Bionic dandelion isothermal amplification system and application thereof

Abstract

The invention belongs to the technical field of cell detection, and particularly relates to a bionic dandelion isothermal amplification system and application thereof. The bionic dandelion isothermal amplification system is composed of a hexapod DNA walker, a nonlinear DNA self-assembly technology and asymmetric carrier-AuFe Janus nanoparticles with high signal probe loading efficiency, and the obvious amplification efficiency is shown. Compared with a traditional isothermal amplification system, the amplification efficiency of the bionic dandelion isothermal amplification system is improved by about 6.72 times.

Description

Technical Field

[0001] The invention belongs to the technical field of cell detection, and particularly relates to a bionic dandelion isothermal amplification system and applications thereof. Background Art

[0002] The detection of circulating tumor cells (CTCs) has important clinical and scientific research value. In terms of tumor metastasis detection, it can capture early signs of tumor cells detaching from the primary tumor site and invading the blood, providing important clues for early assessment of metastasis risk. In terms of treatment plan selection, it can be used to guide individualized treatment and monitor and evaluate the treatment effect in real time so that the treatment plan can be optimized in a timely manner. In terms of prognostic assessment, it can predict the risk of recurrence and determine the patient's survival prognosis. In addition, CTCs also play a key role in elucidating complex metastasis mechanisms, tracking cancer evolution, and promoting innovation in liquid biopsy technology.

[0003] While current CTC detection methods hold great potential, they also face several challenges. For example, these existing technologies lack sufficient sensitivity and often miss low-abundance cells; complex procedures can compromise cell viability and integrity; the lack of standardized protocols further complicates the comparability of results; and high-accuracy CTC detection relies on expensive advanced instrumentation, such as single-cell sequencing platforms and microfluidic chip devices. These factors have limited the widespread adoption of CTC detection in primary healthcare settings and large-scale screening settings.

[0004] CTCs are extremely rare in peripheral blood, so improving the sensitivity of detection methods is one of the key issues that need to be addressed. In order to improve the sensitivity of CTCs detection, isothermal amplification technology has been explored. For example, rolling circle amplification (RCA) combined with surface-enhanced Raman scattering and specific aptamers can achieve ultrasensitive detection and non-destructive recovery of CTCs. 2+ The combination of DNAzyme-based enzyme cleavage technology and parallel catalytic hairpin assembly has significantly improved the sensitivity of CTC detection. CRISPR-Cas-based isothermal amplification technology and other optimized methods and their combinations also show potential. In addition, dynamic DNA technologies (DNA nanomachines and DNA walkers) have become powerful tools for highly sensitive CTC detection due to their excellent controllability and functionality. Summary of the Invention

[0005] The present invention develops a bionic dandelion isothermal amplification system based on nanosynthesis technology and DNAwalker technology. The system uses streptavidin-modified AuFe Janus nanoparticles as carriers of signal probes and 3D tracks for DNAwalker walking. Six-arm PEG is used as a framework to connect DNAwalker single chains through amide bonds to prepare a hexapod DNAwalker. Dandelion seed-like DNA macromolecules are constructed by nonlinear DNA self-assembly technology and combined with CuInS2@ZnS quantum dots to synthesize signal probes. Mg is used as the carrier of the signal probe. 2+ Designed to rely on DNAzyme as the driving force of the DNAwalker, the developed biomimetic dandelion isothermal amplification system has high signal amplification efficiency and high sensitivity in detecting circulating tumor cells.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] The present invention provides a bionic dandelion isothermal amplification system, which includes a closed DNA walker and bionic dandelion nanoparticles. The bionic dandelion nanoparticles include Janus nanoparticles and DNA macromolecules loaded on the Janus nanoparticles. The DNA macromolecules are formed by hybridization of D-RNA, an auxiliary chain 1, and an auxiliary chain 2. The sequences of the D-RNA, the auxiliary chain 1, and the auxiliary chain 2 are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0008] Preferably, in the above-mentioned bionic dandelion isothermal amplification system, the Janus nanoparticles are AuFe Janus nanoparticles, which are asymmetric spherical rod-shaped nanoparticles with a size of 550nm-650nm; the nanoparticles also include Fe, Au and Si, Fe and Au are distributed in the spherical part of the spherical rod-shaped nanoparticles, and Si covers the entire spherical rod-shaped nanoparticles.

[0009] More preferably, in the above-mentioned biomimetic dandelion isothermal amplification system, 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 to 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 to obtain Janus nanoparticles.

[0010] More preferably, 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 aqueous ammonia.

[0011] Preferably, in the above-mentioned bionic dandelion isothermal amplification system, the loading method of the DNA macromolecules loaded on the Janus nanoparticles includes: uniformly coating the surface of the Janus nanoparticles with polyethyleneimine, then covalently linking streptavidin to the surface of the polyethyleneimine-coated Janus nanoparticles through an amide bond, and then combining the streptavidin-modified Janus nanoparticles with the biotin-labeled DNA macromolecules through the interaction between biotin and streptavidin.

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

[0013] Preferably, in the above-mentioned bionic dandelion isothermal amplification system, the DNA walker is a hexapod DNA walker.

[0014] In another aspect, the present invention provides a circulating tumor cell detection product, which includes the bionic dandelion isothermal amplification system of the present invention, wherein the closed DNA walker also includes an EpCAM aptamer, and the EpCAM aptamer specifically recognizes circulating tumor cells.

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

[0016] Preferably, the above-mentioned detection product also includes LFIA test strips.

[0017] The beneficial effects of the present invention include:

[0018] (1) Compared with the traditional isothermal amplification system, the bionic dandelion isothermal amplification system designed in the present invention has an amplification efficiency increased by nearly 6.72 times.

[0019] (2) The circulating tumor cell detection product provided in the present invention is not only easy to operate but also has ultra-high sensitivity for detecting circulating tumor cells, with a limit of detection (LOD) as low as 1.58 cells / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the working principle of the biomimetic dandelion isothermal amplification system; (a) is a schematic diagram of the synthesis of BDNPs; (b) is a schematic diagram of the preparation of the hexapod 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) is the TEM image of Fe3O4NPs; (b) is the TEM image of AuFe JNPs; (c) is the elemental map of AuFe JNPs; (d) is the high-resolution XPS spectrum of C, Fe, Au and Si elements in AuFe JNPs; (e) is the XRD spectrum of Fe3O4NPs and AuFe JNPs; (f) is the hysteresis loop; (g) is the nitrogen adsorption-desorption isotherm;

[0022] Figure 3 Energy spectrum analysis diagram of element distribution in AuFe JNPs;

[0023] Figure 4 XPS spectra of Fe3O4NPs and AuFe JNPs; (a) is the XPS spectra of Fe3O4NPs; (b) is the XPS spectra of AuFe JNPs;

[0024] Figure 5 Actual picture of magnetic adsorption of Fe3O4NPs and AuFe JNPs;

[0025] Figure 6 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 650nm; (d) is the absorbance quantitative analysis of the TMB colorimetric reaction catalyzed by Fe3O4NPs and AuFe JNPs;

[0026] Figure 7 Characterization of BDNPs; (a) is a schematic diagram of the self-assembly of biomimetic dandelion seeds; (b) is a gel electrophoresis image; (c) is D-RNA; (d) is an AFM image of the biomimetic dandelion seeds; (e) is an enlarged image of the green frame part in Figure d; (f) is the UV-visible absorption spectrum of BDNPs; (g) is the dynamic light scattering spectrum of AuFe JNPs, AuFe JNPs-PEI, AuFe JNPs-SA and BDNPs; (h) is the Zeta potential of AuFe JNPs, AuFe JNPs-PEI, AuFe JNPs-SA and BDNPs;

[0027] Figure 8 Schematic diagram of the preparation of BDNPs;

[0028] Figure 9The amplification performance verification of BDIAS; among them, (a) is a comparative schematic diagram of BDIAS and TIAS; (b) is the fluorescence kinetic curve of BDIAS after adding 0pM or 10pM hexapod DNA walker; (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 map of CuInS2@ZnS quantum dots; (g) is the excitation spectrum and emission spectrum of CuInS2@ZnS quantum dots; (h) is the fluorescence intensity of BDIAS based on CuInS2@ZnS quantum dots after adding four different concentrations of hexapod DNA walker (0, 2.5, 5 and 10pM);

[0029] Figure 10 Energy spectrum element distribution data of CuInS2@ZnS quantum dots;

[0030] Figure 11 The optimized experimental conditions are shown in Figure 2. (a) represents pH, (b) represents temperature, and (c) represents reaction time.

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

[0032] Figure 13 The performance verification of the cell sensor in detecting clinical samples; among them, (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 picture of the fluorescent test strips corresponding to 24 clinical blood samples; (e) is a heat map of the fluorescence intensity of the test strips corresponding to 24 clinical blood samples; (f) is a box plot of the fluorescence intensity of the test strips corresponding to 24 clinical blood samples. DETAILED DESCRIPTION

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

[0034] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "comprise" and the like are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0035] An embodiment of the present invention provides a bionic dandelion isothermal amplification system, which includes a closed DNA walker and bionic dandelion nanoparticles. The bionic dandelion nanoparticles include Janus nanoparticles and DNA macromolecules loaded on the Janus nanoparticles. The DNA macromolecules are formed by hybridization of D-RNA, auxiliary chain 1 and auxiliary chain 2. The sequences of D-RNA, auxiliary chain 1 and auxiliary chain 2 are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0036] It should be noted that compared with spherical carriers, Janus nanoparticles have a larger specific surface area, which can significantly improve the loading efficiency of fluorescent signal probes (FSPs). In addition, D-RNA is a multiply modified D-RNA (DNA-RNA chimera) containing functional tags and nucleotide modifications. Its sequence is as follows: "GTTGATGAAGCTGAGATTGCACTTGCTGGTTTGTCATGrArUGGGGGTTGGC-biotin". In addition, DNA macromolecules generated by nonlinear DNA self-assembly technology (nLDSaT) are similar to dandelion seeds and can further improve the loading rate of FSPs (fluorescent signal probes).

[0037] In some specific examples, in the bionic dandelion isothermal amplification system, the auxiliary chain 1 and the auxiliary chain 2 are further connected with fluorescent materials.

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

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

[0040] It should be noted that magnetic iron oxide nanoparticles (e.g., Fe3O4NPs) act as magnetic cores to provide magnetic responsiveness, facilitating subsequent purification of products by magnetic separation. They may also act as substrates to guide the deposition of gold nanoparticles (AuNPs) on or near their surfaces to form heterogeneous structures; while chloroauric acid (HAuCl4) acts as a gold source (Au 3+ ) is reduced to gold nanoparticles (AuNPs) under the action of a reducing agent, which then combines with Fe3O4 to form an Au@Fe heterostructure. Surfactants, adsorbed on the particle surface, prevent aggregation and form micelles or interface templates, guiding the asymmetric growth of the subsequent silica (SiO2) shell and promoting the formation of Janus nanoparticles. Furthermore, tetraethyl orthosilicate (TEOS) is hydrolyzed under ammonia catalysis to produce silica (SiO2), which coats the Au@Fe3O4NPs to form a shell. By controlling the adsorption and reaction conditions of CTAB, the selective deposition of SiO2 on the particle surface can be achieved, ultimately forming the Au-Fe3O4 / SiO2 Janus structure, i.e., the Janus nanoparticles of the present invention.

[0041] In some specific examples, in the above-mentioned biomimetic dandelion isothermal amplification system, 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 to 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 to 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 aqueous ammonia.

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

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

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

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

[0047] It is important to note that the combination of BDNPs and a hexapod DNA walker designed using insect biomimetic technology releases a large number of FSPs after a single enzyme digestion. Furthermore, this biomimetic design, resembling the multi-legged structure of insects, improves the walking dynamics and continuity of the DNA walker without requiring elaborate DNA track design. Compared with the traditional isothermal amplification system (TIAS) based on the DNA walker, the amplification efficiency of BDIAS is increased by nearly 6.72 times. Furthermore, due to the synergistic effect of the high CuInS2@ZnS quantum dot loading rate of the biomimetic dandelion seeds (DNA macromolecules), the strong fluorescent signal probe (FSP) loading rate of the AuFe JNPs, and the high enzyme digestion efficiency of the hexapod DNA walker, the amplification efficiency of BDIAS is 6.72 times higher than that of TIAS.

[0048] It should also be noted that existing DNA walkers rely on random, disordered tracks with limited walking dynamics and continuity, or highly organized, complex DNA tracks (such as long synthetic substrates or DNA origami). Furthermore, current DNA walkers typically produce only one or a few signal probes per enzyme digestion, leaving room for improving amplification efficiency. The hexapod DNA walker in this invention addresses these issues.

[0049] An embodiment of the present invention further provides a circulating tumor cell detection product, which includes the bionic dandelion isothermal amplification system of the present invention, wherein the closed DNA walker also includes an EpCAM aptamer, and the EpCAM aptamer specifically recognizes circulating tumor cells.

[0050] In some specific examples, in the above detection product, the sequence of the EpCAM aptamer is shown as SEQ ID NO: 5.

[0051] It should be noted that during the detection, first, under the action of a magnetic field, MB-labeled anti-EpCAM monoclonal antibodies were used to enrich and separate circulating tumor cells from the blood of red blood cell lysis. After the circulating tumor cells were mixed with the bionic dandelion isothermal amplification system, the circulating tumor cells and the EpCAM aptamers in the bionic dandelion isothermal amplification system competitively bound to each other, thereby releasing the aptamer-enclosed hexapod DNA walker. 2+ With the assistance of , it can act as a DNAzyme to specifically cut the RNA bases of D-RNA and efficiently release dandelion seed-like DNA macromolecules (i.e., BDIAS amplification products). This process is very similar to the natural phenomenon of dandelion seeds drifting away, so the system is named the bionic dandelion isothermal amplification system. The fluorescence intensity of the BDIAS amplification product is tested after magnetic separation, so that a connection with the content of circulating tumor cells can be established, thereby realizing the qualitative and / or quantitative detection of circulating tumor cells. It should be noted that the hexapod DNAwalker, in Mg 2+ With the assistance of a DNAzyme, it can specifically cleave the RNA bases of D-RNA and efficiently release dandelion seed-like DNA macromolecules (i.e., BDIAS amplification products). This process is very similar to the natural phenomenon of dandelion seed dispersion, hence the name of the system, the biomimetic dandelion isothermal amplification system. Furthermore, there is a background signal before cleavage, but after cleavage, magnetic separation is performed. During detection, the fluorescence signal in the supernatant is detected, ensuring that the detected signal is released by the cleaved product, eliminating background signal interference and directly detecting the fluorescence signal.

[0052] In some specific examples, the above-mentioned detection products also include LFIA test strips.

[0053] It should be noted that lateral flow immunochromatography (LFIA) is a widely used point-of-care testing tool with significant advantages. It is simple to operate, does not require specialized training or complex instruments, is suitable for self-testing at home, and lowers the threshold for testing. In addition, in application scenarios such as pregnancy testing and COVID-19 antigen testing, its ultra-fast speed of completing the test in only 5 to 30 minutes has been proven to be extremely valuable, saving time for subsequent diagnosis and management. Cost-effectiveness, room temperature stability, and portability further enhance its applicability in resource-limited environments. If the LFIA platform can replace the expensive instruments currently used for CTCs detection, it will significantly reduce costs and promote its widespread application in primary health care institutions and large-scale screening scenarios.

[0054] It should also be noted that in the present invention, after the biomimetic dandelion isothermal amplification system (BDIAS) of the present invention is combined with the LFIA platform, the test strip labeled with anti-digoxigenin antibody can fix the product generated by BDIAS, and the T-line emits fluorescence (the fluorescence intensity can be measured after about 15 minutes), and 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 Detection of circulating tumor cells based on this assay is not only easy to operate but also has ultra-high sensitivity, with a limit of detection (LOD) as low as 1.58 cells / mL.

[0055] In order to better understand the present invention, the content of the present invention is further explained below with reference to specific examples, but the content of the present invention is not limited to the following examples.

[0056] In the following examples, the oligonucleotide sequences used are shown in Table 1.

[0057] Table 1 Oligonucleotide sequences

[0058]

[0059] In the following example, the cell culture and passaging 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% (10,000 U / mL) penicillin-streptomycin (PS) in a 37°C, 5% CO2 incubator.

[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 the cells are observed to be rounded under a microscope, add an equal amount of high-glucose DMEM medium containing serum to terminate the digestion. Use a pipette to detach the cells.

[0064] (4) The mixture was transferred to a centrifuge tube and centrifuged (500 rpm × 5 minutes), and the supernatant was discarded;

[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% (10,000 U / mL) penicillin-streptomycin (PS) in a 37°C, 5% CO2 incubator.

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

[0069] In the following example, the agarose gel electrophoresis experiment steps are as follows: the running 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 (Biyuntian) are mixed and injected into the gel sample tank, and electrophoresis is carried out at 110V for 30 minutes; the gel is then photographed under UV light and analyzed using a gel imaging analysis system (Bio-Rad, USA).

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

[0071] In the following example, the structure and usage of the wireless test strip analyzer are as follows: The wireless test strip analyzer mainly consists of four components: a highly integrated circuit board, a test strip slot, a battery, and a protective case. The circuit board integrates a 365nm UV LED, an XYZ true color sensor, a Bluetooth 5.1 system-level chip, and a 3.3V power supply. The user controls the fluorescence detector through wireless commands sent by a smartphone. After receiving the command, the Bluetooth chip system can activate the UV LED and fluorescence sensor to measure the fluorescence intensity of the T-line of 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 uses an 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 ultraviolet 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) UV light source activation

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

[0076] (3) Sensor communication and data acquisition

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

[0078] The sensor's core function is to convert captured light signals into hexadecimal values ​​based on the International Commission on Illumination's 1931 color space standard, enabling precise quantification of fluorescence intensity. Its integrated signal processing unit transmits the processed digital light signals directly to a Bluetooth Low Energy (BLE) MCU via an I2C interface.

[0079] (4) Low power mode startup

[0080] After data collection is completed, the BLE MCU will instruct the color sensor to enter the power-down state and turn off the UV 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, and users can use this data to correlate fluorescence intensity with CTCs concentration;

[0083] Through this design, the analyzer uses 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] 1. Construction and characterization of the bionic dandelion isothermal amplification system

[0085] (1) Synthesis and characterization of AuFe JNPs

[0086] synthesis:

[0087] (1) Disperse 5 mg of Fe3O4 NPs (about 300 nm) and 100 μL of 1 M HAuCl4 solution in 3 mL of deionized water and stir for 10 minutes to obtain a dispersion;

[0088] (2) adding 400 μL of 0.1 M NaBH4 ice water solution dropwise to the dispersion and stirring 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) The Au@Fe NPs were washed three times with deionized water and ethanol and resuspended in 3 mL of deionized water;

[0091] (5) After Au@Fe NPs were uniformly dispersed in deionized water, 100 mg of CTAB (cetyltrimethylammonium bromide) was added and ultrasonicated (30 W) for 30 min;

[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 mixture was allowed to react for 36 hours to obtain the reactant.

[0093] (7) The reaction mixture was centrifuged (13000 rpm, 5 min) to collect the AuFe JNPs and washed three times with deionized water and ethanol;

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

[0095] Characterization:

[0096] (1) Transmission electron microscopy characterization

[0097] Transmission electron microscopy (TEM) was used to characterize the morphology of Fe3O4NPs and AuFe JNPs. Figure 2 a and Figure 2 As shown in (b), the results show that Fe3O4NPs are spherical with a diameter of about 300nm. In contrast, AuFe JNPs are asymmetric spherical-rod-shaped Janus nanoparticles with a size of about 600nm.

[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 silica shells, and two basic elements, C and O ( Figure 2 c and Figure 3 ). In addition, it can be observed that Fe and Au elements are mainly distributed in the spherical part of the nanoparticles, while Si element covers the entire nanoparticles. This structure and asymmetric distribution of elements are 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 of the elements in the AuFe JNPs, as shown in Figure 2. Figure 2 d and Figure 4 The results show that the electron binding energy position of the C element at 284.8eV corresponds to CC / CH, accounting for 52.26%; the electron binding energy position at 286.08eV corresponds to CO, accounting for 43.28%; the electron binding energy position at 288.71eV corresponds to C=O, accounting for 4.45%. The electron binding energy positions of the Si element at 99.92eV and 100.53eV correspond to Si's 2p3 / 2 and 2p1 / 2, respectively, accounting for 22.80%; the binding energy position at 103.07eV corresponds to Si-O, accounting for 77.20%. The electron binding energy positions of Fe element at 709.63eV and 723.43eV correspond to 2p3 / 2 and 2p1 / 2 of Fe2+, respectively, accounting for 58.05%; the electron binding energy positions of 711.70eV and 724.50eV correspond to 2p3 / 2 and 2p1 / 2 of Fe3+, respectively, accounting for 41.95%; the electron binding energy positions of 714.55eV, 719.09eV, 727.35eV, and 733.04eV are satellite peaks of Fe. The electron binding energy positions of Au element at 83.72eV and 87.40eV correspond to 4f7 / 2 and 4f5 / 2 of Au0, respectively, accounting for 100%.

[0102] (4) X-ray diffraction characterization

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

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

[0105] (5) Magnetic adsorption experiment

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

[0107] (6) Nitrogen adsorption experiment

[0108] In this study, AuFe JNPs were used as a carrier to support biomimetic dandelion seeds. Therefore, the specific surface area of ​​AuFe JNPs is a crucial property. This was analyzed through nitrogen adsorption experiments. Specifically, the sample was degassed at 50°C for 8 hours. Once the required vacuum level was reached, the sample was transferred to an analysis station for adsorption and desorption testing. Physical adsorption isotherms were recorded at 77 K. The specific surface area was calculated using the Brunauer-Emmett-Teller method, and the total pore volume and average pore diameter were derived using the Brunauer-Joyner-Halena pore 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 load more bionic dandelion seeds.

[0110] (7) Enzyme activity analysis

[0111] The enzymatic activity of AuFe JNPs was also analyzed. Specifically, peroxidase (POD)-like activity was detected using 3,3',5,5'-tetramethylbenzidine (TMB). Dynamic monitoring was performed: 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 minutes using a microplate reader. POD-like activity was compared: Fe3O4 NPs and AuFe JNPs at different concentrations (31.25, 62.5, 125, and 250 μg / mL) were mixed with TMB working solution and incubated at 25°C for 30 minutes. The absorbance of each sample at 650 nm was then measured.

[0112] The results are as follows Figure 6 As shown, the results showed that AuFe JNPs exhibited good peroxidase-like activity, which was significantly higher than that of Fe3O4 NPs.

[0113] (2) Preparation of FSPs

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

[0115] (2) H1 or H2 (20 μL, 20 μM) was then added, and the mixture was stirred at 4°C for 12 h to obtain a mixture;

[0116] (3) The mixture was purified by centrifugation to obtain FSPs1 and FSPs2, which were 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 bionic dandelion seeds

[0120] In order to improve the sensitivity of the constructed cell sensor, a biomimetic dandelion seed ( Figure 7 a), and immobilized them on the surface of AuFe JNPs to prepare BDNPs, as follows: 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; in addition, agarose gel electrophoresis (mass fraction 3%) was used to experimentally demonstrate the feasibility of biomimetic dandelion seed self-assembly. Figure 7 As shown in b, lanes 4-6 show that D-RNA, auxiliary chain 1 (H1) and auxiliary chain 2 (H2) can hybridize to form DNA macromolecules. In addition, as the concentrations of H1 and H2 increase, the self-assembly efficiency and molecular weight also increase. In addition, atomic force microscopy (AFM) further confirms the formation of DNA macromolecules. Figure 7 c. Figure 7 d and Figure 7 As shown in Figure 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) BDNPs construction

[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): 100 μL of biotin-labeled D-RNA (10 μmol / L), FSPs1 (20 μmol / L), and FSPs2 (20 μmol / L) were mixed in 1× TE buffer containing 0.5 mol / L sodium chloride and 0.2 mol / L potassium chloride and incubated at 37°C for 2 h. The self-assembled DNA macromolecules were stored at 4°C until use.

[0124] 2) AuFe JNPs (1 mL, 0.1 mg / mL) were then mixed with 0.2 mg of PEI and vigorously sonicated for 30 min to prepare PEI-coated AuFe JNPs (AuFeJNPs@PEI). The AuFe JNPs@PEI was separated by magnetic separation, and the precipitate was washed twice with 1 mL of deionized water and redispersed. The resulting AuFe JNPs@PEI dispersion was mixed with 0.6 mg of streptavidin (SA), 10 mg of NHS, and 20 mg of EDC, and the mixture was incubated at 37°C for 2 h 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 redispersed.

[0125] 3) The pre-prepared biomimetic dandelion seeds were mixed with AuFe JNPs@PEI@SA and incubated at 37°C for 2 h to prepare BDNPs (bionic dandelion nanoparticles). The BDNPs were separated by magnetic force, precipitated and washed twice, and then dispersed in 1 mL of 1×TE buffer and stored at 4°C until use.

[0126] 4) BDNPs (1 mL, 0.1 mg / mL) were mixed with the DNA macromolecules obtained by incubation in step (1), and the mixture was stirred at 4°C for 12 hours to obtain a mixture; the mixture was magnetically separated to obtain biomimetic nano-dandelions, and the biomimetic nano-dandelions were washed three times with deionized water to remove free DNA macromolecules, and then 1% BSA solution was added, and the mixture was gently stirred at room temperature for 3 hours to block the unbound active sites on the surface of AuFe JNPs to obtain a reaction solution; the reaction solution was magnetically separated to obtain BDNPs, and the BDNPs were dispersed in 1×TE buffer and stored at 4°C for future use.

[0127] BDNPs characterization:

[0128] The process was characterized by UV-visible absorption spectroscopy, dynamic light scattering (DLS) spectroscopy and Zeta potential. The UV-visible absorption spectrum showed that BDNPs had the characteristic peaks of AuFe JNPs, SA and DNA ( Figure 7 f). In addition, Figure 7g shows that the size of AuFe JNPs gradually increased with the sequential modification of PEI and SA. In addition, after combining with biomimetic dandelion seeds, the size increased by about 167nm. At the same time, the surface potential of AuFe JNPs also changed significantly ( 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 Hexapod DNA Walker

[0130] (1) Carboxylated DNA walker strand (5 μL, 20 μM) was mixed with NHS (10 mg / mL) and EDC (20 mg / mL) at room temperature for 30 minutes;

[0131] (2) 6-arm polyethylene glycol-amino (6ARM-PEG-NH2) solution (1 mL, 2 mg / mL) was then added and stirred at 37°C for 2 h to obtain the hexapod DNA walker;

[0132] (V) Preparation of LFIA test strips

[0133] The test strip consists of five components: 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 and the conjugate pad are coated with anti-digoxigenin 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 fully dry before being assembled onto a plastic backing along with the sample pad and absorbent pad. Finally, the card is cut into 5 mm wide test strips and stored in a sealed bag with desiccant until ready for use.

[0134] 2. BDIAS Feasibility and Performance Verification

[0135] In the following example, the preparation method of the traditional isothermal amplification system TIAS includes: In the TIAS, Cy3 and biotin dual-labeled FSPs (100 μL, 10 μM) are mixed with 1 mL of SA / PEI layer-modified MBs at a concentration of 0.1 mg / mL. 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 until use. During the amplification process, DNA walkers and Mg in the working solution 2+ The concentrations of β-actin and β-actin were set to 10 pM and 100 mM, respectively.

[0136] In order to verify the amplification ability of BDIAS, a comparative analysis was conducted with TIAS (a traditional isothermal amplification system (single enzyme digestion only releases a single or a few signal probes)). The schematic diagrams of their different principles are shown in the figure 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 chains) are used to connect to magnetic beads (MBs) to construct a traditional three-dimensional spherical track; subsequently, a single-stranded DNA walker is introduced to construct the TIAS.

[0138] For BDIAS (Bionic Dandelion Isothermal Amplification System), Cy3-labeled biomimetic dandelion seeds were connected to AuFeJNPs to prepare biomimetic dandelion-like nanoparticles (BDNPs), and then hexapod DNA walker was added to construct BDIAS. 10 pM DNA walker and 100 mM Mg were introduced into two isothermal amplification systems, respectively. 2+ After that, DNAwalker can be used in Mg 2+ With the assistance of , it exhibits DNAzyme activity, thereby hydrolyzing the RNA bases of the D-RNA substrate chain and releasing Cy3-labeled FSPs or Cy3-labeled bionic dandelion seeds.

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

[0140] In addition, the feasibility of BDIAS was evaluated by fluorescence kinetics experiments using commercial Cy3-FSPs (Shanghai Sangon Biotech Co., Ltd.). Figure 9 As shown in b, after adding 10 pM hexapod DNA walker, the fluorescence intensity gradually increased over time, indicating that it can efficiently cut and release Cy3-labeled biomimetic dandelion seeds; in addition, after 100 minutes of continuous reaction 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 is significantly higher than that of TIAS ( Figure 9 c).

[0141] In addition, the amplification efficiency of BDIAS was further analyzed by performing fluorescence endpoint experiments using the constructed FSPs1 (CuInS2@ZnS quantum dots-H1) and FSPs2 (CuInS2@ZnS quantum dots-H2). Figure 9 d). Specifically, 0, 2.5 pM, 5 pM and 10 pM of DNAwalker and 100 mM of Mg were introduced into the isothermal amplification system.2+ After that, DNAwalker 2+ With the help of , DNAzyme activity was demonstrated, hydrolyzing the RNA bases of the D-RNA substrate chain and releasing the biomimetic dandelion seeds labeled with CuInS2@ZnS quantum dots. The fluorescence intensity after 100 minutes of reaction was measured using a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, USA).

[0142] Among them, TEM image ( Figure 9 e) It was confirmed that the size of CuInS2@ZnS quantum dots is between 3-10nm. Elemental mapping and EDS showed that CuInS2@ZnS quantum dots are mainly composed of Cu, In, S and Zn ( Figure 9 f and Figure 10 In addition, the excitation and emission spectrum scanning curves show that the optimal excitation and emission wavelengths of CuInS2@ZnS quantum dots are 370nm and 650nm, respectively ( Figure 9 g).

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

[0144] From the above, we can see that BDIAS is not only highly feasible, but also has a much higher amplification efficiency than TIAS.

[0145] 3. Performance Analysis of Cell Sensors

[0146] In order to achieve the best detection performance of this wireless cell sensor, we optimized the experimental conditions in several key steps. For the isothermal amplification process, the pH value of the buffer solution (3-10), incubation temperature (5-55°C), and reaction time (5-80 minutes) were optimized. The results showed that when the pH value of the buffer solution was 7.5 and the incubation temperature was 37.5°C, the fluorescence signal of the detection system reached its maximum value ( Figure 11 a and 11b). As the reaction time increases, the fluorescence signal of the detection system increases until it reaches 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] (1) Sensitivity analysis

[0148] To evaluate the sensitivity of the wireless cell sensor, a series of MCF-7 cells with different concentrations were tested. The corresponding LFIA test strip images and quantitative results are shown in the figure. Figure 12a and 12b. The fluorescence intensity increased with the increase of MCF-7 cell concentration, which proved the effectiveness and feasibility of the wireless cell sensor. In addition, the results also showed that there was 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, R 2 =0.995. The detection limit was 1.58 cells / mL. These results demonstrate that this wireless cell sensor has ultra-high sensitivity.

[0149] (2) Specificity analysis

[0150] To analyze the specificity, the 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 and mixed cell groups produced significant fluorescence signals that were positively correlated with cell concentration. However, the fluorescence signals of the EpCAM-negative cell groups (HeLa cells, HUVEC cells, HL-60 cells, and K562 cells) were not significantly different from those of the blank group. This result demonstrates the high specificity of the wireless cell sensor.

[0151] (III) Inter-batch and intra-batch reproducibility

[0152] To investigate the reproducibility of the developed wireless cell sensor, inter-batch and intra-batch testing was performed. The wireless cell sensor was used to test samples with concentrations of 20, 200, and 1000 cells / ml. The results are shown in Figure 2. Figure 12 As shown in Figure d, the relative standard deviations (RSDs) of inter- 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] In order to evaluate the potential of the developed new wireless cell sensor in clinical applications, clinical whole blood samples were collected and tested using the wireless cell sensor. Figure 13 As shown in a, specifically including:

[0155] First, MCF-7 cells at different concentrations were added to whole blood samples from healthy individuals and detected using a cell sensor to perform a spike-in experiment. Figure 13As shown in (b), the recovery rates of MCF-7 cells at three concentrations (10, 200, and 1000 cells / mL) were 90%, 112%, and 102%, respectively, 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] In addition, HeLa cells, HUVEC cells, HL-60 cells, K562 cells, MCF-7 cells, and a mixture of these cells were added to healthy human whole blood and detected using the cell sensor to evaluate its anti-interference ability and practicality in clinical sample detection. Figure 13 As shown in Figure c, compared with blank whole blood samples and whole blood samples supplemented with EpCAM-negative cells, only the whole blood samples supplemented with MCF-7 cells and the whole blood samples containing MCF-7 cells mixture showed a significant increase in fluorescence signal. Therefore, this wireless cell sensor has strong anti-interference ability in clinical sample detection.

[0157] To further demonstrate the potential of wireless cell sensors in clinical applications, whole blood samples were collected from 8 healthy individuals and 16 breast cancer patients and tested using the developed wireless cell sensor. Figure 13 d. The T-lines of the LFIA test strips in the healthy subjects showed no visible fluorescent bands to the naked eye, whereas the T-lines in the breast cancer patients showed fluorescent bands of varying intensities. The T-lines of each test strip were quantitatively analyzed using a wireless test strip analyzer. Figure 13 The heatmaps and boxplots in e and f showed 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 are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be covered by the scope of the claims of the present invention.

Claims

1. A bionic dandelion isothermal amplification system, characterized in that: The invention comprises a closed DNA walker and biomimetic dandelion nanoparticles. The biomimetic dandelion nanoparticles comprise Janus nanoparticles and DNA macromolecules loaded on the Janus nanoparticles. The DNA macromolecules are formed by hybridization of D-RNA, auxiliary chain 1 and auxiliary chain 2. The sequences of D-RNA, auxiliary chain 1 and auxiliary chain 2 are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

2. The bionic dandelion isothermal amplification system according to claim 1, characterized in that: The Janus nanoparticles are AuFe Janus nanoparticles, which are asymmetric spherical rod-shaped nanoparticles with a size of 550nm-650nm; the nanoparticles also include Fe, Au and Si, Fe and Au are distributed in the spherical part of the spherical rod-shaped nanoparticles, and Si covers the entire spherical rod-shaped nanoparticles.

3. The bionic dandelion isothermal amplification system according to claim 2, 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 to obtain Au@Fe3O4 nanoparticles; (2) mixing the Au@Fe3O4 nanoparticles with a surfactant to obtain a mixture; and (3) adding an alkaline catalyst and tetraethyl orthosilicate to the mixture to react to obtain Janus nanoparticles.

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

5. The bionic dandelion isothermal amplification system according to any one of claims 1 to 4, characterized in that: The loading method of DNA macromolecules 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 through an amide bond, and then combining the streptavidin-modified Janus nanoparticles with the biotin-labeled DNA macromolecules through the interaction between biotin and streptavidin.

6. The bionic dandelion isothermal amplification system according to any one of claims 1 to 4, characterized in that: The sequence of DNA walker is shown in SEQ ID NO:

4.

7. The bionic dandelion isothermal amplification system according to claim 6, characterized in that: DNAwalker is a six-legged DNAwalker.

8. A circulating tumor cell detection product, characterized in that: The bionic dandelion isothermal amplification system comprises any one of claims 1 to 7, wherein the closed DNA walker further comprises an EpCAM aptamer, and the EpCAM aptamer specifically recognizes circulating tumor cells.

9. The detection product according to claim 9, characterized in that: The sequence of the EpCAM aptamer is shown in SEQ ID NO:

5.

10. The detection product according to claim 8 or 9, characterized in that: Also includes LFIA test strips.

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