each group, with 100 μL in each well. The wells were shaken and incubated at the first working temperature of 37°C for 30 min, and the fluorescence intensity of each well was detected. The test results are shown in Figure 2 .

[0070] like Figure 2 As shown, adding EDTA to the system can enhance the fluorescence intensity of the system, that is, the free nucleic acid increases, indicating that the nucleic acid in the black phosphorus / manganese ion composite nanosensor sample to be tested is dissociated from the carrier, and as the amount of EDTA added to the system increases, the fluorescence intensity of the system gradually increases, that is, the amount of nucleic acid dissociation increases with the increase of the added EDTA concentration, because EDTA can chelate with manganese ions, which proves that the nucleic acid in the black phosphorus / manganese ion composite nanosensor sample to be tested is adsorbed on the black phosphorus surface through the coordination of divalent manganese ions. When PBS is added to the black phosphorus / manganese ion composite nanosensor sample to be tested, the fluorescence intensity of the system remains unchanged, proving that phosphate does not promote the desorption of nucleic acid, that is, the binding site of nucleic acid and black phosphorus is not the phosphate skeleton. When urea is added to the black phosphorus / manganese ion composite nanosensor sample to be tested, the fluorescence intensity of the system remains unchanged, because urea can destroy hydrogen bonds, so this result proves that the binding of nucleic acid and black phosphorus does not rely on the force of hydrogen bonds.

[0071] In summary, this example verifies that the adsorbed site of nucleic acid on the black phosphorus / manganese ion composite nanosensor sample to be tested is the base, and the adsorption mainly depends on the coordination effect of divalent manganese ions.

[0072] Example 3 Detection specificity of the constructed black phosphorus / manganese ion composite nanosensor

[0073] In order to verify the detection specificity of the black phosphorus / manganese ion composite nanosensor, a nucleic acid sequence solution that is completely complementary to the A1 probe nucleic acid sequence is used as the t1 nucleic acid solution, a single-base mismatch sequence solution that is partially complementary to the A1 probe nucleic acid sequence is used as the M1 nucleic acid solution, a double-base mismatch sequence solution that is partially complementary to the A1 probe nucleic acid sequence is used as the M2 nucleic acid solution, and a triple-base mismatch sequence solution that is partially complementary to the A1 probe nucleic acid sequence is used as the M3 nucleic acid solution. The black phosphorus / manganese ion composite nanosensor system prepared in step S2.1 is as follows:

[0074] S3.1: dilute t1, M1, M2, and M3 nucleic acid solutions with 10 mM HEPES buffer to a final concentration of 200 nM to obtain t1, M1, M2, and M3 nucleic acid solutions to be tested;

[0075] S3.2: Add the black phosphorus / manganese ion / composite nanosensor prepared in step S2.1 of Example 2 to each well of a 96-well plate, 100 μL per well, and shake and stabilize at the first working temperature of 37° C. for 30 min;

[0076] S3.3: Add 100 μL of the nucleic acid solutions t1, M1, M2, and M3 prepared in S3.1 to the wells to which the black phosphorus / manganese ion composite nanosensor to be tested prepared in step S2.1 of Example 2 has been added, respectively, to each well, with three replicate wells in each group, and incubate at the first working temperature of 37°C for 30 min with shaking, and detect the fluorescence intensity of each well, which is recorded as F;

[0077] S3.4: Add 10 mM HEPES to the wells to which the black phosphorus / manganese ion composite nanosensor prepared in step S2.1 of Example 2 has been added as a blank control, with 100 μL per well, and incubate at the first working temperature of 37°C for 30 min with shaking. Detect the fluorescence intensity of the corresponding wells, record it as F0, and calculate the change in fluorescence value [Calculation formula: -1(λex / λem=490 / 570 nm)】, the test results are as follows Figure 3 ; The nucleic acid sequences of t1, M1, M2 and M3 are shown in Table 1.

[0078] like Figure 3 As shown in the figure, compared with t1, the fluorescence intensity of M1, M2, and M3 is significantly reduced; with the increase of mismatched base pairs, the fluorescence response gradually decreases, and the difference is statistically significant. This result proves that the black phosphorus / manganese ion composite nanosensor has good specificity and can specifically detect complementary DNA.

[0079] Example 4 Black phosphorus / manganese ion composite nanosensor for detecting plasma-derived exosome concentrations

[0080] In order to explore the linear range of the black phosphorus / manganese ion composite nanosensor to detect the concentration of exosomes, the specific protein CD63 present on exosomes and the specific protein EpCAM of exosomes derived from colorectal cancer were used to detect the linear relationship between the concentration of exosomes derived from the plasma of colorectal cancer patients and the fluorescence intensity, and to determine the reliable linear range of the detection, as follows:

[0081] S4.1 Construction of black phosphorus / manganese ion / CD63 nanosensor: 250 μL of 10 mM manganese chloride solution, 50 μL of 0.2 mg / mL black phosphorus nanosheet dispersion prepared in step S1.0, and 100 μL of 1 μM Alexa Fluor 555 fluorescent group-labeled CD63 aptamer were added to 100 μL of 10 mM HEPES buffer in sequence, mixed, and shaken at the first working temperature of 37°C for 3 h to obtain a black phosphorus / manganese ion / CD63 nanosensor;

[0082] S4.2 Construction of black phosphorus / manganese ion / EpCAM nanosensor: Add 250 μL of 10 mM manganese chloride solution, 50 μL of 0.2 mg / mL black phosphorus nanosheet dispersion prepared in step S1.0, and 100 μL of 1 μM Alexa Fluor 647 fluorescent group-labeled EpCAM aptamer to 100 μL of 10 mM HEPES buffer in sequence, mix well, and shake at the first working temperature of 37°C for 3 h to obtain the black phosphorus / manganese ion / EpCAM nanosensor.

[0083] S4.3 Extraction of exosomes: When extracting exosomes, thaw the plasma, add 200 μL of plasma to 1 mL of 10 mM HEPES for dilution, centrifuge the dilution at 300 g for 10 min at the second working temperature of 4°C, and collect the supernatant for the first time; centrifuge the supernatant collected for the first time at 2000 g for 20 min at the second working temperature of 4°C, and collect the supernatant for the second time; centrifuge the supernatant collected for the second time at 10000 g for 45 min at the second working temperature of 4°C, and collect the supernatant for the third time;

[0084] After filtering the supernatant collected for the third time through a microporous filter membrane (pore size of 0.22 μm), the filtrate was subjected to the first ultracentrifugation at 160000 g for 2 h at the second working temperature of 4°C. After the first ultracentrifugation, the supernatant was discarded and the exosome precipitate was blown with 10 mM HEPES solution for the first time. The second ultracentrifugation was performed at 160000 g for 2 h at the second working temperature of 4°C. After the second ultracentrifugation, the supernatant was discarded and the exosome precipitate was blown with 10 mM HEPES solution for the second time. The exosome suspension obtained was stored at -20°C.

[0085] S4.4 Preparation of exosome suspensions of different concentrations: After determining the initial concentration using a particle size analyzer, dilute the exosome suspension obtained in S4.3 with 10 mM HEPES buffer to achieve Figure 4 , Figure 5 Set the target concentration.

[0086] S4.5 Detect the fluorescence intensity of exosome suspensions of different concentrations: add the black phosphorus / manganese ion / CD63 nanosensor prepared in step S4.1 and the black phosphorus / manganese ion / EpCAM nanosensor prepared in step S4.2 to a 96-well plate, 100 μL per well, shake and incubate at the first working temperature of 37°C for 30 min, then add the exosome suspensions of different concentrations prepared in step S4.4 to the wells with nanosensors added, three replicate wells per group, 100 μL per well, shake at the second working temperature of 4°C for 30 min, detect the fluorescence intensity of each well, and the test results are shown in the table below. Figure 4 , Figure 5 ; The CD63 aptamer sequence labeled with Alexa Fluor 555 fluorescent group and the EpCAM aptamer sequence labeled with Alexa Fluor 647 fluorescent group are shown in Table 1.

[0087] Figure 4 and Figure 5 All showed that with the increase of the concentration of plasma exosomes from patients with colon cancer (0-2.00×10 4 / μL), the fluorescence value of the system gradually increased. This proves that the surface protein CD63 of exosomes derived from plasma of patients with colon cancer can bind to the CD63 aptamer in the black phosphorus / manganese ion / CD63 nanosensor and detach from the black phosphorus surface, so that it can recover fluorescence, and the surface specific protein EpCAM of exosomes derived from plasma of patients with colon cancer can bind to the EpCAM aptamer in the black phosphorus / manganese ion / EpCAM nanosensor and detach from the black phosphorus surface, so that it can recover fluorescence. The linear range of detection is 5.00×10 2 -2.00×10 4 Pieces / μL.

[0088] Example 5 Confocal imaging of exosomes from normal cells and colorectal cancer cells

[0089] At different working temperatures, exosomes from normal cells and exosomes from colorectal cancer cells were used as test samples, and nanosensors were labeled with different fluorescent markers for fluorescence photography. The fluorescence of exosome-specific proteins and tumor-related miRNAs inside the exosomes was used to verify whether the black phosphorus / manganese ion / CD63 nanosensor and the black phosphorus / manganese ion / miR-21 nanosensor could play a corresponding fluorescence quenching-recovery effect, thereby determining the specific detection effect of the black phosphorus / manganese ion composite nanosensor on exosomes from colorectal cancer cells. In addition, by controlling the temperature, the action positions of the nanosensor and the exosomes can be controlled to be different, thereby realizing the detection of specific proteins on the surface of exosomes and internal miRNA-21, as follows:

[0090] S5.1 Preparation of black phosphorus / manganese ion / miR-21 nanosensor: 200 μL of 10 mM manganese chloride solution, 40 μL of 0.2 mg / mL black phosphorus nanosheet dispersion prepared in step S1.0, and 80 μL of 1 μM Alexa Fluor 647 fluorescent group-labeled miR-21 recognition probe were added to 80 μL of 10 mM HEPES buffer in sequence and mixed. After shaking at the first working temperature of 37°C for 3 h, centrifuged at room temperature (25°C) and 14,000 rpm for 30 min, removed 380 μL of the supernatant after centrifugation, and then added 50 μL of 10 mM HEPES solution and mixed to prepare the black phosphorus / manganese ion / miR-21 nanosensor.

[0091] S5.2 Preparation of black phosphorus / manganese ion / CD63 nanosensor: Add 200 μL of 10 mM manganese chloride solution, 40 μL of 0.2 mg / mL black phosphorus nanosheet dispersion prepared in step S1.0, and 80 μL of 1 μM Alexa Fluor 555 fluorescent group labeled CD63 aptamer solution to 80 μL of 10 mM HEPES buffer, and mix well. After shaking at the first working temperature of 37°C for 3 h, centrifuge at room temperature (25°C) and 14000 rpm for 30 min, remove 380 μL of the supernatant after centrifugation, add 50 μL of 10 mM HEPES solution and mix well to prepare the black phosphorus / manganese ion / CD63 nanosensor;

[0092] S5.3 Nanosensor and exosome co-incubation system at the second working temperature: to a concentration of 1.00×10 625 μL of black phosphorus / manganese ion / CD63 nanosensor and 25 μL of black phosphorus / manganese ion / miR-21 nanosensor were added to the exosome suspension derived from SW480 cells and the exosome suspension derived from NCM460 cells, respectively, and the mixture was shaken at the second working temperature of 4°C for 30 min.

[0093] S5.4 Co-incubation system of nanosensor and exosomes at the first working temperature: to a concentration of 1.00×10 6 25 μL of black phosphorus / manganese ion / CD63 nanosensor and 25 μL of black phosphorus / manganese ion / miR-21 nanosensor were added to the exosome suspension derived from SW480 cells and the exosome suspension derived from NCM460 cells, respectively, and the mixture was shaken at the first working temperature of 37°C for 30 min.

[0094] S5.5 Nanosensor and exosome co-incubation system at the second and first working temperatures: to a concentration of 1.00×10 6 25 μL of black phosphorus / manganese ion / CD63 nanosensor and 25 μL of black phosphorus / manganese ion / miR-21 nanosensor were added to the exosome suspension derived from SW480 cells and the exosome suspension derived from NCM460 cells at 1 μL / μL, respectively, and shaken for 30 min at the second working temperature of 4°C. Then, 25 μL of black phosphorus / manganese ion / miR21 nanosensor was added to the two systems respectively, and shaken for 30 min at the first working temperature of 37°C.

[0095] Take 20 μL of the co-incubation system after different conditions, drop it in the middle of the slide, cover the slide, and take an upright confocal fluorescence photo. The test results are as follows: Figure 6 ; The miR-21 recognition probe sequence labeled with Alexa Fluor 647 fluorescent group and the CD63 aptamer sequence labeled with Alexa Fluor 555 fluorescent group are shown in Table 1.

[0096] The black phosphorus / manganese ion / CD63 nanosensor and the black phosphorus / manganese ion / miR-21 nanosensor were added to the exosomes and incubated for 30 min at the second working temperature of 4°C. The confocal microscopy results are shown in Figure 2. Figure 6A and 6B. The red fluorescence signal of Alexa Fluor 555 fluorescent group appeared in both SW480 and NCM460 cell-derived exosomes, indicating that the black phosphorus / manganese ion / CD63 nanosensor can interact with the protein CD63 on the surface of the exosomes, produce CD63 aptamer-protein complex, and detach from the black phosphorus surface; however, the blue fluorescence signal of miR-21 recognition probe labeled with Alexa Fluor 647 fluorescent group in both SW480 and NCM460 cell-derived exosomes was very weak, which may be due to the small amount of nanosensor penetrating the exosome membrane at the second working temperature of 4°C.

[0097] After the exosomes and the two nanosensors were incubated at the first working temperature of 37°C for 30 min, the confocal microscopy results were as follows: Figure 6 C and 6D. Compared with the incubation condition at the second working temperature of 4°C, the blue fluorescence is significantly enhanced. This shows that under the first working temperature of 37°C, the membrane permeability of the exosomes increases, and the number of nanosensors penetrating the exosome membrane increases. This is because after the black phosphorus / manganese ion / miR-21 nanosensor penetrates into the exosomes, its recognition probe binds to miR-21 to form a double-stranded structure, and detaches from the black phosphorus surface, the quenched fluorescence is restored, and the fluorescence value of the system is enhanced; but at this time the red fluorescence signal is relatively weak, indicating that under the first working temperature of 37°C incubation conditions, the nanosensor will enter the exosome more, so the black phosphorus / manganese ion / CD63 nanosensor binds less to the exosome surface protein CD63, so the red fluorescence is weaker.

[0098] The black phosphorus / manganese ion / CD63 nanosensor was added to the SW480 exosomes and NCM460 exosomes system and incubated at the second working temperature of 4°C for 30 min. Then the black phosphorus / manganese ion / miR-21 nanosensor was added to the sample and incubated at the first working temperature of 37°C for 30 min. The results were as follows: Figure 6E and 6F. Confocal microscopy showed that the red fluorescence signal (CD63 aptamer labeled with Alexa Fluor 555 fluorescent group) and the blue fluorescence signal (miR-21 recognition probe labeled with Alexa Fluor 647 fluorescent group) appeared in both exosomes, and the two signals overlapped. The fluorescence intensity of SW480 exosomes was higher than that of NCM460 exosomes. The above results prove that the nanosensor can play a detection role in different parts of exosomes by adjusting the incubation temperature with exosomes. When incubated at the first working temperature of 37°C, the black phosphorus / manganese ion / miR-21 nanosensor can enter the exosomes to detect miRNAs. When incubated at the second working temperature of 4°C, the black phosphorus / manganese ion / CD63 nanosensor can detect the protein CD63 on the surface of exosomes.

[0099] Example 6 Quantitative detection of miR-21 in exosomes

[0100] The concentration of exosomes from the plasma of patients with colon cancer was detected by using black phosphorus / manganese ion / miR-21 nanosensor. A linear model was established with the measured fluorescence value and the exosome concentration, and the minimum detection limit was determined as follows:

[0101] S6.1 Preparation of black phosphorus / manganese ion / miR-21 nanosensor: Add 250 μL of 10 mM manganese chloride solution, 50 μL of 0.2 mg / mL black phosphorus nanosheet dispersion prepared in step S1.0, and 100 μL of 1 μM Alexa Fluor 647 fluorescent group-labeled miR-21 recognition probe to 100 μL of 10 mM HEPES buffer, mix well, shake at the first working temperature of 37°C for 3 h, and centrifuge at room temperature (25°C) and 14,000 rpm for 30 min. After centrifugation, remove 450 μL of the supernatant, add 450 μL of 10 mM HEPES solution and mix well to prepare the black phosphorus / manganese ion / miR-21 nanosensor.

[0102] S6.2 Quantitative detection of miR-21 in exosomes: The black phosphorus / manganese ion / miR-21 nanosensor prepared in S6.1 was added to a 96-well plate, 100 μL per well, and after shaking and incubating at the first working temperature of 37°C for 30 min, the following were added: Figure 7 Different concentrations of exosome suspension were prepared in three replicate wells per group, with 100 μL per well. The wells were shaken at the first working temperature of 37°C for 30 min, and the fluorescence intensity of each well was detected. The test results were as follows: Figure 6 .

[0103] Figure 7A The test results showed that after the black phosphorus / manganese ion / miR-21 nanosensor was co-incubated with exosomes at the first working temperature of 37°C, an increase in the concentration of plasma exosomes from colon cancer patients (0-1.00×10 6 / μL), and the measured fluorescence value gradually increased. This proves that the black phosphorus / manganese ion / miR-21 nanosensor can enter the plasma-derived exosomes, recognize that the probe binds to miR-21 and detaches from the black phosphorus surface, causing it to recover fluorescence. Figure 7 As shown in B, the linear range of the black phosphorus / manganese ion / miR-21 nanosensor for detecting exosomes from plasma of patients with colon cancer is 1.00×10 5 - 1.00×10 6 / μL, the minimum detection limit was 2.52×10 4 It has the characteristics of simple and quick operation, high detection accuracy and strong specificity, and does not require complicated processing of the samples to be tested.

[0104] Table 1 Nucleic acid, aptamer, and recognition probe sequences

[0105]

[0106] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A black phosphorus / manganese ion composite nanosensor, characterized in that: The invention is a liquid nanosensor system prepared by fully dispersing black phosphorus nanosheets with an average sheet diameter of 50-100 nm in a liquid medium with biological buffer, divalent manganese ion solution, nucleic acid aptamer solution labeled with a fluorescent group and nucleic acid probe solution labeled with a fluorescent group. The nucleic acid probe labeled with a fluorescent group is a miR-21 recognition probe labeled with a first fluorescent group, the nucleic acid aptamer labeled with a fluorescent group is a CD63 aptamer labeled with a second fluorescent group and an EpCAM aptamer labeled with a third fluorescent group, the mass ratio of the miR-21 recognition probe labeled with the first fluorescent group to the black phosphorus nanosheet is 1:(8-18); the mass ratio of the CD63 aptamer labeled with the second fluorescent group to the black phosphorus nanosheet is 1:(5-15); the mass ratio of the EpCAM aptamer labeled with the third fluorescent group to the black phosphorus nanosheet is 1:(2-12); At a non-first operating temperature, the base of the nucleic acid probe labeled with the fluorescent group is bound to the black phosphorus surface through the coordination of the divalent manganese ion, so that the fluorescence is quenched; After being incubated with the exosomes to be detected at a first working temperature, the nucleic acid probe binds to the exosome miR-21 to be detected, so that the nucleic acid probe is detached from the black phosphorus surface and the quenched fluorescence is restored; At a temperature other than the second operating temperature, the base of the nucleic acid aptamer labeled with the fluorescent group is bound to the black phosphorus surface through the coordination of the divalent manganese ion, so that the fluorescence is quenched; After being incubated with the exosomes to be tested at the second working temperature, the nucleic acid aptamer binds to the protein CD63 and the protein EpCAM on the surface of the exosomes to be tested, so that the nucleic acid aptamer is detached from the black phosphorus surface and the quenched fluorescence is restored.

2. The black phosphorus / manganese ion composite nanosensor according to claim 1, characterized in that: The biological buffer is selected from one of 4-hydroxyethylpiperazineethanesulfonic acid buffer and tris(hydroxymethylaminomethane) hydrochloride buffer solution; the divalent manganese ion solution is selected from any one of manganese chloride solution, manganese phosphate solution and manganese carbonate solution; the black phosphorus nanosheets are added in the form of a black phosphorus nanosheet dispersion and dispersed in the liquid medium.

3. The black phosphorus / manganese ion composite nanosensor according to claim 2, characterized in that: The biological buffer is a 4-hydroxyethylpiperazineethanesulfonic acid buffer, the divalent manganese ion solution is a manganese chloride solution, and the mass ratio of 4-hydroxyethylpiperazineethanesulfonic acid, black phosphorus nanosheets, and manganese chloride is (10-50):1:(20-50).

4. The black phosphorus / manganese ion composite nanosensor according to claim 3, characterized in that: The first operating temperature is 37°C; the second operating temperature is 4°C.

5. The method for preparing the black phosphorus / manganese ion composite nanosensor as claimed in any one of claims 1 to 4, characterized by the following steps: S1: In an ice bath, a suspension of black phosphorus nanosheets with an average sheet diameter of 100 nm-5 μm is ultrasonically exfoliated by an ultrasonic cell crusher to obtain a dispersion of black phosphorus nanosheets with an average sheet diameter of 50-100 nm and fully dispersed; S2: Adding divalent manganese ion solution, black phosphorus nanosheet dispersion, fluorescent group labeled nucleic acid aptamer solution and fluorescent group labeled nucleic acid probe solution to the biological buffer in sequence and mixing them well, the black phosphorus / manganese ion composite nanosensor is obtained.

6. The method for preparing the black phosphorus / manganese ion composite nanosensor according to claim 5, characterized by the following steps: S1: In an ice bath, 0.2 mg / mL black phosphorus nanosheet suspension was subjected to intermittent sonication for 30-45 min using an ultrasonic cell crusher at a power of 200 W in a mode of sonication for 3-5 seconds and pause for 3-5 seconds to obtain a dispersion of black phosphorus nanosheets with an average sheet diameter of 50-100 nm and fully dispersed; S2: The mixing is as follows: after sufficient shaking at the first working temperature or the second working temperature, centrifugation is performed at 10000-14000 rpm for 30-60 min at room temperature, the supernatant is removed after centrifugation, and then an equal amount of biological buffer solution is added to the supernatant.

7. Use of the black phosphorus / manganese ion composite nanosensor as described in any one of claims 1 to 4 in the preparation of a kit for detecting cancer cell markers.

8. Use of the black phosphorus / manganese ion composite nanosensor as claimed in claim 4 in the preparation of a kit for quantitative detection of colorectal cancer exosome markers.

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