A homogeneous fluorescence instant liquid biopsy strategy for tumor based on DNA functionalized nanospheres

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a homogeneous fluorescence instant tumor liquid biopsy strategy based on DNA functionalized nanospheres. 2+ Multifunctional integrated nanospheres. Based on the functional integration of the nanospheres, one-step detection of circulating tumor cells (CTCs) or tumor-derived exosomes (TDEs) is realized, and in combination with a test strip and a smartphone red, green and blue (RGB) analysis technology, a variety of analysis modes including fluorescence detection and smartphone-RGB are provided, a rapid and sensitive tumor liquid biopsy instant detection method is constructed, convenient analysis of "drop and see" is realized, and the rapid and convenient detection demand of tumor liquid biopsy is met, and an effective means is provided for early disease screening.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection, and particularly relates to a homogeneous fluorescence instant tumor liquid biopsy strategy based on DNA functionalized nanospheres. BACKGROUND

[0002] Tumor is still a major disease threatening human health and is the main cause of death. Liquid biopsy is an attractive alternative to pathological biopsy due to its minimally invasive nature, which helps early screening, real-time monitoring and efficacy evaluation of tumors. Circulating tumor cells (CTCs), tumor cell-derived exosomes (TDEs) and circulating tumor DNA are the most commonly detected biomarkers in liquid biopsy. CTCs can reflect the phenotype and genetic composition of the primary tumor, and the change in their concentration level is parallel to the evolution of the tumor. TDEs are relatively early secreted into the blood, and their abundance is higher than that of CTCs, and their stability is higher than that of circulating tumor DNA. Therefore, CTCs and TDEs have become important targets for liquid biopsy in the field of malignant tumors. However, the analysis of CTCs and TDEs faces many challenges, including their extremely low concentration at the early stage of tumor occurrence, high heterogeneity, and interference from normal host cells and a large number of exosomes secreted. Traditional detection methods, such as polymerase chain reaction (PCR), immunofluorescence, flow cytometry, nanoparticle tracking analysis and Western blotting, often rely on large and precise instruments, signal labeling techniques and time-consuming operation steps, which limit the widespread popularity and applicability in community medical care and resource-limited areas. Therefore, there is an urgent need to develop a rapid and simple instant sensing strategy for tumor liquid biopsy to accelerate the development of precision medicine.

[0003] In recent years, various quantitative detection methods for CTCs and TDEs have been reported. The current detection process often involves multi-step reactions and complex immobilization and modification at the non-homogeneous interface, which limits the simplicity and popularity of the method. Developing a homogeneous one-step reaction strategy can save labor-intensive experimental operation processes, shorten the detection time, reduce the dependence on professionals, and improve the practicability. In addition, this one-step sensing strategy can be integrated into various miniaturized and portable detection instruments in different application scenarios, which has broad medical application potential. For example, integrating one-step reactions on paper through deposition, screen printing and other methods can make the sensing strategy have multiple advantages such as no threshold for operation, environmental friendliness and easy handling, which is expected to promote the development of point-of-care testing (POCT). However, due to the limited sensitivity of colorimetric paper-based POCT, it cannot meet the needs of tumor liquid biopsy. SUMMARY

[0004] In order to solve the above problems existing in the prior art, the present application provides a homogeneous fluorescence instant tumor liquid biopsy strategy based on DNA functionalized nanospheres.

[0005] The present application first synthesizes a protein scaffold DNA-Cu by a one-step method at room temperature 2+ functional nanoballs, the composite nanoballs combine functional nucleic acids, protein-DNA and DNA-metal ion nanotechnologies, realize the integration of target recognition, signal amplification and conduction. The "one ball multi-use" of the nanoballs realizes a one-step reaction of tumor CTCs and TDEs within 1 hour. Selecting CdTe quantum dots (QDs) with excellent optical performance as signal molecules, combining test strips and smartphone red, green and blue (RGB) analysis technology, providing multiple analysis modes including fluorescence detection and smartphone-RGB, a rapid and sensitive tumor liquid biopsy real-time detection method is constructed. Using tumor A549 cells and ultracentrifugation method to separate and enrich A549 cell-derived exosomes as research models, the effectiveness and robustness of the strategy are proved by fluorescence and smartphone-RGB multi-mode analysis. In summary, the present application provides a rapid and convenient CTCs and TDEs fluorescence real-time detection strategy, which will provide a scheme for rapid analysis of tumor liquid biopsy.

[0006] The technical scheme adopted by the present application is:

[0007] A homogeneous fluorescence real-time tumor liquid biopsy strategy based on DNA functionalized nanoballs, comprising the following steps:

[0008] (1) biotinylated DNA hairpins are combined with streptavidin (SA) to form a cross-shaped protein scaffold-DNA tetramer;

[0009] PD-L1 aptamer, Cu 2+ and buffer are added to the cross-shaped protein scaffold-DNA tetramer, and after mixing and reaction, DNA functionalized nanoballs are obtained;

[0010] (2) high expression PD-L1 tumor liquid biopsy markers are added to the DNA functionalized nanoballs obtained in step (1), and after reaction at room temperature, a fluorescence probe recognizing Cu 2+ is added, the reaction solution is dropped on a test strip, and mobile phone red, green and blue (RGB) color analysis is performed under ultraviolet irradiation, which is the real-time detection of tumor liquid biopsy.

[0011] In step (1), the biotinylated DNA hairpins include biotin-H1, biotin-H2, biotin-H3 and biotin-H4.

[0012] In step (1), the cross-shaped protein scaffold-DNA tetramer is composed of streptavidin-biotin-H1, streptavidin-biotin-H2, streptavidin-biotin-H3 and streptavidin-biotin-H4.

[0013] The nucleotide sequences of the four hairpins H1, H2, H3 and H4 in the biotinylated DNA hairpin are shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4, respectively.

[0014] In step (1), the nucleotide sequence of the PD-L1 aptamer is shown in SEQ ID No. 5.

[0015] In step (1), the buffer is 3-(N-morpholino)propanesulfonic acid (MOPS) buffer.

[0016] In step (1), the mixing reaction is carried out at 20-28℃ for 2-6 hours.

[0017] In step (2), the tumor liquid biopsy marker with high expression of PD-L1 is circulating tumor cells (CTCs) or tumor cell-derived exosomes (TDEs). The room temperature reaction is carried out at 22-25℃ for 30-60 minutes.

[0018] The CTCs are A549 cells.

[0019] In step (2), the fluorescent probe for recognizing Cu 2+ is any one of QDs, calcein and Cu 2+ -probe.

[0020] The specific operation of fluorescence detection is as follows: the fluorescent probe for recognizing Cu 2+ is added, and after 75 seconds of reaction, fluorescence detection is carried out under 365nm laser wavelength.

[0021] Based on the previous research work, the inventors of the present application are inspired by protein-DNA nanotechnology, functional nucleic acids and metal-DNA coordination effect, and prepare a DNA-Cu 2+ composite nanosphere with streptavidin-biotin as a protein scaffold, which can realize the multifunctional integration of target recognition, signal transduction and amplification. Based on the functional integrated nanosphere, further combined with the self-designed detection test strip, a rapid tumor liquid biopsy strategy with convenient operation is developed. The PD-L1 highly expressed by tumor cells is selected as the detection target, and the corresponding biotin-coupled DNA hairpin is designed according to the sequence of the PD-L1 aptamer. Streptavidin (SA) is combined with biotin with high affinity, and the DNA hairpin is anchored on SA to form a cross-shaped protein scaffold-DNA tetramer. The PD-L1 aptamer is used as a trigger chain to trigger the bidirectional cross-linking hybridization chain reaction among multiple DNA tetramers. Under the coordination of Cu 2+ and DNA base pairs, the protein scaffold DNA-Cu 2+Nanosphere. Further, the present inventors have found that, when the nanosphere encounters tumor cells or exosomes with high expression of PD-L1, the PD-L1 on the surface of the tumor cells or exosomes induces the aptamer in the nanosphere to fold into a specific secondary structure, and specifically binds to the PD-L1 with high affinity. As the content of tumor cells or exosomes increases, a large number of aptamers in the nanosphere bind to the PD-L1, causing the nanosphere to gradually disassemble and release a large amount of Cu 2+ . The released Cu 2+ can be recognized by QDs. In order to improve the ease of operation, the present inventors use inkjet printing technology to make a test strip. Dropping 10 μL of the reaction solution on a 1 cm diameter circular detection area on the test strip, the color change corresponding to the concentration of tumor cells or exosomes can be observed with the naked eye under ultraviolet light. In addition, the color can be converted into RGB values by taking a picture with a smartphone, and more accurate quantitative analysis can be performed.

[0022] It should be noted that the protein scaffold DNA-Cu 2+ nanosphere can be replaced by other nucleic acid structures.

[0023] The RGB analysis mode can also be replaced by other color digital conversion technologies such as HSV.

[0024] The present application has the following beneficial effects:

[0025] The present application provides a homogeneous fluorescence instant tumor liquid biopsy strategy based on DNA functionalized nanosphere. First, by integrating PD-L1 aptamer, protein scaffold, hybridization chain reaction and DNA-Cu 2+ interaction, a protein scaffold DNA-Cu 2+ composite nanosphere is constructed; then CTCs or TDEs are added to the DNA-Cu 2+ composite nanosphere, after reaction, CdTe QDs with excellent optical performance are selected as signal molecules, and test strips and smartphone-RGB analysis technology are combined to provide a variety of analysis modes including fluorescence detection and smartphone-RGB, and a rapid and sensitive tumor liquid biopsy instant detection method is constructed. Using tumor A549 cells and A549 cell-derived exosomes enriched by ultracentrifugation as research models, the effectiveness and stability of the detection method are proved by fluorescence and smartphone-RGB multi-mode analysis. In summary, the detection method of the present application, as a rapid and convenient CTCs and TDEs fluorescence instant detection strategy, will provide a new method for rapid analysis of tumor liquid biopsy. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0027] Figure 1 The present application is based on a protein scaffold DNA-Cu 2+ The schematic diagram of the rapid one-step universal detection strategy of tumor CTCs by functional nanoballs is shown.

[0028] Figure 2 The optimization of PD-L1 analysis conditions is shown. (a)-(b) are the optimization of Cu 2+ concentration in nanoballs; (c)-(d) are the optimization of the amount of signal molecule QDs; (e) is the optimization of the reaction time of signal molecule QDs.

[0029] Figure 3 The analysis performance of PD-L1 is shown. (a) is the change trend of QDs molecular fluorescence spectrum depending on the concentration of PD-L1; (b)-(c) are the change of QDs fluorescence peak value and the linear range of detection depending on the concentration of PD-L1; (d) is the selectivity and anti-interference verification.

[0030] Figure 4 The fluorescence and smartphone-RGB analysis performance of CTCs are shown. (a)-(b) are the decrease of QDs fluorescence intensity with the increase of A549 cell concentration; (c) is the good linear relationship between QDs fluorescence and the logarithm of cell concentration in the range of 10 2 -10 5 cells / mL; (d) is the semi-quantitative visualization of detection results, with the color of test strip changing from magenta to indigo with the increase of tumor cell concentration; (e) is the good linear relationship between R / B ratio and the logarithm of A549 cell concentration in the range of 100 to 10 5 cells / mL.

[0031] Figure 5 The present application is based on a protein scaffold DNA-Cu 2+ The schematic diagram of the rapid one-step universal detection strategy of TDEs by functional nanoballs is shown.

[0032] Figure 6Extraction and characterization of TDEs are shown; wherein (a) is the separation and enrichment of exosomes by ultracentrifugation; (b) is the nanoparticle tracking analysis (NTA) to characterize the concentration and particle size distribution of TDEs; (c) is the analysis of characteristic proteins CD63, CD9, CD81 and target protein PD-L1 of exosomes by capillary immunoblotting assay; (d) is the negative staining transmission electron microscopy (TEM) to characterize the size and morphology of TDEs;

[0033] Figure 7 Analysis performance of TDEs is shown; wherein (a)-(c) are the fluorescence signals decrease with the increase of TDEs concentration, which have good linear relationship in the range of 10 4 -10 7 particles / mL; (d) in the range of 10 4 -10 7 particles / mL, good linear relationship between R / B ratio and TDEs concentration logarithm value is observed; (e) is to verify the good selectivity of the detection method by comparing the fluorescence signal changes of exosomes from different cell sources. DETAILED DESCRIPTION

[0034] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions of the present application will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0035] The protein scaffold DNA-Cu 2+ functional nanospheres described in the present application realize a rapid one-step universal detection strategy for tumor CTCs, as shown in Figure 1 . First, by combining PD-L1 aptamer, streptavidin-biotin coupled DNA hairpin and Cu 2+ , protein scaffold DNA-Cu 2+ functional nanospheres are synthesized by one-step method at room temperature. PD-L1, which is highly expressed by tumor cells, is selected as the detection target. According to the sequence of PD-L1 aptamer, the corresponding biotin-coupled DNA hairpin is designed (Table 1). Streptavidin (SA) binds to biotin with high affinity, and the DNA hairpin is anchored on SA to form a cross-shaped protein scaffold-DNA tetramer. The PD-L1 aptamer is used as a trigger chain to trigger the bidirectional cross-linking hybridization chain reaction between multiple DNA tetramers. Under the coordination of Cu 2+ and DNA base pairs, protein scaffold DNA-Cu 2+ nanospheres are self-assembled.

[0036] When the nanospheres encounter tumor cells or exosomes with high expression of PD-L1, the PD-L1 on the surface of the tumor cells or exosomes induces the aptamers in the nanospheres to fold into a specific secondary structure, and specifically binds to it with high affinity. As the content of tumor cells or exosomes increases, a large number of aptamers in the nanospheres bind to PD-L1, causing the nanospheres to gradually depolymerize and release a large amount of Cu 2+ . The released Cu 2+ can be recognized by QDs. In order to improve the operation convenience, a test strip was prepared using inkjet printing technology. 10 μL of the reaction solution was dropped on the 1 cm diameter circular detection area of the test strip, and under ultraviolet light, the color change corresponding to the concentration of tumor cells or exosomes can be observed with the naked eye. In addition, a smartphone is used to take a picture, convert the color to RGB value, and perform more accurate quantitative analysis.

[0037] Materials and reagents

[0038] Oligonucleotides of different sequences were synthesized and purified by Shenguo Biotech Co., Ltd. (Shanghai, China), and the sequences are shown in Table 1. Copper sulfate pentahydrate (CuS04·5H20), sodium tellurite (Na2Te03), and 3-mercaptopropionic acid (MPA) were ordered from Aladdin Reagent Co., Ltd. (Shanghai, China). Sodium chloride (NaCl), trisodium citrate dihydrate (Na3C6H507·2H20), cadmium chloride (CdCl2), potassium borohydride (KBH4), hydrochloric acid (HC1), sodium hydroxide (NaOH), magnesium chloride (MgCl2) were purchased from Cologne Chemical Reagent Co., Ltd. (Chengdu, China). Recombinant human programmed cell death ligand 1 (PD-L1) protein, streptavidin (SA), cortisol, and insulin-like growth factor-1 (IGF-1) were purchased from Shanghai Shenguo Biotechnology Co., Ltd. Human serum albumin (HSA), trypsin, glucose oxidase (GOD), immunoglobulin G (IgG), pepsin, prostate specific antigen (PSA), transferrin, interferon-gamma (IFN-g), papain, glypican-3 (GPC3), lysozyme, thrombin, and human immunodeficiency virus (HIV) p24 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human mucin 1 (MUC1) protein was ordered from Sino Biological (Beijing, China). 3-(N-morpholino)propanesulfonic acid (MOPS) was purchased from Solarbio Technology Co., Ltd. (Beijing, China). All working solutions were prepared with MOPS buffer solution (10 mM MOPS, 150 mM NaCl, 50 mM MgCl2, pH 7.4). Phosphate buffered solution (PBS) was ordered from Corning (New York, USA). Dulbecco’s medium (DMEM / F12), fetal bovine serum (FBS), penicillin / streptomycin, and syringe filter (0.22 pm) were purchased from Gibco Invitrogen (California, USA). Ultra-clean centrifuge tubes (25 x 89 mm, 38.5 mL, sterile, open thin wall) were purchased from Beckman Coulter (Indianapolis, Indiana, USA). Anti-CD9, anti-CD63, anti-CD81, and anti-PD-L1 antibodies were purchased from R&D systems (Minneapolis, USA). All reagents used in this application were analytical grade or higher grade reagents and did not require further purification when used. All experimental water was obtained from Chengdu Supure Technology Co., Ltd. (Chengdu, China) with a resistivity of 18.25 MΩ·cm. Human lung cancer cells (A549), human umbilical vein endothelial cells (HUVEC), human embryonic kidney 293T cells (HEK-293T), human liver cells (L02), and human normal lung epithelial cells (BEAS-2B) were provided by the West China Hospital Research Base. All solutions were stored in a refrigerator at 4 °C before use.

[0039] Table 1 - Oligonucleotide sequences of aptamer and four DNA hairpins

[0040]

[0041]

[0042] Synthesis of CdTe QDs

[0043] The general procedure for the synthesis of CdTe QDs has been reported previously. The main procedure is as follows: First, a 50 mL solution containing CdCl2(0.5 mM) and trisodium citrate (0.2 g) was prepared. Then, immediately, mercaptopropionic acid (MPA, 52 μί) was added to the above solution, and the pH of the solution was adjusted to 10.5 with NaOH. Subsequently, Na2Te03(0.1 mM) and KBH4(50 mg) were added to the above solution, and CdTe QDs were obtained by refluxing in portions. Subsequently, the high-purity CdTe QDs were obtained by precipitation with n-propanol and centrifugation (11000 rpm). The purified red CdTe QDs were redispersed in ultrapure water before use.

[0044] Cell recovery, culture and passaging

[0045] Cell recovery: The cryopreserved A549 cells were taken out from the liquid nitrogen tank, quickly thawed at 37 °C, and then transferred to a centrifuge tube. 5 mL of DMEM / F-12 medium containing 10% (v / v) FBS was added, and centrifuged at 1000 rpm for 3 minutes. The supernatant was discarded, 5 mL of medium was added, and then transferred to a culture dish. The cells were incubated at 37 °C, 5% CO2.

[0046] Cell culture: A549 cells were cultured in 1640 medium containing 10% (v / v) FBS and incubated at 37 °C and 5% CO2. When the cells were in the logarithmic growth phase, they were digested into a single cell suspension and counted. Finally, the cells were inoculated in a culture dish.

[0047] Cell passaging: When the cells were in the logarithmic growth phase, the old medium was removed and washed with PBS buffer. Subsequently, trypsin was used for digestion and centrifuged at 800 rpm for 3 minutes to collect the cells. Then, fresh medium was added. Finally, the cells were inoculated in a new culture dish for further culture.

[0048] Isolation of exosomes

[0049] After the cells reached 70-80% confluency, they were washed twice with DMEM medium. All cell culture supernatants were collected into a 50 mL sterile conical tube for ultracentrifugation to isolate exosomes. First, centrifuge at 500 g for 10 min at 4°C to pellet cells and cell debris. Then centrifuge the supernatant at 2000 g for 20 min at 4°C to further remove cell debris and apoptotic bodies. The resulting supernatant was centrifuged at 10000 g for 30 min at 4°C to remove large extracellular vesicles. The supernatant was then filtered with a 0.22 μm filter membrane to reduce microparticle contamination. The filtrate was ultracentrifuged at 150000 g for 2 hours at 4°C. The resulting particles were resuspended in PBS and re-centrifuged under the same conditions. Finally, the isolated exosomes were resuspended in 200 μL PBS and stored at -80°C.

[0050] Example 1

[0051] This example provides a homogeneous fluorescence instant tumor liquid biopsy strategy based on DNA functionalized nanospheres, and the specific operation is as follows:

[0052] 10 μM of PD-L1 aptamer (5 μL), 10 μM of streptavidin-biotin-H1 to H4 (5 μL), and 65 μM of Cu 2+ (10 μL) were mixed with 70 μL of 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (10 mM MOPS, 150 mM NaCl, 50 mM MgCl2, pH 7.4), and incubated at room temperature for 2 hours. Then 1 mL of A549 cells or A549 cell-derived exosomes of different concentrations were added, and reacted at room temperature for 45 minutes. Subsequently, 4 μL of QDs solution (concentration was 1 / 10 of the original solution) was added, and reacted for 75 seconds. Fluorescence detection was performed under 365 nm excitation wavelength.

[0053] The pattern and size of the test strip were designed using Adobe Illustrator 2021, and Whatman chromatography paper was prepared into corresponding test strips using inkjet printing technology, and then sealed in a plastic bag and placed in a dark environment at room temperature for drying. According to the above experimental procedure, 10 μL of reaction solution was dropped on the test strip and irradiated under ultraviolet light (UV). The color change corresponding to different marker concentrations was observed with the naked eye. A smartphone was used to take a picture, and the RGB value was analyzed using the colorpicker application (version 0.0.20).

[0054] Example 2

[0055] This example provides a homogeneous fluorescence instant tumor liquid biopsy strategy based on DNA functionalized nanospheres, and the specific operation is as follows:

[0056] PD-L1 aptamer (5 μL), 20 μM streptavidin-biotin-H1 to H4 (5 μL) and 70 μM Cu 2+ (10 μL) were mixed with 70 μL 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (10 mM MOPS, 150 mM NaCl, 50 mM MgCl2, pH 7.4) and incubated at room temperature for 4 hours. Then 1 mL of A549 cells or A549 cell-derived exosomes with different concentrations were added and reacted at room temperature for 30 minutes. Subsequently, 5 μL of QDs solution (concentration was 1 / 10 of the original solution) was added and reacted for 60 seconds. Fluorescence detection was performed under 365 nm excitation wavelength.

[0057] The pattern and size of the test strip were designed using Adobe Illustrator 2021, and Whatman chromatography paper was prepared into corresponding test strips using inkjet printing technology, and then sealed in plastic bags and placed in a dry, dark environment at room temperature. According to the above experimental procedure, 11 μL of reaction solution was dropped on the test strip and irradiated under ultraviolet light (UV). The color change corresponding to different marker concentrations was observed with the naked eye. A smartphone was used to take a picture, and the RGB value was analyzed using the colorpicker application (version 0.0.20).

[0058] Example 3

[0059] This example provides a homogeneous fluorescence instant tumor liquid biopsy strategy based on DNA functionalized nanospheres, and the specific operation is as follows:

[0060] PD-L1 aptamer (5 μL), 20 μM streptavidin-biotin-H1 to H4 (5 μL) and 70 μM Cu 2+ (10 μL) were mixed with 70 μL 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (10 mM MOPS, 150 mM NaCl, 50 mM MgCl2, pH 7.4) and incubated at room temperature for 4 hours. Then 1 mL of A549 cells or A549 cell-derived exosomes with different concentrations were added and reacted at room temperature for 30 minutes. Subsequently, 5 μL of QDs solution (concentration was 1 / 10 of the original solution) was added and reacted for 60 seconds. Fluorescence detection was performed under 365 nm excitation wavelength.

[0061] The patterns and sizes of test strips were designed with Adobe Illustrator 2021, and Whatman chromatography paper was prepared into corresponding test strips using inkjet printing technology, and then sealed in plastic bags and placed in a dark environment at room temperature for drying. According to the above experimental procedure, 12 μL of reaction solution was dropped on the test strip and placed under ultraviolet (UV) irradiation. The color change corresponding to different marker concentrations was observed with the naked eye. Photographs were taken using a smartphone, and the RGB values were analyzed using the colorpicker application (version 0.0.20).

[0062] Experimental Example

[0063] 1. Optimization of PD-L1 detection conditions and evaluation of analytical performance

[0064] Before constructing a rapid liquid biopsy strategy for tumor CTCs or TDEs, the key analysis conditions were first optimized using PD-L1 protein, and the analytical performance of the method was evaluated, as shown in Figure 2 In the analysis conditions, according to the previous research work of the team, in order to obtain the best results, special attention was paid to the Cu 2+ concentration ( Figure 2 a-b) and the amount of QDs ( Figure 2 c-d), 10 pg / mL PD-L1 experimental group compared with blank group, 65 μM Cu 2+ The signal-to-noise ratio is the highest. In order to shorten the detection time as much as possible, the reaction kinetics after the addition of quantum dots was studied. After the addition of quantum dots, the quantum dot signal changed rapidly within 60 seconds, and the quantum dot signal gradually stabilized after 75 seconds. Finally, 75 seconds was selected as the balance point between the complete reaction of quantum dots and Cu 2+ cations and the signal-to-noise ratio of the system ( Figure 2 e).

[0065] After optimizing the key detection conditions, the analytical performance of PD-L1 protein detection was evaluated. As the concentration of PD-L1 protein gradually increased, the fluorescence signal gradually weakened ( Figure 3 a-b). Especially in the concentration range of 1-10 4 fg / mL, the fluorescence signal showed a good linear relationship with the logarithmic value of PD-L1 concentration ( Figure 3c) The detection limit of this assay was 0.6 fg / mL (calculated based on the method of three times signal-to-noise ratio) within 46 min, realizing the rapid and sensitive detection of PD-L1. Compared with the recently reported PD-L1 detection methods, the detection time was shortened by 4.5-7.8 times. To evaluate the specificity, 17 potential interfering proteins were tested. Low concentrations of PD-L1 (100 fg / mL and 10 pg / mL) caused significant signal changes compared with the blank solution, while high concentrations of 17 interferents (10 ng / mL) showed comparable signals to the blank group, showing excellent selectivity Figure 3 d) In general, the good analytical performance of this method for detecting PD-L1 protein laid a solid foundation for subsequent detection of CTCs or TDEs.

[0066] 2, Analytical performance of tumor A549 cells

[0067] A549 cells were used as a research model to evaluate the analytical performance of this strategy for detecting CTCs. Under the optimal experimental conditions, the fluorescence intensity of QDs decreased with the increase of A549 cell concentration Figure 4 a-b) The fluorescence signal was in good linear relationship with the logarithmic value of cell concentration in the range of 10 2 -10 5 cells / mL Figure 4 c) The linear equation was Y = -268LogC + 2185, R 2 = 0.992, and the detection limit (LOD) based on three times signal-to-noise ratio was 75 cells / mL. Compared with the recently reported methods, the detection time of this method was shorter, and the sensitivity was comparable.

[0068] Subsequently, test strips were prepared by inkjet printing technology according to the pre-designed pattern to further improve the portability and economy of the method. 10 μL of the reaction solution was dropped on the circular detection area of the test strip, and the A549 cell concentration-dependent color change was observed under the ultraviolet lamp box. With the increase of tumor cell concentration, the color of the test strip gradually changed from magenta to indigo, realizing the semi-quantitative visualization of the detection results Figure 4 d) The color corresponding to different A549 cell concentrations was converted to RGB values based on the smart phone RGB analysis mode, further improving the accuracy of quantitative detection. Analysis of the RGB values showed that there was a good linear relationship between the R / B ratio and the logarithmic value of A549 cell concentration in the range of 100 to 10 5 cells / mL Figure 4 e), which indicated that this method could realize rapid and convenient detection of CTCs.

[0069] 3, Detection feasibility of tumor cell-derived exosomes

[0070] After verifying the effect of the detection method of the present application on tumor cell detection, tumor cell-derived exosomes were used as another important liquid biopsy marker to evaluate the universality of the detection method of the present application for detection of different types of liquid biopsy markers, such as Figure 5 .

[0071] Before evaluating the analytical performance of the method for detecting tumor cell-derived exosomes, exosomes were first extracted from A549 cell culture supernatant and characterized to prove the purity and integrity of the extracted exosomes for subsequent analytical performance evaluation. The specific operation is as follows: ultracentrifugation method was used to separate and enrich exosomes Figure 6 a). Subsequently, the extracted exosomes were characterized in terms of morphology, size, particle size distribution and characteristic protein expression. First, nanoparticle tracking analysis (NTA) characterized the concentration and particle size distribution of TDEs, which was 1.4×10 8 particles / mL, with an average particle size of 123.6 nm Figure 6 b). Capillary immunoblotting assay was used to analyze the characteristic proteins of exosomes such as CD63, CD9, CD81 and target protein PD-L1 Figure 6 c). By negative staining transmission electron microscopy (TEM), the characteristic cup-shaped morphology was found Figure 6 d), with an average size of 118.5 nm, which was consistent with the NTA result. These results showed that the intact high-purity TDEs were successfully separated.

[0072] 4. Analytical performance of TDEs

[0073] After proving the separation of intact high-purity tumor A549 cell-derived exosomes, the ability of the detection method of the present application for detecting TDEs was evaluated under the optimal analysis condition. Subsequently, the analytical performance of the method for detecting TDEs was evaluated, and the results showed that the fluorescence signal decreased with the increase of TDEs concentration, which had a good linear relationship in the range of 10 4 -10 7 particles / mL Figure 7 a-c). The linear equation was Y=-203LogC+2261, R 2 =0.992, and the detection limit (LOD) based on three times the signal-to-noise ratio was 8.8×10 3 particles / mL. The color corresponding to different TDEs concentrations was analyzed using a smart phone-RGB mode. In the range of 10 4 -10 7 particles / mL, a good linear relationship between the R / B ratio and the TDEs concentration was observed Figure 7d) The sensitivity of the detection method of the present application is comparable to the latest TDEs detection method in the prior art, but the detection time is greatly shortened, which indicates that it has the potential for rapid liquid biopsy application. By comparing the fluorescence signal changes of TDEs, blank control, human embryonic kidney 293T cells (HEK-293T), human normal lung epithelial cells (BEAS-2B), human normal liver cells (L02) and human umbilical vein endothelial cells (HUVEC) derived exosomes, the good selectivity of the method is verified Figure 7 e) In summary, the method of the present application has good versatility and reliability in tumor cell and extracellular vesicle biosensing.

[0074] The detection method of the present application synthesizes protein scaffold DNA-Cu by one-step method at room temperature by combining PD-L1 aptamer, protein-DNA and DNA-metal ion 2+ Multifunctional integrated nanospheres. Based on the functional integration of the nanospheres, one-step detection of CTCs or TDEs is realized, and combined with test strip and smart phone RGB analysis technology, the "drop and see" convenient analysis is realized, which can meet the rapid and convenient detection demand of tumor liquid biopsy, and provide an effective means for early disease screening.

[0075] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited to this, any skilled person in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A homogeneous fluorescent instant tumor liquid biopsy strategy based on DNA functionalized nanospheres, characterized by: The steps include: (1) Biotinylated DNA hairpin binds to streptavidin to form a cross-shaped protein scaffold-DNA tetramer; Add PD-L1 aptamer and Cu to the cross-shaped protein scaffold-DNA tetramer 2+ and buffer solution, and after mixed reaction, DNA functionalized nanospheres were obtained; The cross-shaped protein scaffold-DNA tetramer is composed of streptavidin-biotin-H1, streptavidin-biotin-H2, streptavidin-biotin-H3 and streptavidin-biotin-H4; The nucleotide sequences of the four hairpins H1, H2, H3, and H4 in the biotinylated DNA hairpin are shown as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, respectively; The nucleotide sequence of the PD-L1 aptamer is shown in SEQ ID No. 5; (2) Add a tumor liquid biopsy marker that highly expresses PD-L1 to the DNA functionalized nanospheres obtained in step (1), react at room temperature, and then add the recognition Cu 2+ The fluorescent probe is used to drop the reaction liquid on the test strip, and the mobile phone is used to perform RGB color analysis under ultraviolet irradiation, which is an instant detection of tumor liquid biopsy.

2. The homogeneous fluorescence instant tumor liquid biopsy strategy according to claim 1, characterized in that: In step (1), the biotinylated DNA hairpin includes biotin-H1, biotin-H2, biotin-H3 and biotin-H4.

3. The homogeneous fluorescence instant tumor liquid biopsy strategy according to claim 1, characterized in that: In step (1), the buffer is 3-(N-morpholino)propanesulfonic acid buffer.

4. The homogeneous fluorescence instant tumor liquid biopsy strategy according to claim 1, characterized in that: In step (1), the mixing reaction is carried out at 20-28°C for 2-6 hours.

5. The homogeneous fluorescence instant tumor liquid biopsy strategy according to claim 1, characterized in that: In step (2), the tumor liquid biopsy marker that highly expresses PD-L1 is circulating tumor cells or tumor cell-derived exosomes, and the reaction at room temperature is 22-25°C for 30-60 minutes.

6. The homogeneous fluorescence instant tumor liquid biopsy strategy according to claim 5, characterized in that: The tumor cells are A549 cells.

7. The homogeneous fluorescence instant tumor liquid biopsy strategy according to claim 1, characterized in that: In step (2), the identification Cu 2+ The fluorescent probes are QDs, calcein and Cu 2+ -Any of the probes.

Images