Homogeneous fluorescence instant tumor liquid biopsy strategy based on DNA functionalized nanospheres

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a protein scaffold DNA-Cu < 2 + > multifunctional integrated nanosphere synthesized by combining a programmed death ligand 1 (PD-L1) aptamer, protein-DNA and DNA-metal ions through a room-temperature one-step method based on a homogeneous fluorescence instant tumor liquid biopsy strategy of a DNA functionalized nanosphere. Based on the function integration of the nanospheres, one-step detection of circulating tumor cells (CTCs) or tumor-derived exosomes (TDEs) is realized, a plurality of analysis modes including fluorescence detection and smartphone-RGB are provided by combining a test strip and smartphone red, green and blue (RGB) analysis technologies, and a rapid and sensitive tumor liquid biopsy instant detection method is constructed. The method realizes convenient analysis of'visible while dropping ', can meet the rapid and convenient detection requirements of tumor liquid biopsy, and provides an effective means for early screening of diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and specifically relates to a homogeneous fluorescent instant tumor liquid biopsy strategy based on DNA functionalized nanospheres. Background Art

[0002] Tumors remain a major disease that threatens human health and is the leading cause of death. Liquid biopsy is an attractive alternative to pathological biopsy due to its minimal invasiveness, which helps in 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 primary tumors, and the changes in their concentration levels are parallel to the evolution of tumors. TDEs are secreted into the blood relatively early, with higher abundance than CTCs and higher stability than 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 concentrations in the early stages of tumorigenesis, high heterogeneity, and interference from normal host cells and the large number of exosomes they secrete. Traditional detection methods, such as polymerase chain reaction (PCR), immunofluorescence, flow cytometry, nanoparticle tracking analysis, and Western blotting, often rely on large precision instruments, signal labeling techniques, and cumbersome and time-consuming operation steps, which limits their widespread popularity and applicability in community medical care and resource-limited areas. Therefore, there is an urgent need to build a fast 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 fixation and modification of heterogeneous interfaces, which limits the simplicity and popularity of the method. The development of a homogeneous one-step reaction strategy can eliminate labor-intensive experimental operation processes, shorten detection time, reduce dependence on professionals, and improve practicality. In addition, this one-step sensing strategy can be integrated into various miniaturized and portable detection instruments in different application scenarios, and has broad medical application potential. For example, integrating a one-step reaction on paper by deposition, screen printing and other methods can give the sensing strategy multiple advantages, such as no threshold for operation, environmental friendliness and easy handling, which is expected to promote the development of bedside testing (POCT). However, due to the limited sensitivity of colorimetric paper-based POCT, it cannot meet the needs of tumor liquid biopsy. Summary of the invention

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

[0005] The present invention first synthesizes the protein scaffold DNA-Cu by a one-step process at room temperature. 2+ Functional nanospheres, the composite nanospheres combine multiple nanotechnologies such as functional nucleic acids, protein-DNA and DNA-metal ions, realizing the integration of target recognition, signal amplification and conduction. The "one ball for multiple uses" of the nanospheres realizes the one-step reaction of tumor CTCs and TDEs within 1 hour. CdTe quantum dots (QDs) with excellent optical properties are selected as signal molecules, and combined with test strips and smartphone red, green and blue (RGB) analysis technology, multiple analysis modes including fluorescence detection and smartphone-RGB are provided to construct a rapid and sensitive tumor liquid biopsy instant detection method. Tumor A549 cells and A549 cell-derived exosomes separated and enriched by ultracentrifugation are used as research models, and the effectiveness and robustness of the strategy are demonstrated by fluorescence and smartphone-RGB multi-mode analysis. In summary, the present invention provides a fast and convenient CTCs and TDEs fluorescence instant detection strategy, which will provide a solution for the rapid analysis of tumor liquid biopsy.

[0006] The technical solution adopted by the present invention is:

[0007] A homogeneous fluorescent instant tumor liquid biopsy strategy based on DNA functionalized nanospheres, comprising the following steps:

[0008] (1) Biotinylated DNA hairpin binds to streptavidin (SA) to form a cross-shaped protein scaffold-DNA tetramer;

[0009] Adding programmed death ligand 1 (PD-L1) aptamer, Cu 2+ and buffer, and after mixed reaction, DNA functionalized nanospheres are obtained;

[0010] (2) Adding a tumor liquid biopsy marker that highly expresses PD-L1 to the DNA functionalized nanospheres obtained in step (1), reacting at room temperature, and adding a Cu 2+ The fluorescent probe is used to drop the reaction liquid on the test strip, and the red, green, and blue (RGB) color analysis is performed by the mobile phone under ultraviolet light, which is the instant detection of tumor liquid biopsy.

[0011] In step (1), the biotinylated DNA hairpin includes 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 as 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-morpholinyl)propanesulfonic acid (MOPS) buffer;

[0016] In step (1), the mixing reaction is carried out at 20-28° C. 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 reaction at room temperature is 22-25° C. for 30-60 minutes.

[0018] The CTCs are A549 cells.

[0019] In step (2), the identification Cu 2+ The fluorescent probes are QDs, calcein and Cu 2+ -Any of the probes.

[0020] The specific operation of the fluorescence detection is: adding recognition Cu 2+ The fluorescent probe was reacted for 75 seconds and fluorescence detection was performed under a 365nm laser wavelength.

[0021] Based on previous research, the inventors of this application prepared a DNA-Cu nanoparticle with streptavidin-biotin as the protein scaffold, inspired by protein-DNA nanotechnology, functional nucleic acids and metal-DNA coordination effects. 2+ Composite nanospheres can realize the multifunctional integration of target recognition, signal transduction and amplification. Based on the functionally integrated nanospheres, and further combined with the independently designed test strips, a rapid tumor liquid biopsy strategy with convenient operation has been developed. PD-L1, which is highly expressed in tumor cells, was selected as the detection target, and the corresponding biotin-coupled DNA hairpin was designed according to the PD-L1 aptamer sequence. Streptavidin (SA) binds to biotin with high affinity, and the DNA hairpin is anchored to SA to form a cross-shaped protein scaffold-DNA tetramer. The PD-L1 aptamer is used as the trigger chain to trigger a bidirectional cross-linking hybridization chain reaction between multiple DNA tetramers. In Cu 2+ Self-assembly of protein scaffold DNA-Cu under coordination with DNA base pairs 2+Nanospheres. The inventors of this application have further found that after the nanospheres encounter tumor cells or exosomes that highly express 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 bind to them with high affinity and specificity. 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 disaggregate 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 inventors of this application used inkjet printing technology to make test strips. 10μL of reaction solution was dropped on the circular detection area with a diameter of 1cm on the test strip, and the color change corresponding to the concentration of tumor cells or exosomes could be observed by naked eyes under ultraviolet light. In addition, taking pictures with a smartphone and converting the colors into RGB values ​​can be performed for more accurate quantitative analysis.

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

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

[0024] The beneficial effects of the present invention are:

[0025] The present invention provides a homogeneous fluorescent instant tumor liquid biopsy strategy based on DNA functionalized nanospheres, which first integrates PD-L1 aptamers, protein scaffolds, hybridization chain reactions and DNA and Cu 2+ Interaction, construction of protein scaffold DNA-Cu 2+ Composite nanospheres; then to the DNA-Cu 2+ CTCs or TDEs are added to the composite nanospheres. After the reaction, CdTe QDs with excellent optical properties are selected as signal molecules, and combined with test strips and smartphone-RGB analysis technology, multiple analysis modes including fluorescence detection and smartphone-RGB are provided to construct a rapid and sensitive tumor liquid biopsy instant detection method. Tumor A549 cells and A549 cell-derived exosomes separated and enriched by ultracentrifugation are used as research models, and the effectiveness and stability of the detection method are demonstrated by fluorescence and smartphone-RGB multiple mode analysis. In summary, the detection method of the present invention, as a fast and convenient CTCs and TDEs fluorescence instant detection strategy, will provide a new method for the rapid analysis of tumor liquid biopsy. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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 or the description of the prior art 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.

[0027] Figure 1 The invention shows that the protein scaffold DNA-Cu 2+ Schematic diagram of a rapid one-step universal detection strategy for tumor CTCs using functional nanospheres;

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

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

[0030] Figure 4 Figure 3 shows the fluorescence and smartphone-RGB analysis performance of CTCs; (a)-(b) shows the decrease in the fluorescence intensity of QDs with the increase in A549 cell concentration; (c) shows the fluorescence intensity of QDs at 10 2 -10 5 The logarithm of the cell concentration in the range of 100 cells / mL showed a good linear relationship; (d) as the tumor cell concentration increased, the color of the test strip gradually changed from magenta to indigo, achieving semi-quantitative visualization of the test results; (e) in the range of 100 to 10 5 In the range of cells / mL, there was a good linear relationship between the R / B ratio and the logarithm of A549 cell concentration;

[0031] Figure 5 The invention shows that the protein scaffold DNA-Cu 2+ Schematic diagram of the rapid one-step universal detection strategy for TDEs using functional nanospheres;

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

[0033] Figure 7 Shows the analytical performance of TDEs; (a)-(c) shows that the fluorescence signal decreases with the increase of TDEs concentration. 4 -10 7 There is a good linear relationship in the range of particles / mL; (d) 4 -10 7 In the particle / mL range, a good linear relationship was observed between the R / B ratio and the logarithmic value of the TDEs concentration; (e) The good selectivity of the detection method of the present invention was verified by comparing the changes in the fluorescence signals of exosomes from different cell sources. DETAILED DESCRIPTION

[0034] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0035] The protein scaffold DNA-Cu 2+ Schematic diagram of a rapid one-step universal detection strategy for tumor CTCs using functional nanospheres Figure 1 First, by combining PD-L1 aptamer, streptavidin-biotin-coupled DNA hairpin and Cu 2+ , a one-step synthesis of protein scaffold DNA-Cu at room temperature 2+ Functional nanospheres. PD-L1, which is highly expressed in tumor cells, was selected as the detection target, and the corresponding biotin-coupled DNA hairpin was designed according to the PD-L1 aptamer sequence (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 a bidirectional cross-linking hybridization chain reaction between multiple DNA tetramers. 2+ Self-assembly of protein scaffold DNA-Cu under coordination with DNA base pairs 2+ Nanospheres.

[0036] When the nanosphere encounters tumor cells or exosomes that highly express PD-L1, the PD-L1 on the surface of the tumor cells or exosomes induces the aptamers in the nanosphere to fold into a specific secondary structure and bind to it with high affinity and specificity. 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 disaggregate and release a large amount of Cu 2+ The released Cu 2+ It can be recognized by QDs. In order to improve the ease of operation, the test strips were made using inkjet printing technology. 10μL of the reaction solution was dropped on the circular detection area with a diameter of 1cm on the test strip, and the color change corresponding to the concentration of tumor cells or exosomes can be observed by naked eyes under ultraviolet light. In addition, taking pictures with a smartphone can convert the color into RGB values ​​for more accurate quantitative analysis.

[0037] Materials and Reagents

[0038] Oligonucleotides with different sequences were synthesized and purified by Sangon Biotechnology Co., Ltd. (Shanghai, China), and their sequences are shown in Table 1. Copper sulfate pentahydrate (CuSO 4 ·5H 2 O), sodium tellurite (Na 2 TeO 3 ) and 3-mercaptopropionic acid (MPA) were ordered from Aladdin Reagent Co., Ltd. (Shanghai, China). Sodium chloride (NaCl), trisodium citrate dihydrate (Na 3 C 6 H 5 O 7 ·2H 2 O), cadmium chloride (CdCl 2 ), potassium borohydride (KBH 4 ), hydrochloric acid (HCl), sodium hydroxide (NaOH), magnesium chloride (MgCl 2) were purchased from Cologne Chemical Reagent Company (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 Shenggong Biotechnology Co., Ltd. Human serum albumin (HSA), trypsin, glucose oxidase (GOD), immunoglobulin G (IgG), pepsin, prostate-specific antigen (PSA), transferrin, interferon-γ (IFN-γ), papain, glypican protein 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 (10 mM MOPS, 150 mM NaCl, 50 mM MgCl 2 , pH 7.4). Phosphate buffered saline (PBS) was ordered from Corning (New York, USA). Dulbecco's medium (DMEM / F12), fetal bovine serum (FBS), penicillin / streptomycin and syringe filters (0.22 μm) were purchased from GibcoInvitrogen (California, USA). Ultraclean centrifuge tubes (25×89 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 the present invention are analytical grade or higher and do not require further purification when used. All experimental water uses the water purification system of Chengdu Ultrapure 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 hepatocytes (L02), and human normal lung epithelial cells (BEAS-2B) were provided by the Research Base of West China Hospital. All solutions were stored in a refrigerator at 4°C before use.

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

[0040]

[0041]

[0042] Synthesis of CdTe QDs

[0043] The common synthesis process of CdTe QDs has been reported previously. The main operation is as follows: first, prepare a mixture containing CdCl 2 (0.5 mM) and trisodium citrate (0.2 g) in 50 mL solution. Then, mercaptopropionic acid (MPA, 52 μL) was immediately added to the above solution, and the pH of the solution was adjusted to 10.5 with NaOH. 2 TeO 3 (0.1 mM) and KBH 4 (50 mg) was refluxed in batches to obtain CdTe QDs. Subsequently, it was precipitated with n-propanol and centrifuged (11000 rpm) to obtain high-purity CdTe QDs. The purified red CdTe QDs were redispersed in ultrapure water before use.

[0044] Cell recovery, culture and passaging

[0045] Cell recovery: Take the frozen A549 cells out of the liquid nitrogen tank, thaw quickly at 37°C, and transfer to a centrifuge tube. Add 5 mL of DMEM / F-12 medium containing 10% (v / v) FBS and centrifuge at 1000 rpm for 3 minutes. Discard the supernatant, add 5 mL of medium, and transfer to a culture dish. Place the cells at 37°C, 5% CO 2 Cultivated under conditions.

[0046] Cell culture: A549 cells were cultured in 1640 medium containing 10% (v / v) FBS and maintained at 37°C and 5% CO. 2 When the cells are in the logarithmic growth phase, they are digested into a single cell suspension and counted. Finally, the cells are plated in a culture dish.

[0047] Cell passaging: When the cells are in the logarithmic growth phase, remove the old medium and wash with PBS buffer. Subsequently, digest with trypsin and centrifuge at 800 rpm for 3 minutes to collect the cells. Then add fresh medium. Finally, inoculate the cells in a new culture dish and continue to culture.

[0048] Isolation of exosomes

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

[0050] Example 1

[0051] This embodiment provides a homogeneous fluorescent instant tumor liquid biopsy strategy based on DNA functionalized nanospheres, and the specific operations are as follows:

[0052] 10 μM PD-L1 aptamer (5 μL), 10 μM streptavidin-biotin-H1 to H4 (5 μL) and 65 μM Cu 2+ (10 μL) and 70 μL 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (10 mM MOPS, 150 mM NaCl, 50 mM MgCl 2 , pH 7.4) and incubated at room temperature for 2 hours. Then, 1 mL of A549 cells or exosomes from A549 cells at 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 at an excitation wavelength of 365 nm.

[0053] The pattern and size of the test strips were designed using Adobe Illustrator 2021, and the Whatman chromatography paper was prepared into the corresponding test strips using inkjet printing technology, which were sealed in a plastic bag and placed in a dry, dark environment at room temperature. According to the above experimental process, 10 μL of the reaction solution was dropped on the test strip and placed under ultraviolet (UV) irradiation. The color changes corresponding to different marker concentrations were observed with the naked eye. Photos were taken using a smartphone, and the RGB values ​​were analyzed using the colorpicker application (version 0.0.20).

[0054] Example 2

[0055] This embodiment provides a homogeneous fluorescent instant tumor liquid biopsy strategy based on DNA functionalized nanospheres, and the specific operations are as follows:

[0056] 15 μM PD-L1 aptamer (5 μL), 15 μM streptavidin-biotin-H1 to H4 (5 μL) and 50 μM Cu 2+ (10 μL) and 70 μL 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (10 mM MOPS, 150 mM NaCl, 50 mM MgCl 2 , pH 7.4) and incubated at room temperature for 4 hours. Then, 1 mL of A549 cells or exosomes from A549 cells at 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 at an excitation wavelength of 365 nm.

[0057] The pattern and size of the test strips were designed using Adobe Illustrator 2021, and the Whatman chromatography paper was prepared into the corresponding test strips using inkjet printing technology, which were sealed in a plastic bag and placed in a dry, dark environment at room temperature. According to the above experimental process, 11 μL of the reaction solution was dropped on the test strip and placed under ultraviolet (UV) irradiation. The color changes corresponding to different marker concentrations were observed with the naked eye. Photos were taken using a smartphone, and the RGB values ​​were analyzed using the colorpicker application (version 0.0.20).

[0058] Example 3

[0059] This embodiment provides a homogeneous fluorescent instant tumor liquid biopsy strategy based on DNA functionalized nanospheres, and the specific operations are as follows:

[0060] 20 μM PD-L1 aptamer (5 μL), 20 μM streptavidin-biotin-H1 to H4 (5 μL) and 70 μM Cu 2+ (10 μL) and 70 μL 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (10 mM MOPS, 150 mM NaCl, 50 mM MgCl 2 , pH 7.4) and incubated at room temperature for 6 hours. Then, 1 mL of A549 cells or exosomes from A549 cells at different concentrations were added and reacted at room temperature for 60 minutes. Subsequently, 8 μL of QDs solution (concentration was 1 / 10 of the original solution) was added and reacted for 90 seconds. Fluorescence detection was performed at an excitation wavelength of 365 nm.

[0061] The pattern and size of the test strips were designed using Adobe Illustrator 2021, and the Whatman chromatography paper was prepared into the corresponding test strips using inkjet printing technology, which were sealed in a plastic bag and placed in a dry, dark environment at room temperature. According to the above experimental process, 12 μL of the reaction solution was dropped on the test strip and placed under ultraviolet (UV) irradiation. The color changes corresponding to different marker concentrations were observed with the naked eye. Photos 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 analytical conditions were first optimized using the PD-L1 protein and the analytical performance of the method was evaluated, such as Figure 2 In the analysis conditions, according to the previous research work of the present invention team, special attention was paid to Cu in order to obtain the best results. 2+ concentration( Figure 2 ab) and QDs dosage ( Figure 2 cd), 10pg / mL PD-L1 experimental group compared with the blank group, 65μM Cu 2+ 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 of reaction time, and the quantum dot signal gradually stabilized after 75 seconds. Finally, 75 seconds was selected as the reaction time between quantum dots and Cu 2+ The equilibrium point between the complete cation exchange reaction 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 ab). Especially in 1-10 4 fg / mL concentration range, the fluorescence signal and the logarithmic value of PD-L1 concentration showed a good linear relationship ( Figure 3c). The detection method achieved a detection limit of 0.6fg / mL in 46 minutes (based on the three-fold signal-to-noise ratio calculation method), achieving rapid and sensitive detection of PD-L1. Compared with the most recent PD-L1 detection method, the detection time was shortened by 4.5-7.8 times. In order to evaluate the specificity, the effects of 17 potential interfering proteins on the detection system were tested. Compared with the blank solution, low concentrations of PD-L1 (100fg / mL and 10pg / mL) caused significant signal changes, while the high concentration of 17 interferents (10ng / mL) The signal was comparable to the blank group, showing excellent selectivity ( Figure 3 d). In general, the good analytical performance of this method for detecting PD-L1 protein lays a solid foundation for subsequent CTCs or TDEs detection.

[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 ab). Fluorescence signal at 10 2 -10 5 There is a good linear relationship between the logarithm of cell concentration and the cell concentration in the range of cells / mL ( Figure 4 c). The linear equation is Y = -268LogC + 2185, R 2 =0.992, and the detection limit (LOD) based on a three-fold signal-to-noise ratio is 75 cells / mL. Compared with the latest method proposed in the prior art, the detection method of the present invention has a shorter detection time and comparable sensitivity.

[0068] Subsequently, the test strips were prepared according to the pre-designed pattern using inkjet printing technology 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 color changes dependent on the concentration of A549 cells were observed with the naked eye under a UV light box. As the concentration of tumor cells increased, the color of the test strip gradually changed from magenta to indigo, achieving semi-quantitative visualization of the test results ( Figure 4 d). The colors corresponding to different A549 cell concentrations were converted into RGB values ​​using the smartphone-based RGB analysis mode, further improving the accuracy of quantitative detection. Analysis of RGB values ​​showed that the concentration of A549 cells in the range of 100 to 10 5 cells / mL, and there was a good linear relationship between the R / B ratio and the logarithmic value of A549 cell concentration ( Figure 4 e), which indicates that this method can achieve rapid and convenient CTCs detection.

[0069] 3. Feasibility of Detection of Tumor Cell-derived Exosomes

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

[0071] Before evaluating the analytical performance of this method for detecting tumor cell-derived exosomes, exosomes were first extracted from A549 cell culture supernatant and a series of characterizations were performed to prove the purity and integrity of the extracted exosomes for subsequent analytical performance evaluation. The specific operations were as follows: ultracentrifugation 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). Exosome characteristic proteins such as CD63, CD9, CD81 and target protein PD-L1 were analyzed using capillary immunoblotting assay ( Figure 6 c). Negative staining transmission electron microscopy (TEM) revealed its characteristic cup-shaped morphology ( Figure 6 d), the average size was 118.5 nm, which is consistent with the NTA results. These results indicate that intact and high-purity TDEs were successfully isolated.

[0072] 4. TDEs analysis performance

[0073] After proving the isolation of complete and highly pure exosomes from tumor A549 cells, the ability of the detection method of the present invention to detect TDEs was evaluated under optimal analytical conditions. 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 TDE concentration, and at 10 4 -10 7 There is a good linear relationship in the range of particles / mL ( Figure 7 ac). The linear equation is Y = -203LogC + 2261, R 2 =0.992, and the detection limit (LOD) based on three times the signal-to-noise ratio is 8.8×10 3 The color corresponding to different TDEs concentrations was analyzed using smartphone-RGB mode. 4 -10 7 In the range of particles / mL, a good linear relationship was observed between the R / B ratio and the TDEs concentration ( Figure 7d). The sensitivity of the detection method of the present invention is comparable to the latest TDEs detection method proposed in the prior art, but the detection time is greatly shortened, which indicates that it has the potential for rapid liquid biopsy applications. The good selectivity of the method was verified by comparing the fluorescence signal changes of exosomes derived from 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). Figure 7 e). In summary, the method of the present invention has good versatility and reliability in tumor cell and extracellular vesicle biosensing.

[0074] The detection method of the present invention combines PD-L1 aptamer, protein-DNA and DNA-metal ion to synthesize protein scaffold DNA-Cu in one step at room temperature. 2+ Multifunctional integrated nanospheres. Based on the functional integration of the nanospheres, one-step detection of CTCs or TDEs is achieved, and combined with test strips and smartphone RGB analysis technology, convenient "instant drop, instant results" analysis is achieved, which can meet the needs of rapid and convenient detection of tumor liquid biopsies and provide an effective means for early screening of diseases.

[0075] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A homogeneous fluorescent instant tumor liquid biopsy strategy based on DNA functionalized nanospheres, characterized in that: 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, and after mixed reaction, DNA functionalized nanospheres are obtained; (2) Adding a tumor liquid biopsy marker that highly expresses PD-L1 to the DNA functionalized nanospheres obtained in step (1), reacting at room temperature, and adding a 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 light, 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 2, characterized in that: 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.

4. The homogeneous fluorescence instant tumor liquid biopsy strategy according to claim 2 or 3, characterized in that: 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.

5. The homogeneous fluorescence instant tumor liquid biopsy strategy according to claim 1, characterized in that: In step (1), the nucleotide sequence of the PD-L1 aptamer is shown in SEQ ID No.

5.

6. 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.

7. The homogeneous fluorescent 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.

8. The homogeneous fluorescent 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.

9. The homogeneous fluorescent instant tumor liquid biopsy strategy according to claim 8, characterized in that: The tumor cells are A549 cells.

10. The homogeneous fluorescent 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.

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