A biosensor for capturing and detecting CTCs and a preparation method thereof

Abstract

The present invention discloses a biosensor for capturing and detecting CTCs and a preparation method, and belongs to the technical field of biosensors. The present invention solves the problem of how to further improve the sensitivity and specificity of capturing and detecting circulating tumor cells. The present invention adopts composite nanomaterial AuNPs-G / GDY as a modified sensing interface substrate, and nanogold ions can be anchored with the thiol group of tetrahedral DNA through Au-S bond binding, while the hanging chain of TDN uses cDNA as a template, and a multivalent aptamer network layer is generated through RCA, and the multivalent aptamer network layer can capture low trace CTCs. On this basis, the aptamer of the circulating tumor cells on the PdPtCuRu-aptamer probe is further used to specifically bind to the circulating tumor cells, and then the circulating tumor cells can be detected and quantified by catalyzing the H2O2 reduction reaction to generate an electrochemical signal value, thereby sandwiching the circulating tumor cells between the multivalent aptamer network layer and the PdPtCuRu-aptamer probe, realizing two-way sandwich capture of cells and enhancing the signal.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors, and in particular relates to a biosensor for capturing and detecting CTCs and a preparation method thereof. Background Art

[0002] Circulating Tumor Cells (CTCs) are tumor cells that fall off from primary tumor tissue and invade the human blood circulation system during tumor metastasis. Recently, they have been recognized as a new biomarker for cancer liquid biopsy. Liquid biopsy based on CTCs has the advantages of easy access, painless, non-invasive, and the ability to identify primary / metastatic tumor sites. It has great application potential in the early diagnosis, treatment and monitoring of breast cancer. The number of CTCs in the blood circulation system is extremely small, about 1-100 cells / mL, while there are a large number of red blood cells (109 cells / mL), white blood cells (106 cells / mL), platelets (106 cells / mL) and proteins in the blood. Therefore, it is very difficult to detect CTCs from hundreds of millions of normal blood cells. In addition, the heterogeneity of CTCs is also an important factor affecting the accurate detection of CTCs.

[0003] The CellSearch system is the gold standard for clinical quantification of CTCs, but this gold standard faces problems such as high instrument cost, complex operation, and poor sensitivity, which seriously limit its clinical application. Compared with traditional tissue biopsy, liquid biopsy for CTCs analysis is non-invasive and more easily accepted by patients.

[0004] However, the isolation and counting of CTCs in whole blood remains a challenging task due to the extreme rarity of CTCs in the blood and the interference caused by non-targeted blood cells. In addition, medical imaging has also been integrated into clinical practice to achieve accurate and non-invasive diagnosis of cancer. Fluorescence imaging, as a traditional imaging method, has been widely used in CTCs analysis. However, most fluorescence imaging methods lack specificity. Usually, fluorophores or fluorescent substances are attached to nanomaterials only through the targeting properties of nanomaterials to image CTCs, while the fluorescence of non-targeted or mis-targeted nanomaterials will affect the imaging and counting of CTCs. Summary of the invention

[0005] In order to solve the problem of how to further improve the sensitivity and specificity of capturing and detecting circulating tumor cells in the prior art, the present invention provides a biosensor for capturing and detecting CTCs and a preparation method thereof.

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

[0007] Biosensors for capturing and detecting CTCs include:

[0008] AuNPs-G / GDY coating, wherein the AuNPs-G / GDY coating is disposed on the surface of the electrode;

[0009] A multivalent aptamer network layer, wherein the multivalent aptamer network layer is disposed on the AuNPs-G / GDY coating, and the preparation materials of the multivalent aptamer network layer include tetrahedral DNA, circular DNA template and dNTPs;

[0010] PdPtCuRu-aptamer probe.

[0011] After adopting the technical scheme, the composite nanomaterial AuNPs-G / GDY is used as a modified sensing interface substrate in the present invention, and the functional material is used to modify the electrode to improve the interface activity, which can effectively improve the detection efficiency of the electrochemical sensing platform. The layered nanomaterial graphyne has a non-localized sp and sp2 electronic structure, making it an excellent carbon carrier for metal catalysts. However, its semiconductor properties limit its electrocatalytic activity. Graphene can be used as an epitaxial template for the growth of few-layer graphyne to form a unique few-layer graphene / graphyne heterostructure, which can significantly improve the electrochemical active surface area, charge transfer kinetics and intrinsic activity. The liquid phase van der Waals epitaxial growth method of a few-layer graphene diyne film using graphene as a template is an innovative material preparation technology, which uses graphene as a substrate and grows a high-quality few-layer graphene diyne film on graphene by a solution phase van der Waals epitaxy method. This method can not only prepare a large-area, uniform and continuous film, but also ensure that the film has a high-quality single crystal structure. Gold nanoparticles (AuNPs) are evenly dispersed between graphene and graphyne, and can grow on the surface and between layers of G / GDY to form an effective sensing interface. When AuNPs grow on the composite graphene / graphyne, due to the excellent conductivity of the metal, the electron transfer rate of Au-graphene / graphyne is faster, thus providing better electrochemical performance and biosensing sensitivity. In addition, the nanogold ions in the AuNPs-G / GDY coating can be anchored to the thiol group of TDN (tetrahedral DNA) in the multivalent aptamer network layer through Au-S bonds, and the pendant chains of TDN use cDNA as a template to produce a multivalent aptamer network layer through RCA, which can capture low trace amounts of CTCs. On this basis, the aptamers of circulating tumor cells on the PdPtCuRu-aptamer probe are further used to specifically bind to circulating tumor cells, and then the circulating tumor cells can be detected and quantified by generating electrochemical signal values ​​through catalytic H2O2 reduction reaction, thereby sandwiching the circulating tumor cells between the multivalent aptamer network layer and the PdPtCuRu-aptamer probe to achieve two-way sandwich capture of cells and enhance the signal. The present invention combines the multivalent capture effect with the metal nanomaterial catalytic amplification strategy to improve the sensitivity and stability of the electrochemical sensor, thereby achieving high-sensitivity detection of low-concentration CTCs.

[0012] A method for preparing a biosensor for capturing and detecting CTCs, comprising:

[0013] Preparation of AuNPs-G / GDY coating: introducing nano-gold into a mixed solution of graphene and graphyne to obtain AuNPs-G / GDY nanocomposite material, preparing the AuNPs-G / GDY nanocomposite material into a suspension and then dropping the suspension onto the electrode surface to form AuNPs-G / GDY coating;

[0014] Preparation of multivalent aptamer network layer: Add reduced tetrahedral DNA solution to the surface of the electrode with AuNPs-G / GDY coating and incubate. After the incubation, continue to add Mg 2+ , K + , cDNA, DNA polymerase and dNTP solution and perform in situ RCA reaction to obtain a multivalent aptamer network layer;

[0015] Preparation of PdPtCuRu-aptamer probe: The SH2-MUC1 aptamer was attached to the surface of PdPtCuRuMNs via metal-thiol bonds to obtain the PdPtCuRu-aptamer probe.

[0016] Preferably, in the preparation process of AuNPs-G / GDY coating, HAuCl4 is added to a mixed solution of graphene and graphyne, and then sodium citrate solution is added after standing for reaction to obtain nano-gold particles, and the mass ratio of graphene to graphyne is 1:1.

[0017] After adopting this technical solution, the effects of the combination of the three are as follows: 1. Enhanced electrochemical performance: Both graphene and graphyne have excellent conductive properties, and their combination can further improve the conductivity of the electrode material, thereby improving the sensitivity and response speed of the electrochemical sensor. 2. Stable structure: The composite structure of graphene and graphyne can provide a stable support platform, which helps to fix and disperse the nano-gold particles and prevent them from agglomerating, thereby maintaining good electrochemical activity and stability. 3. Synergistic effect: The composite of graphene and graphyne can produce a synergistic effect and improve the overall performance of the material. For example, the porous structure of graphyne can provide more active sites, while the high specific surface area of ​​graphene can increase the effective area of ​​the electrode. 4. Controllable morphology and size: By adjusting the amount of HAuCl4 and sodium citrate added, the morphology and size of the nano-gold particles can be controlled, thereby optimizing the performance of the electrode material. In the process of synthesizing the AuNP-G / GDY composite material, the mass ratio of graphene (G) and graphyne (GDY) was set to 1:1. This ratio is based on the results of experimental optimization, aiming to achieve the best synergistic effect of the two materials. In materials science, the performance of composite materials often depends on the interaction between the components, the uniformity of dispersion, and the properties of each component. By changing the ratio of graphene to graphyne, the electronic structure, mechanical properties, and chemical stability of the composite material can be adjusted. For example, increasing the proportion of graphene may improve the conductivity of the composite material, while increasing the proportion of graphyne may enhance its chemical stability and selective adsorption capacity. Therefore, the optimal ratio of 1:1 was selected.

[0018] Preferably, during the preparation of the multivalent aptamer network layer, four DNA chains are thermally induced to self-assemble to form tetrahedral DNA by a heating-rapid cooling method.

[0019] After adopting this technical solution, the benefits of choosing the heating-rapid cooling method for thermally induced self-assembly to prepare tetrahedral DNA include: 1. Rapid preparation: This method may be faster than the traditional annealing process because it involves rapid temperature changes, which can accelerate the self-assembly process of the DNA chain. 2. Improved yield: Rapid cooling causes the DNA chain to quickly fall into a low-energy state, thereby increasing the yield of tetrahedral DNA. 3. Reduced by-products: Rapid cooling reduces the residence time of the intermediate state, thereby reducing the formation of undesirable by-products. 4. Controlled structure: By precisely controlling the rates of heating and cooling, the structure and size of the tetrahedral DNA can be better controlled. 5. Simplified process: This method may simplify the preparation process because it does not require complex equipment or long-term monitoring.

[0020] Preferably, the preparation method of PdPtCuRu MNs comprises: mixing Pluronic F127 with H2PtCl6, Na2PdCl4, CuCl2, RuCl3 and HCl solution according to a ratio, adding ascorbic acid after ultrasonic dissolution, then storing the mixed solution in an oil bath, and centrifuging the obtained precipitate after removing residual Pluronic F127 to obtain PdPtCuRuMNs.

[0021] After adopting this technical solution, the present invention uses a one-pot thermal reduction method to prepare palladium platinum copper ruthenium mesoporous nanospheres (PdPtCuRu MNs), and loads aptamers of circulating tumor cells on the surface thereof, thereby achieving bidirectional sandwich capture of cells and enhancing signals.

[0022] Preferably, the concentration of AuNPs-G / GDY solution is 1-4 mg / ml. When the effective area of ​​the glassy carbon electrode is 7 mm 2 When the AuNPs-G / GDY solution is used, the amount is 6 μL, the concentration of tetrahedral DNA is 0.3-0.6 μM, the amount is 10 μL, and the concentration of cDNA is 1 μM, the amount is 5-10 μL.

[0023] After adopting this technical solution, the time of in-situ RCA reaction is 30-50 minutes.

[0024] After adopting this technical solution, the multivalent aptamer network leads to an increase in nonspecific interactions within or between molecules, which hinders the binding of the aptamer to the cell.

[0025] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0026] High sensitivity: The sensor can achieve highly sensitive capture and non-destructive detection of circulating tumor cells (CTCs), with a limit of detection (LOD) as low as 7 cell / mL.

[0027] Wide detection range: The sensor exhibits a linear range of 10-106 cell / mL under optimal conditions, which is suitable for cell detection at different concentrations.

[0028] High specificity: Aptamer sensors have higher specificity in target recognition and can effectively distinguish target cells from other cells.

[0029] Good biocompatibility: The sensor has good biocompatibility, can achieve lossless recovery of cells, and has little effect on cell activity.

[0030] Easy operation: Electrochemical sensors usually have simple operation procedures, are easy to use, and are suitable for rapid clinical testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a schematic diagram of the principle of the present invention, wherein Figure 1 A is the preparation principle diagram of AuNPs-G / GDY coating. Figure 1 B is the schematic diagram of the preparation of PdPtCuRu-aptamer probe. Figure 1 C is a schematic diagram of the preparation and use of the biosensor;

[0032] Figure 2 is a characterization diagram of the AuNPs-G / GDY nanocomposite material and PdPtCuRu MNs in the present invention, wherein: Figure 2 A is the SEM image of 1μM G / GDY, Figure 2 B is the SEM image of 200 nm G / GDY. Figure 2 C is the TEM image of 100 nm Au-G / GDY. Figure 2 D is the TEM image of 50 nm Au-G / GDY. Figure 2 EG is the transmission electron microscopy images of PdPtCuRu nanoparticles at different magnifications. Figure 2 H is the surface scanning element distribution map of PdPtCuRu MNs;

[0033] Figure 3 This is a graph showing the electrochemical performance analysis results of the sensing interface sensitizing material of the present invention, wherein: Figure 3 A is the conductivity result diagram of different materials analyzed using CV. Figure 3 B is the conductivity result diagram of different materials analyzed using EIS. Figure 3 C is a graph comparing the catalytic performance of PdPt, PdPtCu, and PdPtCuRu in the presence or absence of H2O2. Figure 3 D is the impedance comparison result of PdPt, PdPtCu, and PdPtCuRu using EIS;

[0034] Figure 4 This is a diagram for the synthesis verification of tetrahedral DNA and DNA network and the layer-by-layer assembly verification of the sensing interface in the present invention, wherein: Figure 4 A is the TDN self-installation process diagram, 4 B is the TDN page diagram, Figure 4 C is the PAGE diagram of the RCA network on TDN. Figure 4 D is a physical picture of the DNA network. Figure 4 E is the CLSM and SEM images of the DNA network. Figure 4 F is the SEM image of the local DNA network. Figure 4 G is the concentration of 10 mmol L -1 0.1 mol L of [Fe(CN)6] -1 CV response curve of 3- / 4- in KCl solution, Figure 4 H is the EIS graph;

[0035] Figure 5 This is a diagram of the electrical signal detection results during the optimization of experimental conditions in the present invention, wherein: Figure 5 A is the electrical signal detection result diagram under the condition of TDN concentration change. Figure 5 B is the result of the electrical signal detection under the condition of RCA time variation. Figure 5 C is a graph showing the detection results of electrical signals when capturing time changes;

[0036] Figure 6 This is a diagram of sensor detection, performance analysis, and cell recovery in the present invention, wherein: Figure 6 A represents different cell concentrations (10×10 6 cell mL −1 ) DPV curve diagram, Figure 6 B is different cell concentrations (10×10 6 cell mL −1 ), Figure 6 C is the reproducibility test diagram of the sensor detecting MCF-7 cells. Figure 6 D is the stability test diagram of the sensor detecting MCF-7 cells. Figure 6 E is the specific detection diagram of the sensor detecting MCF-7 cells. Figure 6 F is the calibration curve of measuring different concentrations of MCF-7 cells in blood using screen-printed electrodes. Figure 6 G is a schematic diagram of DNA network shearing and recycling. Figure 6 H is the release process of benzonase nuclease under bright field microscope. Figure 6 I is a diagram of the cell release process under a bright field microscope after 6 hours of culture. Figure 6 J is a diagram of the cell release process under bright field microscopy after 10 hours of culture. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments.

[0038] A biosensor for capturing and detecting CTCs, comprising:

[0039] AuNPs-G / GDY coating, wherein the AuNPs-G / GDY coating is disposed on the surface of the electrode;

[0040] A multivalent aptamer network layer, wherein the multivalent aptamer network layer is disposed on the AuNPs-G / GDY coating, and the preparation materials of the multivalent aptamer network layer include tetrahedral DNA, circular DNA template and dNTPs;

[0041] PdPtCuRu-aptamer probe.

[0042] This example takes breast cancer MCF-7 cells as an example. Figure 1 As shown, the preparation method of the biosensor for capturing and detecting breast cancer MCF-7 cells is as follows:

[0043] (1) Preparation of AuNPs-G / GDY nanocomposites

[0044] 2.5 mg of G (graphene sheet diameter 0.5-5 μM, thickness 0.8-1.2 μM) and 2.5 mg of GDY (graphene sheet diameter 40-200 nm, number of layers 2 to 10) were dispersed in 50 mL of deionized water, ultrasonically mixed for 1 h, and then 10 mg of HAuCl4 was added. After standing for 30 min, 5 mL of sodium citrate solution (concentration 0.02%) was added, magnetically stirred for 30 min, and heated to 80 ° C for 1 h. Finally, it was washed several times with deionized water. When the supernatant was clear, the washing was completed. The washed product was then centrifuged (7000 rpm) to remove free Au NPs to obtain AuNPs-G / GDY nanocomposite materials. The final AuNPs-G / GDY nanocomposite materials were placed in a 60 ° C oven for 12 h and then collected and stored.

[0045] Scanning electron microscopy (SEM) was used to characterize the morphology of the prepared G / GDY. Figure 2A, B), the results show that G / GDY is lamellar and has multiple layers of interwoven wrinkled surfaces. The smooth plane indicates that G / GDY has a high specific surface area and a layered two-dimensional structure, providing sites for loading Au NPs. Transmission electron microscopy (TEM) was used to analyze G / GDY loaded with Au NPs. The results showed that Au NPs showed a regular small nano-morphology and could grow independently on the two-dimensional heterostructure film sheet ( Figure 2 C, D).

[0046] (2) Preparation of PdPtCuRu-aptamer probe

[0047] 50.0 mg Pluronic F127 was mixed with 1.5 mL H2PtCl6 (20.0 mM), 1.5 mL Na2PdCl4 (20.0 mM), 0.75 mL CuCl2 (20 mM), 0.75 mL RuCl3 (20 mM) and 0.2 mL HCl solution (6.0 M) in a 50 mL round-bottom flask and sonicated until Pluronic F127 was dissolved, and 2.0 mL ascorbic acid (0.1 M) was added. Then, the mixed solution was stored in an oil bath at 105 °C for 4 h. The residual Pluronic F127 was removed by continuous washing / centrifugation with deionized water several times, and the precipitate was collected after centrifugation to obtain PdPtCuRu MNs. The synthesis method of the trimetallic PdPtCu MNs and the bimetallic PdPt MNs used in the comparative example was the same as that of PdPtCuRu MNs, excluding RuCl3 and / or CuCl2. TCEP (tris(2-chloroethyl) phosphate) was added to 40 μL SH2-MUC1 aptamer solution (10 μM) for 30 min to reduce the thiol bond, and then added to 100 μL PdPtCuRu MNs solution and mixed at 4 °C for 12 h to attach the SH2-MUC1 aptamer to the surface of PdPtCuRu MNs through the metal-thiol bond. Subsequently, 0.5% BSA was added and mixed (4 °C, 2 h). Finally, the dispersion was centrifuged and repeatedly washed to remove the residual aptamer. The precipitate was dispersed in 100 μL ultrapure water for further use, and the concentration of the obtained PdPtCuRu-aptamer probe was 2 mg / ml.

[0048] The mesoporous structure of PdPtCuRu MNs was verified by TEM, and well-dispersed and uniformly sized mesoporous nanospheres were observed ( Figure 2 E). The selected area electron diffraction (SAED) pattern shows that the PdPtCuRu MNs are highly polycrystalline ( Figure 2F, G). Elemental mapping EDS image measurements also show that the nanospheres are composed of Pd, Pt, Cu, and Ru elements ( Figure 2 H).

[0049] (3) Assembly of tetrahedral DNA (TDN)

[0050] The four DNA chains were dissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) to a final concentration of 100 µM. Then, 1 µL of each of the four DNA chains was mixed in 45 µL TM buffer (10 mM Tris, 50 mM MgCl2, pH 8.0), and 1 µL TCEP (500 mM) was added to activate the thiol groups. The mixture was heated at 95 °C for 10 minutes and then rapidly cooled to 4 °C in an ice bath for 30 minutes to obtain tetrahedral DNA.

[0051] (4) Preparation of circular DNA template

[0052] CD (2 µL, 100 µM), Primer (2 µL, 100µM), NaCl solution (2 µL, 800 mM) and 14 µL enzyme-free water were added to form a 20 µL reaction system. The mixture was subjected to programmed circularization operation: 95 ℃ 2 min; 65 ℃ 2 min; 60 ℃ 5 min 30 s; 40 ℃ 2 min; 20 ℃ 2 min; 4 ℃ 10 min. The ligation reaction was carried out overnight after adding 25 U T4 DNA ligase (containing ligase buffer), and the final prepared cDNA should be stored at 4 ℃ for future use.

[0053] (5) Synthesis of multivalent aptamer networks

[0054] TDN (10 μL, 1 μM), cDNA (20 μL, 1 μM), dNTPs (10 μL 10 mM), BSA solution (5 μL, 10 mg / ml), phi29 DNA polymerase buffer (10 μL, 10×), phi29 DNA polymerase (2 μL, 10 U / μL), NaCl solution (10 μL, 5 mM), enzyme-free water (43 μL), mixed evenly. Place in a thermostatic mixer (400 rpm, 37 ℃) and continue the reaction for 10 hours; after the reaction is completed, immediately transfer to a metal bath (65 ℃) and keep for 10 minutes to heat inactivate phi29 DNA polymerase in this step. Finally, the multivalent aptamer network was stored at -20 ℃ for future use.

[0055] The gene sequences used in this example are shown in Table 1:

[0056] Table 1

[0057]

[0058] (6) Step-by-step construction of electrochemical sensors

[0059] First, the glassy carbon electrode (GCE) was polished to a bright mirror surface. Subsequently, 6 μL of AuNP-G / GDY suspension was dropped on the GCE surface and dried in an oven (37 °C). TDN (0.3 μM) was reduced with TCEP for 30 min and then dropped onto the electrode surface, and the Au-SH bonds were incubated at 37 °C for 1 h. MCH was added and incubated for 30 min to block nonspecific sites. 5 μL of Mg-containing 2+ (100 mM), K + (660 mM), cDNA (1 μM), phi29 DNA polymerase (10 U), and dNTP (1 mM) were added to the solution and subjected to in situ RCA reaction on the electrode surface for 40 min to obtain TDN-multivalent capture probe (TDN-PCP). 2+ It is a metal ion essential for the activity of DNA polymerase. It participates in stabilizing the structure of DNA polymerase and promotes the binding of the enzyme to the substrate (dNTPs). In the rolling circle amplification process, Mg 2+ Helps maintain enzyme activity, thereby ensuring continuous DNA strand synthesis. 2+ , the activity of DNA polymerase will decrease, resulting in a decrease in amplification efficiency.

[0060] 2: Potassium ions mainly play an auxiliary role in rolling circle amplification. It can improve the stability of DNA polymerase and help maintain the osmotic pressure balance of the reaction system. Although potassium ions are not necessary for the activity of DNA polymerase, they can optimize the reaction conditions and make the amplification process more efficient.

[0061] In order to verify the performance of the chemical sensor, the pretreated cell suspension (10 μL) of different concentrations was incubated with the treated electrode at 37 °C for 30 min, and then the uncaptured cells were gently rinsed with PBS. 5 μL PdPtCuRu MNs probe suspension was added to the electrode and incubated at 37 °C for 30 min, and the unbound signal probe was washed with PBS and electrochemical detection was performed. All electrochemical experiments were performed using an electrochemical workstation with a conventional three-electrode system (CHI660E, Shanghai, China). In the experiment, the modified GCE was used as the working electrode, the Ag / AgCl electrode soaked in saturated KCl solution was used as the reference electrode, and the platinum wire was used as the auxiliary electrode. Among them, the CV scan rate was 100 mV / s, and the voltage range was -0.2~0.6 V; the frequency of the EIS working voltage was 100 kHz~1 Hz, and the voltage amplitude was 0.005 V. DPV analysis was performed in PBS (0.1 M, 10 mL) containing H2O2, with the potential window set at -0.2~0.6 V and the voltage amplitude at 0.05 V.

[0062] The conductivity of the electrode directly affects the sensitivity of the sensor. CV was used to verify that G / GDY had significant differences in redox peaks compared to independent G and GDY ( Figure 3 A). When Au NPs are grown on the composite G / GDY, the electron transfer rate is faster due to the excellent conductivity of the metal, which provides better electrochemical performance and biosensing sensitivity. The electrochemical properties of the sensor were monitored using EIS ( Figure 3 B), the results are consistent with the results of CV, G / GDY-Au has a smaller impedance. In addition, the comparison of the catalytic H2O2 and impedance of four-metal nanospheres and trimetallic and bimetallic nanospheres was evaluated by CV and EIS. PdPtCuRu MNs showed better catalytic performance and smaller resistance ( Figure 3 C, D), which may be due to the addition of Cu and Ru metals, which adjust the ductility of the mesoporous structure, increase the overall specific surface area, reduce impedance, and increase the rate of electron transfer.

[0063] TDN was characterized by PAGE. The figure shows the schematic diagram of the self-assembly process of TDN ( Figure 4 A). Lanes 2-5 represent four single strands, T1, T2, T3, and T4, respectively. Lane 6 is a three-dimensional structure generated by the four DNA single strands through autonomous hybridization. The electrophoresis speed is the slowest. This is because TDN has the largest molecular weight and there is a steric hindrance effect, so the band closest to the loading point appears ( Figure 4 B). At the same time, PAGE was used to characterize the preparation of the multivalent aptamer network ( Figure 4C). Band 2 is Primer, band 3 is CD chain, and band 4 is a complex of Primer and CD chain. There are obvious differences among the three. The migration speed of lane 4 is slower, which indicates that Primer and CD chain can hybridize to form a product cDNA with a larger molecular weight. TDN is mixed with cDNA, and dNTPs and Phi29 DNA polymerase are added. After enzyme catalysis and incubation to form TDN-PCP, a bright band with a significantly reduced migration speed can be observed in band 6, indicating that RCA has occurred and a long-chain multivalent aptamer network product has been formed. In addition, a hydrogel-like solid also appears in the EP tube when observed with the naked eye ( Figure 4 D). The DNA network was stained with SYBRGreen I and imaged by confocal laser scanning microscopy (CLSM) after lyophilization, and green tangles were observed ( Figure 4 E). In order to observe the network structure more clearly, scanning electron microscopy (SEM) was used to characterize the morphology of the multivalent aptamer network, and a network like a coil of yarn could be observed at different magnifications ( Figure 4 E, F). In summary, the multivalent aptamer network was successfully prepared on TDN.

[0064] The layer-by-layer modification process was verified by CV in 10.0 mM K3[Fe(CN)6] solution ( Figure 4 G). Compared with the bare electrode GCE, the redox peak current increased significantly after modification with Au-G / GDY, which is due to the large effective surface area of ​​the two-dimensional nanomaterial G / GDY and the strong conductivity of Au-G / GDY, which accelerated the electron transfer rate of [Fe(CN)6] 3- / 4-. Further, after the addition of TDN / MCH, the peak current continued to decrease, which is due to their non-conductive properties. The peak current decreased when the RCA reaction was carried out on the interface, which was due to the formation of a large number of multivalent aptamer networks on the interface, which hindered the interaction between the electrode surface and [Fe(CN)6] 3- / 4-, thereby further reducing the electron transfer rate. Next, due to the resistance of the cell membrane, the peak current continued to decrease after incubating MCF-7 cells on the sensor. When the sensing interface was modified with the signal probe S1-PdPtCuRu MNs, the peak current increased significantly, proving that the label material PdPtCuRu MNs has good conductivity and can effectively promote electron transfer.

[0065] To further investigate the impedance of electron transfer, the impedance response was recorded using EIS ( Figure 4H). Compared with the bare electrode GCE, the modification of Au-G / GDY on the bare electrode GCE surface resulted in a significant decrease in Rct, indicating an accelerated electron transfer rate. However, after the addition of TDN / MCH, RCA reaction system, and MCF-7 cells for assembly, the Rct value increased significantly. It is worth noting that after the addition of capture probe S1-PdPtCuRu MNs, the Rct value decreased due to the high conductivity that can significantly promote the electron transfer rate. These results are consistent with the results of CV, indicating that a highly conductive aptamer sensor was successfully prepared.

[0066] The concentration of TDN determines the amount of template for rolling amplification. However, more TDN will increase the steric hindrance of the sensing interface. Here, we study the effect of TDN concentration on the electrical signal ( Figure 5 A), the best electrical signal was generated when the TDN concentration was 0.4 μM, therefore, the optimal concentration of TDN was 0.4 μM.

[0067] The RCA time directly affects the length of the RCA product. Optimizing the RCA time can quickly achieve effective amplification ( Figure 5 B). In this embodiment, the RCA time was increased from 10 minutes to 60 minutes in increments of 10 minutes, and the optimal time was 30 minutes, because after this time, the current response decreased. This is because the multivalent aptamer network caused an increase in nonspecific interactions within or between molecules, which hindered the binding of the aptamer to the cell. The cell incubation time determines the effect of cell capture. Here, the cell incubation time was optimized ( Figure 5 C). As the cell incubation time increased from 20 minutes to 120 minutes, the current response peaked at 80 minutes. Therefore, in subsequent experiments, the cell incubation time was selected to be 80 minutes.

[0068] The performance of the DNA network-modified biosensor in detecting MCF-7 breast cancer cells was analyzed using DPV analysis under optimal conditions. 6 cells per ml, the DPV value gradually increases ( Figure 6 A). At the same time, the linear regression model showed that the current value was related to the logarithmic concentration (10 1 ×10 6 cells mL -1 ) showed a good linear relationship, where the linear equation was Y (μA) = 135.269 + 8.54051 lg C, and the correlation coefficient was 0.9958. Y represents the current response value corresponding to different cell concentrations, and C represents the cell concentration. The detection limit was 7 cells mL -1 ( Figure 6B). Compared with previously reported biosensors (Table 2), the aptamer network sensor developed in this study shows great potential for efficient capture of CTCs. Such efficient detection performance is mainly due to the following factors. First, the pendant chain can produce a multivalent aptamer network through RCA to efficiently and specifically capture cells, and has a multivalent cooperative capture mode, thereby enhancing the affinity for cells. Second, the addition of TDN provides a moist environment and stable support for incubation using the steric effect. Third, Au-G / GDY enhances the electron transfer rate on the electrode surface and increases the fixation of TDN on the electrode surface. In addition, the S1-PtPdCuRu capture probe has excellent electrocatalytic performance and can accurately and effectively capture MCF-7 cells.

[0069] Table 2 Performance comparison of different biosensors

[0070]

[0071] Detection of MCF-7 in human serum samples The potential clinical application of the proposed biosensor was evaluated by recovery experiments, and the biosensor was constructed using disposable screen-printed electrode sheets for spiking experiments ( Figure 6 F). The current signal increased with the increase of MCF-7 cell number (range 10-1×10 6 Cell mL −1 ). Similarly, the electrochemical response was linearly correlated with CTCs (Y = 5.447 + 1.6087 lgC, R2 = 0.9946), indicating that the electrochemical biosensor has good accuracy and high sensitivity and has potential application prospects in CTCs analysis of clinical samples.

[0072] Schematic diagram of the process of RCA forming a multivalent aptamer network to release cells (see Figure 6 G). The release process of cells was observed under bright field by inverted microscope. Benzonase nuclease was used to shear the nucleic acid chain, which resulted in the release of cells, showing a dispersed distribution state ( Figure 6 H). The cell suspension was collected by centrifugation, and a suitable proportion of culture medium was added and transferred to a constant temperature incubator (37 ℃, 5% CO2) for culture. After 6 h of culture, a small number of spindle-shaped single cells were observed to grow attached to the wall ( Figure 6 I). After culturing for 10 h, more cells showed spindle-shaped adherent growth and good cell morphology ( Figure 6 J). This indicates that when MCF-7 cells were restored to culture, they maintained good growth and division and were more suitable for further biological analysis.

[0073] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A biosensor for capturing and detecting CTCs, characterized in that: include: AuNPs-G / GDY coating, wherein the AuNPs-G / GDY coating is disposed on the surface of the electrode; A multivalent aptamer network layer is provided on the AuNPs-G / GDY coating, and the preparation materials of the multivalent aptamer network layer include tetrahedral DNA, circular DNA template and dNTPs. The preparation method includes: dripping a reduced tetrahedral DNA solution on the surface of an electrode provided with the AuNPs-G / GDY coating and incubating the solution, and then dripping a solution containing Mg after the incubation. 2+ , K + , cDNA, DNA polymerase and dNTP solution and perform in situ RCA reaction to obtain a multivalent aptamer network layer; The invention discloses a PdPtCuRu-aptamer probe, and the preparation method thereof comprises: attaching a circulating tumor cell aptamer to the surface of PdPtCuRu MNs through a metal thiol bond to obtain the PdPtCuRu-aptamer probe.

2. A method for preparing a biosensor for capturing and detecting CTCs as claimed in claim 1, characterized in that: include: Preparation of AuNPs-G / GDY coating: introducing nano-gold into a mixed solution of graphene and graphyne to obtain AuNPs-G / GDY nanocomposite material, preparing the AuNPs-G / GDY nanocomposite material into a suspension and then dropping the suspension onto the electrode surface to form AuNPs-G / GDY coating; Preparation of multivalent aptamer network layer: Add reduced tetrahedral DNA solution to the electrode surface with AuNPs-G / GDY coating and incubate. After the incubation, continue to add Mg 2+ , K + , cDNA, DNA polymerase and dNTP solution and perform in situ RCA reaction to obtain a multivalent aptamer network layer; Preparation of PdPtCuRu-aptamer probe: The circulating tumor cell aptamer was attached to the surface of PdPtCuRu MNs through metal-thiol bonds to obtain the PdPtCuRu-aptamer probe.

3. The method for preparing a biosensor for capturing and detecting CTCs according to claim 2, characterized in that: During the preparation of the AuNPs-G / GDY coating, HAuCl4 was added to a mixed solution of graphene and graphyne, and then sodium citrate solution was added to obtain gold nanoparticles, and the mass ratio of graphene to graphyne was 1:

1.

4. The method for preparing a biosensor for capturing and detecting CTCs according to claim 2, characterized in that: During the preparation of the multivalent aptamer network layer, four DNA chains are thermally induced to self-assemble into tetrahedral DNA through a heating-rapid cooling method.

5. The method for preparing a biosensor for capturing and detecting CTCs according to claim 2, characterized in that: The preparation method of PdPtCuRu MNs includes: mixing Pluronic F127 with H2PtCl6, Na2PdCl4, CuCl2, RuCl3 and HCl solutions according to a ratio, adding ascorbic acid after ultrasonic dissolution, then storing the mixed solution in an oil bath, and centrifuging the obtained precipitate after removing residual Pluronic F127 to obtain PdPtCuRu MNs.

6. The method for preparing a biosensor for capturing and detecting CTCs according to claim 2, characterized in that: The concentration of AuNPs-G / GDY solution is 1-4 mg / ml. When the effective area of ​​the glassy carbon electrode is 7 mm 2 When the AuNPs-G / GDY solution is used, the amount is 6 μL, the concentration of tetrahedral DNA is 0.3-0.6 μM, the amount is 10 μL, and the concentration of cDNA is 1 μM, the amount is 5-10 μL.

7. The method for preparing a biosensor for capturing and detecting CTCs according to claim 2, characterized in that: The in situ RCA reaction time is 30-50 minutes.

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