Bionic dandelion isothermal amplification system and application thereof

Abstract

The application belongs to the technical field of cell detection, and particularly relates to a bionic Taraxacum isothermal amplification system and application thereof. The bionic Taraxacum isothermal amplification system is composed of six-legged DNA walker, nonlinear DNA self-assembly technology and an asymmetric carrier, AuFe Janus nanoparticles, with high signal probe loading efficiency, and shows significant amplification efficiency. Compared with a traditional isothermal amplification system, the amplification efficiency of the bionic Taraxacum isothermal amplification system is increased by about 6.72 times.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cell detection, and particularly relates to a biomimetic dandelion isothermal amplification system and application thereof. BACKGROUND

[0002] Detection of circulating tumor cells (CTCs) has important clinical and scientific value. In terms of tumor metastasis detection, it can capture early signs of tumor cells shedding from the primary tumor site and invading the blood, providing important clues for early assessment of metastasis risk. In terms of treatment regimen selection, it can be used to guide individualized treatment and monitor and evaluate treatment effect in real time to optimize treatment regimen in a timely manner. In terms of prognosis evaluation, it can predict recurrence risk and judge the survival prognosis of patients. In addition, CTCs also play a key role in elucidating complex metastasis mechanisms, tracing cancer evolution, and promoting innovation of liquid biopsy technology.

[0003] Although the current CTC detection methods have great potential, they also face some challenges. For example, these existing technologies lack sufficient sensitivity, often missing low-abundance cells; complex procedures can compromise cell viability and integrity; the lack of standardized protocols further complicates the comparability of results; and high-precision CTC detection relies on expensive advanced instruments such as single-cell sequencing platforms and microfluidic chip devices. These factors limit the widespread adoption of CTC detection in primary health care institutions and large-scale screening scenarios.

[0004] CTCs are extremely rare in peripheral blood, so improving the sensitivity of the detection method is one of the key problems to be solved. In order to improve the sensitivity of CTC detection, isothermal amplification technology has been explored. For example, rolling circle amplification (RCA) combined with surface-enhanced Raman scattering and specific aptamers can achieve ultra-sensitive detection and non-destructive recovery of CTCs. Mn 2+ Dependence on DNAzyme enzyme cutting technology combined with parallel catalytic hairpin assembly technology significantly improves the sensitivity of CTC detection. CRISPR-Cas-based isothermal amplification technology and other optimization methods and their combinations also show potential. In addition, dynamic DNA technology (DNA nanomachines and DNA walkers) has become a powerful tool for high-sensitivity CTC detection due to its excellent controllability and functionality. SUMMARY

[0005] The application is based on nanosynthesis technology, DNA walker technology and the like to develop a bionic dandelion isothermal amplification system, which is a signal probe carrier and a 3D track for DNA walker walking by using streptavidin modified AuFe Janus nanoparticles, and a six-legged DNA walker is prepared by using six-armed PEG as a framework to connect DNA walker single strands through an amide bond, a dandelion seed-like DNA macromolecule is constructed by using a nonlinear DNA self-assembly technology, and a signal probe is synthesized by combining CuInS2@ZnS quantum dots, and Mg 2+ The bionic dandelion isothermal amplification system is designed by relying on DNAzyme as a driving force for DNA walker walking.

[0006] In order to achieve the above purpose, the application can adopt the following technical scheme:

[0007] The application provides a bionic dandelion isothermal amplification system, which comprises a closed DNA walker and a bionic dandelion nanoparticle, the bionic dandelion nanoparticle comprises a Janus nanoparticle and a DNA macromolecule loaded on the Janus nanoparticle, and the DNA macromolecule is formed by hybridization of D-RNA, auxiliary strand 1 and auxiliary strand 2, the sequences of the D-RNA, the auxiliary strand 1 and the auxiliary strand 2 are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively.

[0008] Preferably, in the bionic dandelion isothermal amplification system, the Janus nanoparticle is an AuFe Janus nanoparticle, which is an asymmetric nanorod-shaped nanoparticle with a size of 550nm-650nm; the nanoparticle further comprises Fe, Au and Si, the Fe and Au are distributed on the spherical part of the nanorod-shaped nanoparticle, and the Si covers the entire nanorod-shaped nanoparticle.

[0009] More preferably, in the bionic dandelion isothermal amplification system, the preparation method of the Janus nanoparticle comprises the following steps: (1) dispersing magnetic iron oxide nanoparticles and chloroauric acid in deionized water, and then adding a reducing agent to obtain Au@Fe3O4 nanoparticles; (2) mixing the Au@Fe3O4 nanoparticles with a surfactant to obtain a mixture; (3) adding an alkaline catalyst and tetraethyl orthosilicate to the mixture to obtain the Janus nanoparticle.

[0010] More preferably, in the preparation method of the Janus nanoparticle, the reducing agent is sodium borohydride; and / or the surfactant is cetyltrimethylammonium bromide; and / or the alkaline catalyst is ammonia water.

[0011] Preferably, in the above-mentioned biomimetic dandelion isothermal amplification system, the loading mode of the DNA macromolecule loaded on the Janus nanoparticle comprises: uniformly coating polyethyleneimine on the surface of the Janus nanoparticle, then covalently connecting streptavidin to the surface of the polyethyleneimine-coated Janus nanoparticle through an amide bond, and then binding the streptavidin-modified Janus nanoparticle to the biotin-labeled DNA macromolecule through biotin and streptavidin interaction.

[0012] Preferably, in the above-mentioned biomimetic dandelion isothermal amplification system, the sequence of the DNA walker is shown in SEQ ID NO: 4.

[0013] Preferably, in the above-mentioned biomimetic dandelion isothermal amplification system, the DNA walker is a six-legged DNA walker.

[0014] In another aspect of the present application, a circulating tumor cell detection product is provided, which comprises the biomimetic dandelion isothermal amplification system of the present application, wherein the blocking DNA walker further comprises an EpCAM aptamer, and the EpCAM aptamer specifically recognizes the circulating tumor cell.

[0015] Preferably, in the above-mentioned detection product, the sequence of the EpCAM aptamer is shown in SEQ ID NO: 5.

[0016] Preferably, the above-mentioned detection product further comprises an LFIA test strip.

[0017] The present application has the following advantages:

[0018] (1) The biomimetic dandelion isothermal amplification system designed in the present application has an amplification efficiency that is improved by nearly 6.72 times compared with the traditional isothermal amplification system.

[0019] (2) The circulating tumor cell detection product provided in the present application has high sensitivity and a detection limit (LOD) as low as 1.58 cells / mL. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Figure 1 is a schematic diagram of the working principle of the biomimetic dandelion isothermal amplification system; wherein (a) is a schematic diagram of the synthesis of BDNPs; (b) is a schematic diagram of the preparation of a six-legged DNA walker; (c) is a schematic diagram of the biomimetic dandelion isothermal amplification system; magnetic beads (MB), streptavidin (SA);

[0021] Figure 2Characterization of AuFeJNPs: (a) TEM image of Fe3O4NPs; (b) TEM image of AuFeJNPs; (c) elemental spectrum of AuFeJNPs; (d) high-resolution XPS spectra of C, Fe, Au and Si in AuFeJNPs; (e) XRD patterns of Fe3O4NPs and AuFeJNPs; (f) hysteresis loop; (g) nitrogen adsorption-desorption isotherm.

[0022] Figure 3 Energy dispersive spectral analysis of elemental distribution in AuFe JNPs;

[0023] Figure 4 XPS spectra of Fe3O4NPs and AuFe JNPs are shown below; (a) is the XPS spectrum of Fe3O4NPs; (b) is the XPS spectrum of AuFe JNPs.

[0024] Figure 5 The images show the magnetic adsorption properties of Fe3O4NPs and AuFe JNPs.

[0025] Figure 6 The following is a verification of the enzyme activity of AuFe JNPs: (a) is a schematic diagram of the peroxidase (POD)-like activity of Fe3O4NPs and AuFe JNPs; (b) is the absorption spectrum of the TMB colorimetric reaction catalyzed by Fe3O4NPs and AuFe JNPs; (c) is the typical kinetic curve of the TMB colorimetric reaction catalyzed by Fe3O4NPs and AuFe JNPs at 650 nm; and (d) is the quantitative analysis of the absorbance of the TMB colorimetric reaction catalyzed by Fe3O4NPs and AuFe JNPs.

[0026] Figure 7 The following are the characterization results of BDNPs: (a) schematic diagram of biomimetic dandelion seed self-assembly; (b) gel electrophoresis image; (c) D-RNA; (d) AFM image of biomimetic dandelion seeds; (e) magnified image of the green box in figure d; (f) UV-Vis absorption spectrum of BDNPs; (g) dynamic light scattering spectra of AuFe JNPs, AuFe JNPs-PEI, AuFe JNPs-SA and BDNPs; (h) Zeta potential of AuFe JNPs, AuFe JNPs-PEI, AuFe JNPs-SA and BDNPs.

[0027] Figure 8 This is a schematic diagram illustrating the preparation principle of BDNPs.

[0028] Figure 9The amplification performance of BDIAS is verified. Among them, (a) is a schematic diagram comparing BDIAS and TIAS; (b) is the fluorescence kinetic curve of BDIAS after adding 0 pM or 10 pM hexapod DNAwalker; (c) is the relative fluorescence kinetic curve of BDIAS and TIAS; (d) is a schematic diagram of BDNPs based on CuInS2@ZnS quantum dots; (e) is a transmission electron microscope image of CuInS2@ZnS; (f) is the elemental spectrum of CuInS2@ZnS quantum dots; (g) is the excitation and emission spectra of CuInS2@ZnS quantum dots; and (h) is the fluorescence intensity of BDIAS based on CuInS2@ZnS quantum dots after adding four different concentrations of hexapod DNAwalker (0, 2.5, 5 and 10 pM).

[0029] Figure 10 Energy spectral elemental distribution data for CuInS2@ZnS quantum dots;

[0030] Figure 11 The experimental conditions were optimized; where (a) represents pH; (b) represents temperature; and (c) represents reaction time.

[0031] Figure 12 The performance validation of the cell sensor is shown below; (a) images of LFIA test strips corresponding to different concentrations of MCF-7 cells (0, 2, 5, 20, 50, 100, 200, and 1000 cells / mL); (b) T-line fluorescence values ​​corresponding to different concentrations of MCF-7 cells (inset, linear relationship between fluorescence value and logarithm of MCF-7 cell concentration); (c) fluorescence signals of different types and concentrations of cancer cells detected by the wireless cell sensor; (d) fluorescence signals of inter-batch and intra-batch experiments for samples with concentrations of 20, 200, and 1000 MCF-7 cells / mL.

[0032] Figure 13 The following is a performance verification of the cell sensor in clinical samples: (a) is a schematic diagram of the cell sensor used to detect whole blood samples; (b) is the recovery rate of MCF-7 cells detected in whole blood samples; (c) is the specificity analysis of the cell sensor in whole blood samples; (d) is a physical image of the fluorescent test strips corresponding to 24 clinical blood samples; (e) is a fluorescence intensity thermogram of the test strips corresponding to 24 clinical blood samples; and (f) is a box plot of fluorescence intensity of the test strips corresponding to 24 clinical blood samples. Detailed Implementation

[0033] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0034] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0035] This invention provides a biomimetic dandelion isothermal amplification system, which includes a blocked DNAwalker and biomimetic dandelion nanoparticles. The biomimetic dandelion nanoparticles include Janus nanoparticles and DNA macromolecules loaded on Janus nanoparticles. The DNA macromolecules are formed by hybridization of D-RNA, auxiliary strand 1, and auxiliary strand 2. The sequences of D-RNA, auxiliary strand 1, and auxiliary strand 2 are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0036] It should be noted that Janus nanoparticles have a larger specific surface area compared to spherical carriers, which can significantly improve the loading efficiency of fluorescent signal probes (FSPs). Furthermore, the D-RNA is a multimodified D-RNA (DNA-RNA chimera) containing functionalized markers and nucleotide modifications, with the sequence shown below: “GTTGATGAAGCTGAGATTGCACTTGCTGGTTTGTCATGrArUGGGGGTTGGC-biotin”. In addition, the DNA macromolecules generated through nonlinear DNA self-assembly technology (nLDSaT), similar to dandelion seeds, can further improve the loading rate of FSPs (fluorescent signal probes).

[0037] In some specific examples, in the above-mentioned biomimetic dandelion isothermal amplification system, auxiliary strand 1 and auxiliary strand 2 are also connected to fluorescent materials.

[0038] In some specific examples, the fluorescent material in the above-mentioned biomimetic dandelion isothermal amplification system is CuInS2@ZnS quantum dots.

[0039] In some specific examples, in the above-mentioned biomimetic dandelion isothermal amplification system, the Janus nanoparticles are AuFeJanus nanoparticles, which are asymmetric spherical nanoparticles with a size of 550nm-650nm; the nanoparticles also include Fe, Au and Si, with Fe and Au distributed in the spherical part of the spherical nanoparticles and Si covering the entire spherical nanoparticles.

[0040] It should be noted that magnetic iron oxide nanoparticles (e.g., Fe3O4NPs) serve as a magnetic core, providing magnetic responsiveness and facilitating subsequent product purification via magnetic separation. They may also act as a substrate, guiding the deposition of gold nanoparticles (AuNPs) on or near their surface to form heterostructures; while chloroauric acid (HAuCl4) acts as a gold source (Au... 3+ Under the action of a reducing agent, gold nanoparticles (AuNPs) are reduced to gold nanoparticles (AuNPs), which combine with Fe3O4 to form an Au@Fe heterostructure. Surfactants, adsorbed on the particle surface, prevent aggregation and form micelles or interfacial templates, guiding the asymmetric growth of the subsequent silica (SiO2) shell and promoting the formation of Janus nanoparticles. Furthermore, tetraethyl orthosilicate (TEOS) hydrolyzes under ammonia catalysis to generate silica (SiO2), which coats the surface of Au@Fe3O4NPs to form a shell. By controlling the adsorption and reaction conditions of CTAB, selective deposition of SiO2 on the particle surface can be achieved, ultimately forming an Au-Fe3O4 / SiO2 Janus structure, i.e., the Janus nanoparticles of this invention.

[0041] In some specific examples, the preparation method of Janus nanoparticles in the above-mentioned biomimetic dandelion isothermal amplification system includes: (1) dispersing magnetic iron oxide nanoparticles and chloroauric acid in deionized water, and then adding a reducing agent to react and obtain Au@Fe3O4 nanoparticles; (2) mixing Au@Fe3O4 nanoparticles with a surfactant to obtain a mixture; (3) adding an alkaline catalyst and tetraethyl orthosilicate to the mixture to react and obtain Janus nanoparticles.

[0042] In some specific examples, in the above-mentioned method for preparing Janus nanoparticles, the reducing agent is sodium borohydride; and / or the surfactant is hexadecyltrimethylammonium bromide; and / or the alkaline catalyst is ammonia.

[0043] It should be noted that in the above preparation method, the reducing agent, surfactant and alkaline catalyst are all known in the art. For example, the reducing agent can be sodium borohydride (NaBH4), the surfactant can be hexadecyltrimethylammonium bromide (CTAB), and the alkaline catalyst can be ammonia.

[0044] In some specific examples, in the above-mentioned biomimetic dandelion isothermal amplification system, the loading method of DNA macromolecules loaded on Janus nanoparticles includes: uniformly coating the surface of Janus nanoparticles with polyethyleneimine, then covalently linking streptavidin to the surface of the polyethyleneimine-coated Janus nanoparticles via amide bonds, and then binding the streptavidin-modified Janus nanoparticles with biotin-labeled DNA macromolecules through the interaction between biotin and streptavidin.

[0045] In some specific examples, the DNAwalker sequence in the above-mentioned biomimetic dandelion isothermal amplification system is shown in SEQ ID NO: 4.

[0046] In some specific examples, in the above-mentioned biomimetic dandelion isothermal amplification system, the DNAwalker is a hexapod DNAwalker.

[0047] It should be noted that combining BDNPs with a hexapod DNAwalker designed using insect biomimetic technology releases a large number of FSPs after a single enzyme digestion. Furthermore, this insect-like multi-legged structure improves the walking dynamics and continuity of the DNAwalker, and eliminates the need for meticulous DNA track design. Compared to the traditional isothermal amplification system (TIAS) based on the DNAwalker, BDIAS achieves an amplification efficiency that is nearly 6.72 times higher. Additionally, due to the synergistic effect of the high CuInS2@ZnS quantum dot loading rate of the biomimetic dandelion seed (DNA macromolecule), the strong fluorescent signal probe (FSP) loading rate of AuFe JNPs, and the high enzyme digestion efficiency of the hexapod DNAwalker, BDIAS achieves an amplification efficiency 6.72 times higher than TIAS.

[0048] It should also be noted that existing DNAwalkers rely on random, disordered orbits with limited walking dynamics and continuity, or highly organized, complexly designed DNA orbitals (such as long synthetic substrates or DNA origami); furthermore, current DNAwalkers typically produce only one or a few signal probes per enzyme digestion, leaving room for improving amplification efficiency. The hexapod DNAwalker of this invention solves the above problems.

[0049] This invention also provides a product for detecting circulating tumor cells, which includes the biomimetic dandelion isothermal amplification system of this invention, wherein the blocked DNAwalker further includes the EpCAM aptamer, which specifically recognizes circulating tumor cells.

[0050] In some specific examples, the sequence of the EpCAM aptamer in the above-mentioned test products is shown in SEQ ID NO: 5.

[0051] It should be noted that during the detection process, circulating tumor cells (CTCs) are first enriched and isolated from lysed red blood cells using an MB-labeled anti-EpCAM monoclonal antibody under a magnetic field. After mixing the CTCs with a biomimetic dandelion isothermal amplification system, the CTCs and the EpCAM aptamers in the system competitively bind, thereby releasing the aptamer-blocked hexapod DNA walker. 2+ With the assistance of [unclear - possibly a specific technology or method], it can act as a DNAzyme, specifically cleaving the RNA bases of D-RNA and efficiently releasing dandelion seed-like DNA macromolecules (i.e., BDIAS amplification products). This process is very similar to the natural phenomenon of dandelion seed dispersal, hence the system is named the biomimetic dandelion isothermal amplification system. After magnetic separation, the fluorescence intensity of the BDIAS amplification products is measured, thus establishing a correlation with the content of circulating tumor cells, thereby achieving qualitative and / or quantitative detection of circulating tumor cells. It should be noted that the hexapod DNAwalker, in Mg [unclear - possibly a specific technology or method], ... 2+ With the assistance of [unclear - possibly a specific technology or method], it can act as a DNAzyme, specifically cleaving the RNA bases of D-RNA and efficiently releasing dandelion seed-like DNA macromolecules (i.e., BDIAS amplification products). This process is very similar to the natural phenomenon of dandelion seeds dispersing, hence the system is named the biomimetic dandelion isothermal amplification system. Furthermore, there is a background signal before cleavage, but after cleavage, magnetic separation occurs, and the fluorescence signal in the supernatant is detected. This ensures that the detected signal is released by the cleaved product, eliminating background interference and allowing for direct detection of the fluorescence signal.

[0052] In some specific examples, the aforementioned testing products also include LFIA test strips.

[0053] It's important to note that lateral flow immunochromatography (LFIA) is a widely used point-of-care testing tool with significant advantages. Its ease of operation, requiring no specialized training or complex equipment, makes it suitable for home testing, lowering the barrier to entry. Furthermore, its ultra-fast testing speed of only 5 to 30 minutes, demonstrated in applications such as pregnancy testing and COVID-19 antigen testing, has proven invaluable, saving time for subsequent diagnosis and management. Cost-effectiveness, room temperature stability, and portability further enhance its applicability in resource-constrained environments. If the LFIA platform can replace the expensive instruments currently used for CTC detection, it will significantly reduce costs and promote its widespread adoption in primary healthcare facilities and large-scale screening scenarios.

[0054] It should also be noted that in this invention, after the biomimetic dandelion isothermal amplification system (BDIAS) is combined with the LFIA platform, the test strip labeled with anti-digoxigenin antibody can fix the products generated by BDIAS, and the T-line emits fluorescence (the fluorescence intensity can be measured after about 15 minutes). A linear equation between the fluorescence signal intensity and the number of CTCs is constructed to achieve accurate detection of CTCs (the working principle is as follows). Figure 1 (As shown). This detection method for circulating tumor cells is not only easy to operate, but also has extremely high sensitivity, with a detection limit (LOD) as low as 1.58 cells / mL.

[0055] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0056] The oligonucleotide sequences used in the following examples are shown in Table 1 below.

[0057] Table 1 Oligonucleotide Sequences

[0058]

[0059] In the following example, the cell culture and passage steps are as follows:

[0060] For MCF-7 cells, HUVEC cells, and HeLa cells:

[0061] (1) MCF-7 cells, HUVEC cells and HeLa cells were cultured in high glucose DMEM medium (Gibco) containing 10% fetal bovine serum (FBS) and 1% (10000U / mL) penicillin-streptomycin (PS) and placed in an incubator at 37°C and 5% CO2.

[0062] (2) Discard the old culture medium and wash twice with 1× phosphate-buffered saline (PBS);

[0063] (3) Add 0.25% trypsin for digestion. Incubate at 37°C for 30 seconds to 2 minutes. After observing the cells become rounded under a microscope, add an equal volume of high-glucose DMEM medium containing serum to stop digestion, and use a pipette to detach the cells.

[0064] (4) Transfer the mixture to a centrifuge tube and centrifuge (500 rpm × 5 minutes), discarding the supernatant;

[0065] (5) Transfer the cells to fresh high-glucose DMEM medium and continue culturing.

[0066] For HL-60 cells and K562 cells:

[0067] (1) HL-60 cells and K562 cells were cultured in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (FBS) and 1% (10000 U / mL) penicillin-streptomycin (PS) and placed in an incubator at 37°C and 5% CO2.

[0068] (2) Transfer the suspension to a centrifuge tube, centrifuge at 500 rpm for 5 minutes, carefully remove the supernatant, resuspend in fresh RPMI 1640 medium and continue culturing.

[0069] In the following example, the agarose gel electrophoresis experimental procedure is as follows: the electrophoresis buffer is 1×TBE (89mM Tris, 89mM boric acid, 2mM EDTA), the agarose concentration is 3%, and GoldView is used as the fluorescent dye; 5μL of DNA sample and 1μL of DNA loading buffer (Beyotime) are mixed and injected into the gel sample well, and electrophoresis is performed at 110V for 30 minutes; then the gel is photographed under a UV lamp, and the gel images are analyzed using a gel imaging analysis system (Bio-Rad Laboratories, Inc.).

[0070] In the following example, the detection of circulating tumor cells includes: First, preparing the BDIAS system in a sterile centrifuge tube: adding 50 μL of prepared BDNPs, 15 μL of aptamer-blocked hexapod DNAwalker, and Mg 2+ The solution (final concentration 100mM) was brought to a final volume of 100μL with PBS buffer and vortexed to mix. Then, different concentrations of CTCs were added and incubated at 37°C for 40 min. After magnetic separation, the supernatant was dropped onto the sample pad of the test strip. After 15 minutes, the fluorescence intensity was measured.

[0071] The following example illustrates the structure and usage of a wireless test strip analyzer: The wireless test strip analyzer mainly consists of four components: a highly integrated circuit board, a test strip slot, a battery, and a protective casing. The circuit board integrates a 365nm UV LED, an XYZ true-color sensor, a Bluetooth 5.1 system-on-a-chip, and a 3.3V power supply. The user controls the fluorescence detector via wireless commands sent from a smartphone. Upon receiving the command, the Bluetooth chip system activates the UV LED and fluorescence sensor to measure the fluorescence intensity of the T-line on the test strip. The sensor then captures the fluorescence information and outputs it as X, Y, and Z values. The wireless LFIA test strip analyzer utilizes the XYZ true-color sensor to determine the concentration of CTCs by analyzing the fluorescence intensity of the T-line on the LFIA test strip under UV light. Its specific working mechanism is as follows:

[0072] (1) Bluetooth connection and command transmission

[0073] After powering on, the analyzer establishes a Bluetooth connection with the smartphone via a Bluetooth Low Energy microcontroller unit (BLE MCU, model HJ-531IMH). The smartphone wirelessly sends control commands to the MCU.

[0074] (2) Activation by ultraviolet light source

[0075] When the BLE MCU receives the instruction, it will set the general purpose input / output (GPIO) pin to a high level, triggering the N-channel metal-oxide-semiconductor field-effect transistor (MOSFET), thereby turning on the ultraviolet LED;

[0076] (3) Sensor Communication and Data Acquisition

[0077] Subsequently, the BLE MCU establishes communication with the XYZ true color sensor (model AS73211) via the I2C protocol to complete sensor parameter configuration and data acquisition;

[0078] The core function of this sensor is to convert captured light signals into hexadecimal values ​​according to the International Commission on Illumination 1931 color space standard, thereby achieving precise quantification of fluorescence intensity. Its integrated signal processing unit transmits the processed digital light signal directly to the BLE MCU via an I2C interface.

[0079] (4) Low power mode startup

[0080] After data acquisition is complete, the BLE MCU will instruct the color sensor to enter a power-down state and turn off the ultraviolet LED to reduce power consumption;

[0081] (5) Wireless Data Transmission and Result Analysis

[0082] Finally, the collected hexadecimal values ​​are wirelessly transmitted to a smartphone via Bluetooth, allowing users to correlate fluorescence intensity with CTC concentration.

[0083] Through this design, the analyzer utilizes advanced components such as the XYZ true-color sensor and optimized signal processing technology to achieve accurate and efficient fluorescence detection, ensuring the accuracy of result interpretation.

[0084] I. Construction and Characterization of a Biomimetic Dandelion Isothermal Amplification System

[0085] (I) Synthesis and Characterization of AuFe JNPs

[0086] synthesis:

[0087] (1) Disperse 5 mg Fe3O4NPs (about 300 nm) and 100 μL 1 M HAuCl4 solution in 3 mL of deionized water and stir for 10 minutes to obtain a dispersion.

[0088] (2) Add 400 μL of 0.1 M NaBH4 ice water solution dropwise to the dispersion and stir continuously for 1 hour to generate gold nanoparticles in situ to obtain a mixture;

[0089] (3) The mixture was magnetically separated to obtain Au@Fe NPs;

[0090] (4) Wash Au@Fe NPs three times with deionized water and ethanol, and resuspend them in 3 mL of deionized water;

[0091] (5) After Au@Fe NPs were uniformly dispersed in deionized water, 100 mg CTAB (hexadecyltrimethylammonium bromide) was added, and the mixture was ultrasonically treated (30 W) for 30 minutes.

[0092] (6) After ultrasonic treatment, the reaction system was transferred to 40°C, and 1 mL of ammonia and 180 μL of TEOS (tetraethyl orthosilicate) were added dropwise. The reaction was allowed to proceed for 36 hours to obtain the reactant.

[0093] (7) Centrifuge the reactants (13000 rpm, 5 min) to collect AuFe JNPs, and wash them three times with deionized water and ethanol;

[0094] (8) The prepared AuFe JNPs were stored in 20 mL of ethanol for later use.

[0095] Characterization:

[0096] (1) Transmission electron microscopy characterization

[0097] The morphology of Fe3O4NPs and AuFe JNPs was characterized using transmission electron microscopy (TEM), such as... Figure 2 a and Figure 2 As shown in b, the results indicate that Fe3O4NPs exhibit a spherical structure with a diameter of approximately 300 nm. In contrast, AuFe JNPs are asymmetric, ball-and-bar shaped Janus nanoparticles with a size of approximately 600 nm.

[0098] (2) Energy-dispersive X-ray spectroscopy characterization

[0099] Next, energy-dispersive X-ray spectroscopy (EDS) and elemental mapping confirmed that AuFe JNPs mainly contain five elements, including Fe from Fe3O4 NPs, Au from Au nanoparticles, Si from the silica shell, and the two basic elements C and O. Figure 2 c and Figure 3 Furthermore, it can be observed that Fe and Au elements are mainly distributed in the spherical part of the nanoparticles, while Si elements cover the entire nanoparticle. This asymmetric distribution of structure and elements is consistent with the characteristics of Janus nanomaterials.

[0100] (3) X-ray photoelectron spectroscopy characterization

[0101] Next, X-ray photoelectron spectroscopy (XPS) was used to further analyze the composition and valence states of the elements in AuFe JNPs, such as... Figure 2 d and Figure 4 As shown, the results indicate that the electron binding energy position of C at 284.8 eV corresponds to CC / CH, accounting for 52.26%; the electron binding energy position at 286.08 eV corresponds to CO, accounting for 43.28%; and the electron binding energy position at 288.71 eV corresponds to C=O, accounting for 4.45%. For Si, the electron binding energy positions at 99.92 eV and 100.53 eV correspond to 2p3 / 2 and 2p1 / 2 of Si, respectively, accounting for 22.80%; and the binding energy position at 103.07 eV corresponds to Si-O, accounting for 77.20%. The electron binding energies of Fe at 709.63 eV and 723.43 eV correspond to the 2p3 / 2 and 2p1 / 2 of Fe2+, respectively, accounting for 58.05%; the electron binding energies at 711.70 eV and 724.50 eV correspond to the 2p3 / 2 and 2p1 / 2 of Fe3+, respectively, accounting for 41.95%; and the electron binding energies at 714.55 eV, 719.09 eV, 727.35 eV, and 733.04 eV are satellite peaks of Fe. The electron binding energies of Au at 83.72 eV and 87.40 eV correspond to the 4f7 / 2 and 4f5 / 2 of Au0, respectively, accounting for 100%.

[0102] (4) X-ray diffraction characterization

[0103] Furthermore, the crystal structure of AuFe JNPs was characterized by X-ray diffraction (XRD), such as... Figure 2 As shown in Figure e, the results show that AuFe JNPs retain the main diffraction peaks of Fe3O4 NPs, and have two additional crystal planes (111) and (200) due to the presence of Au.

[0104] The results of characterization (1) to (4) above confirm the successful synthesis of AuFe JNPs.

[0105] (5) Magnetic Adsorption Experiment

[0106] To characterize the magnetism of AuFe JNPs, a magnetic adsorption experiment was conducted using a magnetic rack. Specifically, 1 mL of magnetic beads (5 mg / mL) were placed in an EP tube, the EP tube was vertically inserted into the magnetic rack, and the mixture was allowed to stand for 3 minutes until the solution became clear (the magnetic beads were completely adsorbed to the tube wall). The results were then photographed and recorded. Figure 5As shown, both Fe3O4 NPs and AuFe JNPs exhibit good magnetic properties. Furthermore, to gain a deeper understanding, hysteresis loop tests were performed using a vibrating sample magnetometer (VSM), such as... Figure 2 As shown in f, the results show that AuFe JNPs have better magnetism compared to Fe3O4 NPs.

[0107] (6) Nitrogen adsorption experiment

[0108] In this invention, AuFe JNPs are used as a carrier to support biomimetic dandelion seeds. Therefore, the specific surface area of ​​AuFe JNPs is a very important characteristic. This was analyzed using nitrogen adsorption experiments, specifically: the sample was first degassed at 50°C for 8 hours; after reaching the required vacuum level, the sample was transferred to the analysis station for adsorption-desorption testing; the instrument recorded the physical adsorption isotherm at 77 K; the specific surface area was calculated using the Brunauer-Emmett-Teller method; and the total pore volume and average pore size were derived using the Brett-Joyner-Halena porosity analysis method.

[0109] The results are as follows Figure 2 As shown in g, the results show that the specific surface area of ​​AuFe JNPs is significantly larger than that of Fe3O4 NPs, which means that AuFe JNPs can carry more biomimetic dandelion seeds.

[0110] (7) Enzyme activity analysis

[0111] Furthermore, the enzyme activity of AuFe JNPs was analyzed in this invention, specifically including: detecting peroxidase (POD)-like activity using 3,3′,5,5′-tetramethylbenzidine (TMB); dynamic monitoring: Fe3O4 NPs, AuFe JNPs, and deionized water were mixed with TMB working solution. The absorbance of the mixed solution samples at 650 nm was continuously monitored over 10 min using a microplate reader; comparison of POD-like activity: different concentrations (31.25, 62.5, 125, and 250 μg / mL) of Fe3O4 NPs and AuFe JNPs were mixed with TMB working solution and incubated at 25°C for 30 min. Subsequently, the absorbance of each sample at 650 nm was measured.

[0112] The results are as follows Figure 6 As shown, the results indicate that AuFe JNPs exhibit good peroxidase-like activity, which is significantly higher than that of Fe3O4NPs.

[0113] (II) Preparation of FSPs

[0114] (1) CuInS2@ZnS quantum dots (particle size 3nm-10nm, 100μL, 10mg / mL) were activated at 37℃ for 30 minutes with 200μL NHS (N-hydroxysuccinimide) (10mg / mL) solution and EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) solution (20mg / mL).

[0115] (2) Then add H1 or H2 (20 μL, 20 μM) respectively, and stir at 4°C for 12 hours to obtain a mixture;

[0116] (3) The mixture was purified by centrifugation to obtain FSPs1 and FSPs2, and washed three times with deionized water, and then dispersed in 1×TE buffer for later use.

[0117] (III) Preparation and Characterization of BDNPs

[0118] Preparation of BDNPs:

[0119] (1) Preparation of biomimetic dandelion seeds

[0120] To improve the sensitivity of the constructed cell sensor, a biomimetic dandelion seed was designed using nLDSaT. Figure 7 a) Biotin-labeled D-RNA (100 μL, 10 μM), FSPs1 (100 μL, 20 μM), and FSPs2 (100 μL, 20 μM) were mixed and incubated at 37 °C for 2 hours. Furthermore, the feasibility of biomimetic dandelion seed self-assembly was demonstrated using agarose gel electrophoresis (3% by mass). Figure 7 As shown in b, bands 4-6 demonstrate that D-RNA, helper strand 1 (H1), and helper strand 2 (H2) can hybridize to form a DNA macromolecule. Furthermore, with increasing H1 and H2 concentrations, the self-assembly efficiency and molecular weight also increase; moreover, atomic force microscopy (AFM) further confirmed the formation of the DNA macromolecule. Figure 7 c. Figure 7 d and Figure 7 As shown in e, in the presence of only D-RNA, the AFM image appears as scattered small dots, while after the addition of H1 and H2, a macromolecular structure with obvious branching morphology appears, indicating the successful self-assembly of DNA macromolecules.

[0121] (2) Construction of BDNPs

[0122] The construction process of BDNPs is as follows Figure 8 As shown, the details are as follows:

[0123] 1) Preparation of biomimetic dandelion seeds (DNA macromolecules): Mix 100 μL of biotin-labeled D-RNA (10 μmol / L), FSPs1 (20 μmol / L) and FSPs2 (20 μmol / L) in 1×TE buffer containing 0.5 mol / L sodium chloride and 0.2 mol / L potassium chloride, and incubate at 37°C for 2 hours; store the self-assembled DNA macromolecules at 4°C for later use.

[0124] 2) Then, AuFe JNPs (1 mL, 0.1 mg / mL) were mixed with 0.2 mg PEI and subjected to vigorous sonication for 30 minutes to prepare PEI-coated AuFe JNPs (AuFeJNPs@PEI); AuFe JNPs@PEI was separated by magnetic separation, and the precipitate was washed twice with 1 mL of deionized water and then redispersed; the obtained AuFe JNPs@PEI dispersion was mixed with 0.6 mg streptavidin (SA), 10 mg NHS and 20 mg EDC and incubated at 37 °C for 2 hours to obtain AuFe JNPs@PEI@SA; the product was separated by magnetic separation, and the precipitate was washed twice with 1 mL of deionized water and then redispersed.

[0125] 3) The pre-prepared biomimetic dandelion seeds were mixed with AuFe JNPs@PEI@SA and incubated at 37°C for 2 hours to prepare BDNPs (biomimetic dandelion nanoparticles); BDNPs were separated by magnetic force, and after precipitation and washing twice, they were dispersed in 1 mL of 1×TE buffer and stored at 4°C for later use.

[0126] 4) Mix BDNPs (1 mL, 0.1 mg / mL) with the DNA macromolecules obtained from step (1) and stir at 4°C for 12 hours to obtain a mixture; perform magnetic separation on the mixture to obtain biomimetic nano-dandelion, wash the biomimetic nano-dandelion three times with deionized water to remove free DNA macromolecules, then add 1% BSA solution, and gently stir at room temperature for 3 hours to block the unbound active sites on the surface of AuFe JNPs to obtain a reaction solution; perform magnetic separation on the reaction solution to obtain BDNPs, disperse the BDNPs in 1×TE buffer, and store at 4°C for later use.

[0127] Characterization of BDNPs:

[0128] The process was characterized by UV-Vis absorption spectroscopy, dynamic light scattering (DLS) spectroscopy, and Zeta potential. UV-Vis absorption spectroscopy showed that BDNPs simultaneously exhibited characteristic peaks of AuFe JNPs, SA, and DNA. Figure 7 f). Additionally... Figure 7The g-display shows that the size of AuFe JNPs gradually increases with sequential modification by PEI and SA. Furthermore, the size increases by approximately 167 nm after incorporating biomimetic dandelion seeds. Simultaneously, the surface potential of the AuFe JNPs also undergoes a significant change. Figure 7 h). As can be seen from the above, due to the positive charge of PEI and the negative charge of SA and DNA macromolecules, the surface potential first increases and then decreases. These changes provide strong evidence for the successful construction of BDNPs.

[0129] (iv) Preparation of the hexapod DNA walker

[0130] (1) Mix carboxylated DNAwalker strands (5 μL, 20 μM) with NHS (10 mg / mL) and EDC (20 mg / mL) at room temperature for 30 minutes;

[0131] (2) Then add 6-arm polyethylene glycol-amino (6ARM-PEG-NH2) solution (1 mL, 2 mg / mL) and stir at 37°C for 2 hours to obtain hexapod DNAwalker;

[0132] (V) Preparation of LFIA test strips

[0133] The test strip consists of five parts: a sample pad, a conjugate pad, an absorbent pad, a nitrocellulose (NC) membrane, and a polyvinyl chloride (PVC) backing. The C-line and T-line on the NC membrane, as well as the conjugate pad, are coated with anti-digoxin antibody (1.0 mg / mL), SA (1.0 mg / mL), and biotinylated CuInS2@ZnS quantum dots (10 mg / mL), respectively. The antibody-loaded NC membrane is placed in a 37°C oven for at least 4 hours to dry thoroughly, and then assembled with the sample pad and absorbent pad onto the plastic backing. Finally, the card is cut into 5 mm wide test strips and stored in a sealed bag containing desiccant for later use.

[0134] II. Feasibility and Performance Verification of BDIAS

[0135] In the following example, the preparation method of the traditional isothermal amplification system TIAS includes: In TIAS, 100 μL (10 μM) of Cy3 and biotin-labeled FSPs are mixed with 1 mL of 0.1 mg / mL SA / PEI layer-modified MBs. The mixture is incubated at 37 °C for 2 hours. Subsequently, the resulting 3D spherical tracks are washed three times with 1×TE buffer, dispersed in 1 mL of 1×TE buffer, and stored at 4 °C for later use. During amplification, DNAwalkers and Mg in the working solution... 2+ The concentrations were set at 10 pM and 100 mM, respectively.

[0136] To verify the amplification capability of BDIAS, a comparative analysis was conducted with TIAS (a traditional isothermal amplification system (where a single enzyme digestion releases only one or a few signal probes)). The different principles are illustrated in the diagram below. Figure 9 As shown in a, the specific comparative analysis is as follows:

[0137] In the TIAS (Traditional Isothermal Amplification System) system, Cy3-labeled FSPs (which also serve as D-RNA substrate strands) are used to connect with magnetic beads (MBs) to construct a traditional three-dimensional spherical orbital; subsequently, a single-stranded DNA walker is introduced to construct the TIAS.

[0138] For BDIAS (Bionic Dandelion Isothermal Amplification System), bionic dandelion-shaped nanoparticles (BDNPs) were prepared by linking Cy3-labeled bionic dandelion seeds with AuFeJNPs, and then a hexapod DNAwalker was added to construct BDIAS. 10 pM of DNAwalker and 100 mM of Mg were introduced into two isothermal amplification systems, respectively. 2+ Afterwards, DNAwalker can be found in Mg 2+ With the assistance of [unclear], DNAzyme activity is exhibited, thereby hydrolyzing the RNA bases of the D-RNA substrate chain and releasing Cy3-labeled FSPs or Cy3-labeled biomimetic dandelion seeds.

[0139] Fluorescence intensity fluctuations in both systems were monitored using a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, Inc., fluorescence kinetic mode). Finally, the amplification efficiency of both systems was determined based on the increase in fluorescence intensity (ΔF).

[0140] In addition, the feasibility of BDIAS was evaluated using fluorescence kinetic experiments with commercially available Cy3-FSPs (Sangon Biotech (Shanghai) Co., Ltd.), and the results were as follows: Figure 9 As shown in b, after the addition of 10 pM hexapod DNAwalker, the fluorescence intensity gradually increased over time, indicating that it could efficiently cleave and release Cy3-labeled biomimetic dandelion seeds; furthermore, after continuous reaction for 100 minutes under the same conditions, the relative fluorescence intensity of the BDIAS group was 6.72 times that of the TIAS group, indicating that the amplification efficiency of BDIAS was significantly higher than that of TIAS. Figure 9 c).

[0141] Furthermore, the amplification efficiency of BDIAS was further analyzed by using constructed FSPs1 (CuInS2@ZnS quantum dot-H1) and FSPs2 (CuInS2@ZnS quantum dot-H2) for fluorescence endpoint experiments. Figure 9 d). Specifically, this includes introducing 0, 2.5 pM, 5 pM, and 10 pM DNAwalker and 100 mM Mg into the isothermal amplification system, respectively.2+ Afterwards, DNAwalker in Mg 2+ With the assistance of [unclear], DNAzyme activity was exhibited, hydrolyzing the RNA bases of the D-RNA substrate chain and releasing CuInS2@ZnS quantum dot-labeled biomimetic dandelion seeds. Fluorescence intensity was detected 100 minutes after the reaction using a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, Inc.).

[0142] Among them, TEM images ( Figure 9 e) Confirmed that the size of CuInS2@ZnS quantum dots is between 3-10 nm. Elemental spectra and EDS show that CuInS2@ZnS quantum dots are mainly composed of Cu, In, S, and Zn. Figure 9 f and Figure 10 Furthermore, the excitation and emission spectral scans show that the optimal excitation and emission wavelengths for CuInS2@ZnS quantum dots are 370 nm and 650 nm, respectively. Figure 9 g).

[0143] In addition, to further confirm the feasibility of the developed BDIAS, different concentrations of hexapod DNAwalker were added, such as Figure 9 As shown in h, the results showed that the fluorescence signal in the blank group was weak, while the signal gradually increased with the increase of hexapod DNAwalker concentration.

[0144] As can be seen from the above, BDIAS is not only highly feasible, but also has a much higher amplification efficiency than TIAS.

[0145] III. Performance Analysis of Cell Sensors

[0146] To achieve optimal detection performance of this wireless cell sensor, we optimized experimental conditions in several key steps. For the isothermal amplification process, we optimized the pH of the buffer solution (3-10), incubation temperature (5-55℃), and reaction time (5-80 min). The results showed that the fluorescence signal of the detection system reached its maximum value when the pH of the buffer solution was 7.5 and the incubation temperature was 37.5℃. Figure 11 (a and 11b). As the reaction time increased, the fluorescence signal of the detection system increased until it reached a plateau at 40 minutes. Based on the results of the above optimization conditions, the analytical performance of the wireless cell sensor was verified. Figure 11 c).

[0147] (I) Sensitivity Analysis

[0148] To evaluate the sensitivity of the wireless cell sensor, a series of MCF-7 cells at different concentrations were detected. The corresponding LFIA test strip images and quantitative results are shown below. Figure 12As shown in a and 12b, the fluorescence intensity increased with increasing MCF-7 cell concentration, demonstrating the effectiveness and feasibility of the wireless cell sensor. Furthermore, the results also showed a good linear relationship between the logarithm of the MCF-7 cell number and the quantitative value of the fluorescence signal in the range of 5 to 1000 cells / mL. Figure 12 b). The linear regression equation is: Y = 61.18LogC + 49.34, R0 2 =0.995. The detection limit is 1.58 cells / mL. These results demonstrate that this wireless cell sensor possesses extremely high sensitivity.

[0149] (II) Specificity Analysis

[0150] To analyze specificity, this wireless cell sensor was used to detect different types of tumor cells, such as HeLa cells, HUVEC cells, HL-60 cells, K562 cells, MCF-7 cells, and mixtures of these cells. Figure 12 As shown in Figure c, the MCF-7 cell group and the mixed cell group produced significant fluorescence signals, which were positively correlated with cell concentration. However, the fluorescence signals of the EpCAM-negative cell group (HeLa cells, HUVEC cells, HL-60 cells, and K562 cells) showed no significant difference compared to the control group. This result indicates that the wireless cell sensor has high specificity.

[0151] (III) Reproducibility of inter-batch and intra-batch testing

[0152] To test the reproducibility of the wireless cell sensor developed in our institute, inter-batch and intra-batch assays were performed. Samples with concentrations of 20, 200, and 1000 cells / mL were analyzed using the wireless cell sensor. The results are as follows: Figure 12 As shown in d, the relative standard deviations (RSDs) for inter-batch and intra-batch experiments were in the range of 1.5%–2.7% and 1.1%–2.1%, respectively. These results indicate that the wireless cell sensor has acceptable reproducibility.

[0153] IV. Clinical Sample Analysis Performance of Cell Sensors

[0154] To evaluate the potential of the newly developed wireless cell sensor in clinical applications, clinical whole blood samples were collected and analyzed using the wireless cell sensor. The analysis process is as follows: Figure 13 As shown in a, it specifically includes:

[0155] First, different concentrations of MCF-7 cells were added to whole blood samples from healthy individuals, and a cell sensor was used to detect the spiked cells in a spiking experiment. The results are as follows: Figure 13As shown in b, the recoveries of MCF-7 cells at three concentrations (10, 200, and 1000 cells / mL) were 90%, 112%, and 102%, with relative standard deviations of 2.16%, 3.76%, and 4.29%, respectively. Clearly, this cell sensor can accurately detect CTCs in whole blood samples.

[0156] Furthermore, HeLa cells, HUVEC cells, HL-60 cells, K562 cells, MCF-7 cells, and mixtures of these cells were added to whole blood from healthy individuals and detected using a cell sensor to evaluate their anti-interference ability and practicality in clinical sample testing. Results Figure 13 As shown in Figure c, compared with blank whole blood samples and whole blood samples with added EpCAM-negative cells, only whole blood samples with added MCF-7 cells and whole blood samples containing a mixture of MCF-7 cells showed a significant increase in fluorescence signal. Therefore, this wireless cell sensor has strong anti-interference capability in clinical sample testing.

[0157] To further demonstrate the application potential of the wireless cell sensor in clinical settings, whole blood samples were collected from 8 healthy individuals and 16 breast cancer patients, and the developed wireless cell sensor was used for detection. A photo of the test strip is shown below. Figure 13 As shown in d, the T-line of the LFIA test strips in the healthy individual group showed no obvious fluorescent bands to the naked eye, while the T-line of the breast cancer patient group showed fluorescent bands of varying intensities. Quantitative analysis of the T-line of each test strip was performed using a wireless test strip analyzer. Figure 13 The heatmaps and box plots in e and f show that the fluorescence intensity of the T-line in the breast cancer patient group was significantly higher than that in the healthy group (p<0.0001), indicating that the developed wireless cell sensor can effectively distinguish breast cancer patients.

[0158] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A biomimetic dandelion isothermal amplification system, characterized in that, The invention includes a blocked DNAwalker and biomimetic dandelion nanoparticles. The biomimetic dandelion nanoparticles include Janus nanoparticles and a DNA macromolecule loaded on the Janus nanoparticles. The DNA macromolecule is formed by hybridization of D-RNA, auxiliary strand 1 and auxiliary strand 2. The sequences of D-RNA, auxiliary strand 1 and auxiliary strand 2 are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively. DNAwalker is a six-legged DNAwalker; The blocked DNAwalker also includes the EpCAM aptamer, which specifically recognizes circulating tumor cells. The sequence of the EpCAM aptamer is shown in SEQ ID NO:

5. Janus nanoparticles are AuFe Janus nanoparticles, which are asymmetric spherical nanoparticles with a size of 550nm-650nm. The nanoparticles also contain Fe, Au and Si. Fe and Au are distributed in the spherical part of the spherical nanoparticles, while Si covers the entire spherical nanoparticles.

2. The biomimetic dandelion isothermal amplification system according to claim 1, characterized in that, The preparation method of Janus nanoparticles includes: (1) dispersing magnetic iron oxide nanoparticles and chloroauric acid in deionized water, and then adding a reducing agent to react and obtain Au@Fe3O4 nanoparticles; (2) mixing Au@Fe3O4 nanoparticles with a surfactant to obtain a mixture; (3) adding an alkaline catalyst and tetraethyl orthosilicate to the mixture to react and obtain Janus nanoparticles.

3. The biomimetic dandelion isothermal amplification system according to claim 2, characterized in that, The reducing agent is sodium borohydride; and / or the surfactant is hexadecyltrimethylammonium bromide; and / or the alkaline catalyst is ammonia.

4. The biomimetic dandelion isothermal amplification system according to any one of claims 1 to 3, characterized in that, The loading method for DNA macromolecules loaded on Janus nanoparticles includes: uniformly coating the surface of Janus nanoparticles with polyethyleneimine, then covalently linking streptavidin to the surface of the polyethyleneimine-coated Janus nanoparticles via amide bonds, and then binding streptavidin-modified Janus nanoparticles with biotin-labeled DNA macromolecules through the interaction between biotin and streptavidin.

5. The biomimetic dandelion isothermal amplification system according to any one of claims 1 to 3, characterized in that, The sequence of DNAwalker is shown in SEQ ID NO:

4.

6. A product for detecting circulating tumor cells, characterized in that, Includes the biomimetic dandelion isothermal amplification system as described in any one of claims 1 to 5.

7. The testing product according to claim 6, characterized in that, It also includes LFIA test strips.

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