Microfluidic detection system based on MBene nanosheet and functional partition as well as detection method and application thereof

Abstract

The invention relates to a microfluidic detection system based on MBene nanosheets and functional partitions as well as a detection method and application thereof, and belongs to the technical field of biological detection. The method aims at solving the problems that an existing ctDNA detection method is high in cost, complex in operation and insufficient in sensitivity. The invention provides a microfluidic detection system based on MBene nanosheets and functional partitions, which is characterized in that the MBene nanosheets which have high adsorbability to ssDNA and selectivity to ssDNA / dsDNA are combined with an Exo III enzyme-assisted signal amplification strategy to realize ctDNA detection. According to the invention, the detection sensitivity reaches 0.062 pM (solution phase) and 4.125 pM (paper-based chip), and the specificity is gt; 95%; the detection time is shortened to 40 minutes; a lung cancer mouse model verifies that the result is highly consistent with a qPCR result (R2 = 0.991). The invention provides a high-sensitivity and portable solution for early diagnosis and postoperative monitoring of lung cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochip detection, and relates to a microfluidic detection system based on MBene nanosheets and functional partitions, and a detection method and application thereof. Background Art

[0002] Circulating tumor DNA (ctDNA) in the peripheral blood of cancer patients contains information on genomic alterations that are highly specific to the original tumor. Therefore, in order to achieve more personalized diagnosis and treatment, from early detection to postoperative monitoring, ctDNA detection is becoming a prominent non-invasive method. For example, in non-small cell lung cancer (NSCLC), the detection of ctDNA for the most common mutations in the EGFR gene ((exon 19del) and exon 21 (L858R)) has been approved for clinical use. Currently, the main standard methods for detecting ctDNA are next-generation sequencing (NGS) and polymerase chain reaction (PCR), but they still have limitations such as high cost and complex operation. Therefore, the establishment of nanomaterial-based DNA biosensors has attracted widespread attention because many properties of new nanomaterials can be developed and functionalized to achieve low-cost and effective detection.

[0003] Among these materials, single-layer nanosheets have been shown to have efficient DNA molecule sensing capabilities because they selectively adsorb single-stranded nucleic acids (ssDNA or ssRNA) through π-π stacking, hydrogen bonding, or electrostatic interactions between nitrogenous bases and two-dimensional surfaces. Most nanosheet-based DNA sensing strategies are used for optical applications. For example, typical graphene, MXene, and metal sulfide nanosheets (such as MoS2) are used to construct fluorescent DNA biosensors, in which fluorophore-labeled ssDNA probes are adsorbed by the nanosheets and quench fluorescence, but the double-stranded DNA (dsDNA) formed after the fluorescent probe captures the target DNA cannot be adsorbed by the nanosheets, and the fluorescence is restored. The changed fluorescence value can be used to quantify the content of the target DNA. Therefore, it is crucial to explore new nanosheet materials with excellent fluorescence quenching ability and different affinities for ssDNA and dsDNA.

[0004] MBene, a derivative of MXene, is a two-dimensional transition metal boride, with MoB-MBene being the most widely studied. The exposed metal and boron atoms on the surface of MBene serve as active sites, enabling it to exhibit excellent performance in certain catalytic reactions. Common preparation methods include alkaline / acid etching and solvothermal methods. MBene exhibits high mechanical stability, high charge carrier mobility, electrochemical properties, and catalytic performance. As an emerging material, MBene has demonstrated significant potential in certain specific applications and is expected to be widely developed within the MXene field. MBene was first described in 2017, and while research remains limited, its unique properties have already been studied and applied in fields such as energy storage and catalysis. However, the application of MBene nanosheets in biosensing is still in its early stages, with few reports. In particular, the interaction between DNA and MBene nanosheets remains largely unexplored.

[0005] Furthermore, in the construction of biosensing platforms, there is an increasing demand for cost-effective devices with functional designs to simplify complex biochemical reactions and enable lab-on-a-chip testing. Paper-based microfluidic analytical devices (μPADs) have emerged as a promising and powerful candidate due to their attractive advantages, including low cost, ease of operation, and portability. Microfluidic channels can be fabricated on paper-based devices by constructing hydrophobic barriers. Liquids can then flow through the hydrophilic channels, leveraging the inherent capillary forces of the paper. Reaction results output on the paper can be read using handheld devices such as smartphones, making the entire testing process more convenient. Furthermore, μPADs are inexpensive to manufacture and can be easily implemented using common methods such as inkjet printing, wax printing, and screen printing. Due to these advantages, μPADs are widely used in point-of-care (POCT) testing for health diagnosis, environmental monitoring, and food safety. Summary of the Invention

[0006] In light of this, the present invention constructs an MBene-based fluorescent DNA biodetection system on a microfluidic chip for real-time detection of circulating tumor DNA (ctDNA). The present invention aims to provide a microfluidic detection system based on MBene nanosheets and functional zoning. A second objective is to provide a detection method based on this microfluidic detection system. A third objective is to use this microfluidic detection system based on MBene nanosheets and functional zoning for ctDNA detection.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a microfluidic detection system based on MBene nanosheets and functional zoning, the microfluidic detection system comprising a microfluidic chip and a detection reagent, the microfluidic chip comprising two layers, the upper layer being a reaction zone, the lower layer comprising a mixing channel and a delay zone, with a fast channel and a slow channel, the detection reagent comprising MBene nanosheets, fluorescently labeled ssDNA, and exonuclease III;

[0009] Preferably, the preparation method of the MBene nanosheets is as follows:

[0010] S1: Precursor etching treatment: lithium salt and concentrated hydrochloric acid solution are mixed to form an etching system, MoAlB powder is added and stirred at 37-40°C for 20-24 hours. The concentration of the concentrated hydrochloric acid is 8-10M, and the mass ratio of lithium salt to MoAlB is (1.5-1.7):1. A precipitate is obtained by centrifugation;

[0011] S2: Purification treatment: The precipitate obtained in step S1 was washed with deionized water until the pH value was 7, the precipitate was collected by centrifugation at 5000 rpm for 5 min, and dried at 60-80°C for 8-12 hours to obtain MBene powder;

[0012] S3: Nanosheet exfoliation: The purified MBene powder was dispersed in deionized water to prepare a 1-3 mg / mL aqueous solution, which was then treated in a water bath with ultrasonic waves for 20-24 hours at a power of 70-90% of the total instrument power.

[0013] S4: Gradient centrifugation: The ultrasonic product was centrifuged at 3000 rpm to collect the supernatant, and then the supernatant was centrifuged at 12000 rpm to collect the precipitated product, which was then freeze-dried in vacuum to obtain the MBene nanosheets.

[0014] Furthermore, the detection method based on the microfluidic detection system of MBene nanosheets and functional partitioning has the following detection steps:

[0015] S1: Mix the fluorescently labeled ssDNA probe with the target to be detected, then add exonuclease III and incubate at 37°C in the dark for 30 minutes;

[0016] S2: Then, 25 μL of 0.05 mg / mL MBene nanosheets was added and reacted for 10 min. After the reaction was completed, the fluorescence intensity was measured at an excitation wavelength of 480 nm and an emission wavelength of 520 nm.

[0017] Preferably, the molar ratio of the fluorescently labeled ssDNA probe to the target to be detected and exonuclease III is 1:1:1;

[0018] Furthermore, the application of microfluidic detection system based on MBene nanosheets and functional partitioning in the preparation of biosensors;

[0019] Furthermore, the application of microfluidic detection system based on MBene nanosheets and functional partitioning in ctDNA detection;

[0020] Preferably, the detection steps are as follows:

[0021] S1: 25 μL of 0.5 μM FAM-labeled ssDNA probe and 25 μL of ctDNA to be tested were added dropwise to the mixing channel on the microfluidic chip respectively;

[0022] S2: Add 25 μL of 40 U exonuclease III to the fast channel and 25 μL of 0.05 mg / mL MBene nanosheets to the slow channel;

[0023] S3: Incubate the paper-based chip at 37°C in the dark for 40 minutes, then place the paper-based chip in a dark box. Use a blue LED excitation light with a 480 nm filter to illuminate the microfluidic chip, and take a picture through a 520 nm filter under the lens.

[0024] S4: Use Image J software to obtain the RGB values ​​of the reaction area photos and calculate the ctDNA concentration;

[0025] Preferably, the nucleic acid sequence of the ssDNA is shown in SEQ ID NO: 1;

[0026] Preferably, the ctDNA is lung cancer ctDNA.

[0027] The beneficial effects of the present invention are:

[0028] The present invention combines an Exo III-assisted fluorescence signal amplification strategy to construct a fluorescent DNA sensing platform, which achieves sensitive detection of ctDNA with a detection limit as low as 0.062pM, a significant improvement over existing methods. To further advance this detection method in the field of point-of-care (POCT), the present invention designed a paper-based microfluidic chip with mixing-flow regulation functions, which can integrate and program the above-mentioned fluorescence detection process. Ultimately, using the WeChat mini-program "ctDNADetection", sensitive and immediate detection of ctDNA was achieved with a detection limit of 4.125pM. Finally, in terms of actual sample analysis, peripheral blood samples of mice with lung cancer were tested before and after surgery. The present invention used MB@GO to efficiently couple Clutch probes and enrich ctDNA for detection. The test results were consistent with standard qPCR and are expected to be used for further clinical sample analysis.

[0029] 1. Material innovation advantages

[0030] MBene nanosheets, prepared by LiF / HCl etching combined with ultrasonic exfoliation, exhibit high adsorption and selectivity for single-stranded DNA (ssDNA) and can accurately distinguish between ssDNA and double-stranded DNA (dsDNA), providing a core material foundation for high-sensitivity fluorescence detection.

[0031] 2. Ultra-low detection limit and high sensitivity

[0032] The solution-phase fluorescence detection limit reaches 0.062 pM (linear range 0.1-100 pM), which is 5-10 times more sensitive than traditional nanosheet detection methods (such as MXene and MoS2). The paper-based chip has a detection limit of 4.125 pM, which is superior to similar paper-based fluorescence detection technologies.

[0033] 3. Device portability and intelligence

[0034] The integrated design of the paper-based microfluidic chip and the cross-shaped mixing channel improve the reagent mixing efficiency, and the fast / slow channels realize the automatic sequential delivery of reagents.

[0035] 4. Clinical Applicability Verification

[0036] The animal model validated its reliability. Blood samples from tumor-bearing mice showed a significant difference in ctDNA concentration between healthy and lung cancer groups (p<0.001), with a postoperative monitoring sensitivity of 100%. The ROC curve had an AUC of 1, equivalent to qPCR, demonstrating potential for clinical translation.

[0037] 5.Technical scalability

[0038] MBene has diverse application scenarios. This material can be expanded to detect other nucleic acid markers (such as miRNA and methylated DNA), or used in nanopore sequencing and biological imaging.

[0039] Other advantages, objects, and features of the present invention will be described in part in the following description and, in part, will be apparent to those skilled in the art upon examination of the following description or may be learned from practice of the present invention. The objects and other advantages of the present invention may be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below with reference to the accompanying drawings, in which:

[0041] Figure 1 Silk screen printing for designing and printing paper-based chips;

[0042] Figure 2 For the screen printing process and the physical paper-based chip;

[0043] Figure 3 A device for taking photos during immediate testing;

[0044] Figure 4 A is the preparation of MBene NSs, B is the SEM image of multilayer MBene, C is the TEM image of MBene NSs, D is the TEM image of MBene NSs and the corresponding EDS element mapping, E is the high-resolution TEM image of MBene NSs, F is the AFM image of MBene NSs, G, H, and I are the XPS spectra of Mo 3d, Al 2p, and B1s of MoAlB and MBene NSs, respectively;

[0045] Figure 5 is the difference in adsorption energy of DNA bases adsorbed on MBene or MXene;

[0046] Figure 6 is the difference in adsorption energy between nucleobases and base pairs on MBene;

[0047] Figure 7 A is the fluorescence quenching kinetics of FAM-ssDNA and FAM-ssDNA+MBene NSs, B is the fluorescence recovery kinetics of FAM-ssDNA+cDNA+MBene NSs, and C is a simplified schematic diagram of the possible reaction processes of FAM-ssDNA+MBene NSs and FAM-ssDNA+cDNA+MBeneNSs;

[0048] Figure 8 To test the feasibility analysis results;

[0049] Figure 9 A is the optimization of MBene concentration, B is the optimization of ExoⅢ dosage, C is the optimization of hybridization time, and D is the optimization of quenching time;

[0050] Figure 10 A is the fluorescence detection spectrum of different concentrations of ctDNA, B is the corresponding linear fitting graph, C is the specific detection of target ctDNA and other analytes, D is the reproducible detection result, and E is the detection of target ctDNA in spiked serum;

[0051] Figure 11 Schematic diagram of paper-based microfluidic chip;

[0052] Figure 12 A is the flow image of red and blue ink in different mixing channels of paper base, and B is the corresponding concentration distribution of different channels, the unit is mol / m 3 , C is the absolute mixing index (AMI) along the distance of the segmentation point of different channels, D and E are the total flux and velocity fields of different channels, respectively;

[0053] Figure 13 A is an illustration of the smartphone fluorescence detection device, B is the QR code and user interface of the WeChat applet for ctDNA detection, C and D are the fluorescent paper-based detection of different ctDNA concentrations and the 3D ImageJ images of the corresponding paper-based chip, respectively, E is the detection of different analytes, F is the reproducible detection, and G is the detection of target ctDNA in serum samples;

[0054] Figure 14 A and B are SEM images of magnetic beads (MB) and graphene oxide-coated magnetic beads (MB@GO), respectively. C is the evaluation of coupling efficiency (a: stock solution before Clutch 5 probe coupling with different magnetic beads, b: supernatant after coupling with MB, c: supernatant after coupling with MB@GO). D is the evaluation of cDNA removal efficiency (d: stock solution before enrichment, e: supernatant after MB / Clutch5 enrichment, f: supernatant after MB@GO / Clutch 5 enrichment).

[0055] Figure 15 Figures A and B show the qPCR detection of ctDNA at different concentrations and the linear relationship between the Ct value and the ctDNA concentration, respectively. Figure C shows the consistency comparison of ctDNA concentrations in lung cancer cell samples at different dilutions detected by qPCR and the proposed method.

[0056] Figure 16 Preoperative and postoperative images of tumor-bearing nude mice and hematoxylin and eosin staining of the corresponding tumors;

[0057] Figure 17 A is a schematic diagram of the establishment of the tumor-bearing mouse model and surgical treatment. B is the proposed biosensor and qPCR method for detecting ctDNA in healthy mice, tumor-bearing mice, and mice after surgery. C and D are the statistical results of the proposed biosensor and qPCR for measuring ctDNA concentrations in healthy mice, tumor-bearing mice, and mice after surgery (***p < 0.001). E and F are ROC analysis evaluations of the accuracy of the proposed biosensor and qPCR in distinguishing tumor-bearing mice from healthy mice.

[0058] Figure 18 Overview of the real-time fluorescence detection of ctDNA based on MBene nanosheets (MBene NSs) and multifunctional partitioned paper-based microfluidic chips. DETAILED DESCRIPTION

[0059] The following describes the embodiments of the present invention by means of specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic illustrations of the basic concept of the present invention, and the following embodiments and features in the embodiments can be combined with each other without conflict.

[0060] Among them, the accompanying drawings are only for illustrative purposes and represent only schematic diagrams rather than actual pictures, and should not be understood as limiting the present invention. In order to better illustrate the embodiments of the present invention, some parts of the accompanying drawings may be omitted, enlarged or reduced, and do not represent the dimensions of actual products. For those skilled in the art, it is understandable that some well-known structures and their descriptions may be omitted in the accompanying drawings.

[0061] The same or similar numbers in the drawings of the embodiments of the present invention correspond to the same or similar parts; in the description of the present invention, it should be understood that if there are terms such as "upper", "lower", "left", "right", "front", "back", etc. indicating directions or positional relationships, they are based on the directions or positional relationships shown in the drawings. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operate in a specific direction. Therefore, the terms describing the positional relationship in the drawings are only used for illustrative purposes and cannot be understood as limiting the present invention. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0062] Example 1 Preparation of MBene Nanosheets

[0063] First, 1.6g of LiF was added to 20mL of 9M HCl solution under magnetic stirring. Then, 1g of MoAlB powder was slowly added to the mixture and stirred at 37°C for 24h. The etched product was centrifuged at 5000rpm for 5min, and the precipitate was collected. The precipitate was then washed with deionized water until the pH of its aqueous solution was close to 7. Finally, the precipitate was collected by centrifugation at 5000rpm for 5min, and then dried in a 60°C oven for 12h. The powder was collected and stored for later use. MBene nanosheets (MBene NSs) were then synthesized by ultrasonic aqueous exfoliation. Specifically, a 2mg / ml multilayer MBene aqueous solution was prepared and placed in a water bath sonicator at 80% of the instrument's total ultrasonic power for 24h. The supernatant was then collected by centrifugation at 3000rpm. The final precipitated product was collected by centrifugation at 12000rpm and vacuum freeze-dried for 24h.

[0064] Example 2 MBene nanosheet performance test

[0065] (1) Adsorption performance of MBene nanosheets on DNA: ssDNA was labeled with 6-carboxyfluorescein (FAM) fluorophore to study the DNA adsorption of MBene NSs. The excitation and emission wavelengths of the fluorescence test were 480 nm and 520 nm, respectively. A mixed solution of 0.5 μM FAM-ssDNA and buffer was taken as the control group, with a volume ratio of 1:1. A mixed solution of 0.5 μM FAM-ssDNA and 0.1 mg / mL MBene NSs was taken as the experimental group, with a volume ratio of 1:1. The mixture was incubated at room temperature for different times and its fluorescence intensity was measured. By measuring the fluorescence change of the mixed solution of 0.5 μM FAM-ssDNA, 0.5 μM cDNA and 0.1 mg / mL MBene NSs over time, the solution was prepared at a volume ratio of 1:1:1 to study the desorption after double-strand formation. All solutions were prepared in Tris-HCl buffer (20 mM, 10 mM NaCl, pH 7.4).

[0066] (2) MBene nanosheets for ctDNA detection: 25 μL of 0.5 μM FAM-ssDNA probe was mixed with 25 μL of ctDNA of different concentrations, and then 25 μL of 40 U Exo III was added. The final solution was incubated at 37°C in the dark for 30 min. Then, 25 μL of 0.05 mg / mL MBene NSs was added and reacted for 10 min. Finally, the fluorescence intensity was measured at an excitation wavelength of 480 nm and an emission wavelength of 520 nm.

[0067] Example 3 Preparation of paper-based microfluidic chip

[0068] The microfluidic channels on the paper-based chip were designed using AutoCAD, and then the corresponding silk screen wax printing screens were customized, such as Figure 1 A is a mixing channel of two different shapes for comparative analysis. Figure 1 B is a paper-based chip device used for detection. The screen wax printing process is as follows Figure 2 A, make finished products and assemble as Figure 2 As shown in B. In order to reduce the volatilization during the detection process, two PVC boards are cut and Velcro with adhesive is pasted along the edges. The paper-based chip is placed between the two PVC boards and can be sealed by Velcro. Figure 2 As shown in C, volatilization is alleviated and pollution is prevented to a certain extent, and the transparent PVC plate does not affect the observation results.

[0069] Example 4 Detection of ctDNA using a paper-based microfluidic chip

[0070] 25 μL of 0.5 μM FAM-labeled DNA probe and 25 μL of ctDNA of different concentrations were dropped into the mixing channel on the paper-based chip, and then 25 μL of 40 U Exo III was added to the fast channel and 25 μL of 0.05 mg / mL MBene NSs was added to the slow channel. The paper-based chip was placed in a PVC-velcro seal and incubated at 37 °C in the dark for 40 min. The Velcro was torn off and the paper-based chip was placed in a dark box, as shown in FIG. Figure 3 As shown, a blue LED excitation light with a filter (480 nm) was used to illuminate the paper chip, and then a smartphone was used to take a picture through a filter (520 nm) below the lens to obtain the emitted green fluorescence. Finally, Image J software was used to obtain the RGB value of the reaction area and input it into the WeChat applet to read the ctDNA concentration.

[0071] Example 5 Performance test of paper-based microfluidic chip

[0072] In the following tests:

[0073] (1) DFT Theory: Density functional theory (DFT) calculations were performed using the CASTEP package in Materials Studio. The exchange-correlation interaction was studied using the revised Perdew-Burke-Ernzerhof (PBE) parameterization and the generalized gradient approximation (GGA). The cutoff energy of the ultrasoft pseudopotential was 600 eV. In the calculations, a (2 × 2 × 2) k-point grid was used, and the Hellmann-Feynman force was kept at Below, the total energy difference is less than 2×10 -5 eV / atom, inter-ion displacement is lower than In the model, five layers are separated, the bottom three layers are restricted, and A vacuum layer is established at the , which makes the interaction between consecutive unit cells negligible. The adsorption energy (E) between MBeneNSs and DNA is calculated using the following formula ad ):E ad =E base+nanosheet -(E base +E nanosheet ), where E base+nanosheet is the energy of the nanosheet-base adsorption system, E base is the energy of base adsorption, E nanosheet is the adsorption energy of MBeneNSs.

[0074] (2) Numerical simulation: The numerical analysis of the paper-based flow channel was performed in COMSOL 5.5 software. The capillary wicking phenomenon of the filter paper was determined using Darcy's law. The Frck law was used to describe the relationship between the diffusion flux and the concentration gradient. The absolute mixing index (AMI) was calculated as follows: Combining the AMI with the Frck law in the concentration field, the mixing performance of the fluid can be quantified by analyzing the diffusion behavior and mixing uniformity of the concentration field. Therefore, an equation representing the mixing process can be obtained: C i (t) is the local concentration at each node evolving with time according to Fick’s law,<C(t)> is the average concentration at that moment.

[0075] (3) Sample pre-enrichment: Graphene oxide-coated magnetic beads (MB@GO) were used for magnetic pre-enrichment of single-stranded ctDNA. First, MB@GO was prepared: 50 μL of 2 mg / mL GO, 50 μL of 0.1 M EDC / NHS mixture, and 50 μL of amino-modified magnetic beads (0.5%, w / v) were mixed, and then the above solution was slowly rotated and mixed for 12 hours. EDC / NHS acts as a catalyst for the coupling between carboxyl and amino groups, which can promote the formation of amides. EDC increases the reaction rate by producing an activated ester intermediate, while NHS acts as a catalytic enhancer. Finally, the product was centrifuged and washed three times at 10,000 rpm for 3 minutes, and then the precipitate was resuspended in 50 μL of deionized water.

[0076] Before enrichment, MB@GO was first coupled to the Clutch 5 probe (SEQ ID NO: 5): the MB@GO solution was mixed with 0.1M EDC / NHS and the Clutch 5 probe, the mixture was incubated for 60 minutes, and then the precipitate was collected by centrifugation three times at 10,000 rpm for 3 minutes and resuspended in 50 μL Tris-HCl buffer and stored at 4°C until use. The coupling efficiency was evaluated to compare the difference between MB@GO and MB in coupling to the Clutch 5 probe. Specifically, the ultraviolet absorption of the supernatant was measured in the last step. The coupling efficiency was calculated by the formula: Among them, A1 is the ultraviolet absorption of the original probe solution, and A2 is the ultraviolet absorption of the supernatant after coupling. Next is the capture of complementary DNA (cDNA). Unscrew 10μL of the actual sample at 90°C for 2min, then add 10μL of Clutch 3 probe (SEQ ID NO: 6) and 10μL of the above-mentioned MB@GO / Clutch 5 probe, and react at 0°C for 30min. The supernatant is then collected for detection. Then, the capture of cDNA by MB@GO / Clutch 5 probe and MB / Clutch 5 probe, that is, the removal efficiency, is compared and evaluated. Specifically: by measuring the ultraviolet absorption of the supernatant after the reaction, the removal efficiency is calculated using the formula: Among them, A M is the UV absorption of the solution before adding MB@GO / Clutch 5 or MB / Clutch 5, A m It is the UV absorption of the supernatant after cDNA capture.

[0077] Figure 4 In the synthesis of MBene NSs, Figure 4 As shown in A, the Al layer is first etched from MoAlB to obtain multilayer MBene, and then ultrasonic aqueous phase exfoliation is performed to obtain MBene nanosheets. From the SEM results, it can be observed that the original MoAlB exhibits a multilayer morphology after etching ( Figure 4 B). Figure 4 C shows the TEM results of MBene NSs obtained by further ultrasonic treatment, showing a thin sheet morphology with some small wrinkles on the surface.

[0078] TEM X-ray spectroscopy mapping (EDS) ( Figure 4 D) shows that the material contains Mo, B, O, and Al elements, and they are evenly distributed. It can be seen that the content of Al is relatively low, which means that aluminum is etched away to a certain extent, but due to structural defects in the MAB phase material (M is a transition metal element, A is Al, and B is a boron element), there is less Al remaining. Further observation by high-resolution transmission electron microscopy, such as Figure 4 As shown in E, the lattice spacing of MBene NSs is 0.27nm and 0.43nm respectively. The thickness was then analyzed by AFM and the result was 3.77nm ( Figure 4 F)

[0079] The elemental composition and valence changes were studied by XPS, such as Figure 4 As shown in G, the Mo 3d spectra of MoAlB appear at 235.4 eV and 232.2 eV, corresponding to Mo 6+ The oxidation state of MBene NSs is different from that of Mo 3d spectra, and the different peaks appearing in the Mo 3d spectra are attributed to Mo 6+ (236.2eV and 233.1eV), Mo 5+ (234.5eV and 230.8eV) and Mo 4+ (229.8eV). These changes may be attributed to the oxidation reaction during HF etching, in which the oxygen and fluorine atoms appear and cause changes in the electron cloud density of the molybdenum atoms. Figure 4 H shows the changes of Al elements before and after etching. The Al characteristic peak at 71.5eV in MoAlB disappears in the XPS spectrum of MBeneNSs, which indicates that Al in MoAlB is effectively etched, and the peaks at 74.2 and 75.1eV belong to MoAlB and Al2O3, respectively. Figure 4 In I, the peaks of 192.3eV and 187.8eV in MoAlB and the peaks of 193.1eV and 190.1eV in MoAlB all correspond to MoB and B oxide. After etching, the peaks shift to high fields, indicating the appearance of negatively charged oxygen and fluorine atoms. Therefore, the above results all indicate that the preparation of MBene NSs is successful. The formula for calculating the ratio of each element in the figure is: N1 / N2=I1S2 / I2S1, where N is the atomic concentration, I is the integrated area of ​​the peak, and S is the sensitivity factor of the element, which is determined only by the type of element. The number 2 represents the total XPS spectrum of each element, and the number 1 represents each spectrum separated from the total spectrum. From Figure 4 As can be seen from Figure H, the etching of Al during the synthesis process is effective, and the almost complete retention of Al2O3 is due to the amorphous passivation process formed by the presence of the top layer of highly active Al.

[0080] Figure 5 In this study, density functional theory (DFT) was used to compare the interactions between MXene and MBene with DNA. The adsorption energies (E ad ). The calculation results are as follows Figure 5 As shown, the E of MXene-DNA and MBene-DNA interactions ad The calculated results are all negative, indicating the stability of the adsorption calculation system, and E ad The larger the negative charge, the more favorable the adsorption process. Compared with MXene, MBene exhibits lower E for the adsorption of A, G, and C bases. ad The values ​​indicate that the MBene-A, MBene-G, and MBene-C complexes have higher adsorption strengths. However, in the case of T base adsorption, MXene-T exhibits higher adsorption strength than MBene-T. This may be due to the surface charge distribution or polarity of MXene being more compatible with T bases, resulting in stronger electrostatic interactions, and the presence of more functional groups on its surface, which form additional hydrogen bonds or van der Waals forces with T bases, enhancing adsorption strength. Overall, MBene has excellent adsorption properties when interacting with ssDNA molecules.

[0081] Figure 6 In the present study, the difference in the adsorption between ssDNA and dsDNA and MBene was further studied because the probe in DNA biosensors is often ssDNA, which forms dsDNA by Watson-Crick base pairing with the target ssDNA in analytical applications. The DFT model and the corresponding calculation results are shown in Figure 2. Figure 6 As shown, it can be seen that E adThe value is between -6eV and -10eV, and the E of MBene+A / T / G / C ad Lower than E of MBene+AT / GC ad This indicates that MBene binds more strongly to single nucleobases than to base pairs, implying that dsDNA has a weaker affinity for MBene than ssDNA. Therefore, in theory, MBeneNSs can be used as recognition elements to distinguish between ssDNA and dsDNA.

[0082] Figure 7 In this paper, based on the theoretical analysis results above, before studying the detection of ctDNA, we first used ssDNA labeled with the fluorophore FAM to further explore the interaction between MBene NSs and ssDNA and dsDNA. Figure 7 As shown in the fluorescence kinetic analysis, in the two-component system containing FAM-ssDNA and MBene NSs, the fluorescence intensity decreased significantly over time, while the fluorescence intensity of FAM-ssDNA alone remained relatively stable. This result demonstrates the ability of MBene NSs to adsorb and quench FAM-ssDNA fluorescence, thereby proving that its DNA binding affinity is consistent with theoretical results. Then, a three-component system consisting of FAM-ssDNA, cDNA and MBene NSs was studied, in which cDNA can effectively pair with FAM-ssDNA to form dsDNA, as shown in Figure 3. Figure 7 The detection results show an initial decrease in fluorescence intensity followed by recovery. This phenomenon can be attributed to a dynamic process: initial DNA adsorption by MBene NSs, followed by hybridization of FAM-ssDNA with cDNA, ultimately forming dsDNA. This dsDNA exhibits a weaker affinity for MBene NSs, facilitating desorption and subsequent fluorescence recovery. In summary, the fluorescence sensing processes of the two-component and three-component systems exhibit distinct patterns of fluorescence change.

[0083] In order to gain a deeper understanding of the dynamic process of adsorption, six potential reactions were speculated, such as Figure 7 As shown, these reactions are based on three basic mechanisms: Langmuir-Hinshelwood theory, Eley-Rideal theory, and substitution theory. These reactions include: photobleaching of FAM-ssDNA (K1), adsorption of FAM-ssDNA by MBene NSs (K2), hybridization between FAM-ssDNA and cDNA (K3), adsorption of cDNA by MBene NSs (K4), desorption of FAM-ssDNA from MBene NSs after hybridization with cDNA (K5), and the reverse process (K6). Notably, K1 and K2 contribute to the decrease in fluorescence, while K5 indicates that DNA hybridization promotes the transition from fluorescence quenching to recovery.

[0084] Figure 8 In this study, a fluorescent sensor for detecting ctDNA was constructed based on the obvious difference in the affinity of MBene NSs between ssDNA and dsDNA. Figure 8 As shown, in the absence of target, significant fluorescence quenching was observed (P + MBene NSs), indicating that the single-stranded probe was adsorbed by the nanosheet, resulting in fluorescence quenching. In the presence of ctDNA, there was a significant 3.7-fold fluorescence enhancement (P + Target + MBene NSs), indicating that the probe successfully captured the target ctDNA to form a dsDNA structure. To further enhance the signal response and improve analytical sensitivity, a convenient and sensitive signal amplification strategy driven by a nuclease III (Exo III)-assisted target recovery cycle was introduced. This mechanism promotes the hydrolysis of the FAM-labeled DNA probe in the formed dsDNA into monomeric nucleotides and short oligonucleotide fragments modified with the dye. The released target DNA will then participate in the probe capture process to form a double strand, resulting in fluorescence recovery. This cycle amplifies the fluorescence signal by 5.8-fold, further improving the ctDNA fluorescence detection effect.

[0085] Figure 9 In order to improve the detection efficiency, the concentration of MBene NSs was first optimized, as shown in Figure 9 As shown in A, at a concentration of 50 μg / ml, the fluorescence response signal area is stable, so this value is the optimal concentration. Further optimization of the dosage of Exo III, such as Figure 9 As shown in Figure B, when the content reaches 40 U, ​​the response signal is optimal and no longer increases, so the amount of Exo III added in subsequent tests is 40 U. Next, the optimal hybridization time between the probe and the target is optimized. Selecting the most appropriate time will help improve detection efficiency, as shown in Figure 4. Figure 9 As shown in Figure C, as time goes by, the fluorescence response signal no longer increases, indicating that hybridization has reached saturation. Therefore, 30 min is the most suitable reaction time. After the hybridization reaction, MBene NSs needs to be added to quench the unreacted fluorophore-modified probe, as shown in Figure 4. Figure 9 As shown in D, the fluorescence signal stabilized after 10 min, indicating that quenching was completed and 10 min was the most suitable reaction time.

[0086] Figure 10 In the present invention, ctDNA was analyzed based on the above optimized conditions to establish a standard curve. Figure 10 As shown in AB, within the ctDNA concentration range of 0.1 to 100 pM, the fluorescence intensity increased linearly, which is consistent with Y = 0.006X + 0.324 (R 2=0.993) with a limit of detection (LOD) as low as 0.062 pM. The results indicate that MBene NSs can be used to develop fluorescent DNA biosensors for analyzing ctDNA. Comparisons show that their performance is comparable to or superior to other nanosheet-based fluorescent DNA / RNA detection methods. These advantages can be attributed to: 1) the unique physicochemical properties of MBene NSs in their nanosheet structure, including single-layer nanosheets with high surface area to provide more adsorption sites, as well as the high surface energy of the nanosheets and electronic interactions with DNA molecules, such as electrostatic attraction and π-π stacking, which help enhance adsorption; 2) the specific adsorption of MBene NSs to ssDNA and their ability to exhibit differential affinity between dsDNA and ssDNA; and 3) the benefit of the amplification strategy, which effectively promotes more sensitive detection through the exonuclease III-assisted target recovery cycle.

[0087] Afterwards, the specificity and repeatability of the sensor were explored, e.g. Figure 10 As shown in Figure C, there is a significant difference in signal response between the target ctDNA and its sequence analogs (p < 0.001), indicating the good specificity of the biosensor. Figure 10 As shown in D, there is no significant difference in the response signals of the five parallel groups tested, and the relative standard deviation RSD = 3.29%, indicating that the sensor has good reproducibility during the detection process. In order to evaluate its practical applicability, the biosensor was further tested in serum samples with different concentrations of ctDNA. Figure 10 As shown in Figure E, the spiked recoveries were in the range of 98.42% to 101.58%, and the RSDs of the concentration values ​​detected in the three groups of samples were 3.20% to 4.10%, indicating the potential of the biosensor in actual sample analysis.

[0088] Figure 11 In this paper, a corresponding paper-based microfluidic analysis device was further designed with the aim of extending this biosensing strategy to a more user-centric point-of-care (POCT) field. Figure 11 As shown in the figure, the device consists of two different paper-based chip layers: the upper layer is used for reaction, and the lower layer contains a mixing channel and a delay zone, with fast and slow channels for precise distribution and automatic sequential delivery of various fluids. According to the fluorescent sensing strategy explored above, FAM-DNA probes and target ctDNA are introduced into the mixing channel, Exo III is distributed in the fast channel (yellow) with fewer flow barriers, and MBene NSs are distributed through the slow channel (blue). This process allows all reagents to ultimately converge in the reaction zone on the top layer to observe the detection results.

[0089] Figure 12In the process of preparing paper-based chips, in order to optimize the mixing performance, the shape of the mixing channel was evaluated by ink mixing experiments and numerical simulations using COMSOL 5.5 software. Figure 12 As shown in Figure A, two fabricated mixing channels on a paper-based chip—one crisscross and one straight—were observed. Blue and red inks flowed through the channels, mixing to varying degrees in both configurations. The mixing effect was further evaluated through theoretical simulations.

[0090] like Figure 12 As shown in Figure 2, in the concentration distribution field, the original colors are represented by red and blue, and the appearance of green indicates good mixing. It can be observed that the green color appears in both the cross-shaped and straight mixing channels during the fluid flow, indicating effective mixing. For further quantitative evaluation, the absolute mixing index (AMI) is calculated for analysis, and the range of the index is from 0 (indicating perfect mixing) to 1 (indicating no mixing). First, the flow channel is evenly divided. The black line on the flow channel in the figure is the dividing line, and the AMI value is obtained at a total of six dividing points. As shown in Figure 2, the original colors are represented by red and blue, and the appearance of green indicates good mixing. Figure 12 As shown in Figure C, as the flow progresses, a clear difference in the AMI values ​​between the two structures emerges. Notably, the cross-shaped channel exhibits a lower AMI, indicating better mixing ability. This observation is consistent with previous reports that chaotic advection within the mainstream enhances mixing performance and that molecular diffusivity plays a major role in the mixing process when streamlines are parallel. Further analysis of the total flux of the two flow channels ( Figure 12 D), it can be clearly seen that the unique contraction-expansion structure of the cross-shaped channel promotes a significant change in the direction of the fluid, resulting in a significant curved streamline. According to the radial Bernoulli equation: The compression of the fluid in the contraction area produces This indicates that the external air pressure is reduced compared to the internal air pressure. This pressure gradient leads to an increase in the interfacial surface area, which promotes significant mixing within the paper-based chip. In addition, Figure 12 As shown in the E velocity field analysis, the blue streamlines indicate that the flow velocity in the cross-shaped channel is partially reduced, which can, to a certain extent, enhance the interaction between different fluids in the channel. Based on the above analysis, the cross-shaped mixing channel was ultimately selected for fabrication on the paper-based chip.

[0091] Figure 13 Then, the prepared paper-based microfluidic chip was used for fluorescence detection of ctDNA. The fluorescence signal acquisition equipment was as follows. Figure 13 As shown in Figure A, a blue LED light was used as the excitation light source, and a smartphone (Huawei nova 9 Pro) was used to photograph the chip. These images were then processed to extract RGB values ​​using Image J software, which is accessible on both PC and smartphone platforms.

[0092] In order to facilitate users to convert RGB values ​​into ctDNA concentration, a WeChat applet named “ctDNADetection” was developed ( Figure 13 B), enter the extracted RGB values ​​into the corresponding positions and click the green "Calculate" button to display the analysis results directly to the user interface in real time. At the same time, with the continuous development of the Internet of Things, this innovation will have the potential to realize real-time data sharing between patients and doctors, promoting remote analysis and diagnosis, especially in resource-limited environments. Experimental results are as follows Figure 13 C and Figure 13 As shown in the corresponding 3D image in D, it can be observed that as the ctDNA concentration increases, the fluorescence intensity also increases. In the range of 10-200 pM, the ctDNA concentration is linearly related to the G / B value, and the linear regression equation is Y = 0.002X + 0.702 (R 2 =0.991), with a limit of detection (LOD) of 4.125 pM. Compared with other paper-based fluorescence detection methods, this method showed comparable detection performance.

[0093] also, Figure 13 Figure E shows the specificity study results. The detection signal of the target showed a significant difference (p < 0.01) compared with the non-target substance, indicating the good specificity of the method. Figure 13 In F, the five parallel tests showed good reproducibility, and the relative standard deviation (RSD) of the response signals between the groups was 2.45%. Figure 13 G), the recoveries were 97.30% to 100.26% and the RSDs were 3.05% to 3.97%, indicating the potential of this method in the detection of real samples.

[0094] Figure 14 In order to further improve the detection efficiency of actual samples, graphene oxide-coated magnetic beads (MB@GO) are used to modify more clutch probes Clutch 5 and increase the capture sites. Figure 14 Figures AB show SEM images of magnetic beads before and after being coated with graphene oxide. It can be observed that the surface of the magnetic beads after coating is wrinkled and flake-like, indicating the synthesis of MB@GO and a significant increase in the surface area of ​​the magnetic beads. MB and MB@GO were further used to couple Clutch 5 DNA probes, and the coupling efficiency was evaluated by UV absorption measurement. Figure 14As shown in Figure C, the UV absorption of the original solution before coupling is shown in curve a. After coupling with magnetic beads MB, the UV absorption of the supernatant is measured as shown in curve b, with a coupling efficiency of 74.68%. Curve c is the UV absorption of the supernatant after coupling with MB@GO, with a coupling efficiency of 87.43%. By comparison, it was found that the UV absorption of nucleic acids in the supernatant decreased more after coupling with MB@GO, indicating that the coupling efficiency was improved by 1.17 times. The complexes after different magnetic beads coupled with probes were used for pre-enrichment evaluation and comparison, as shown in Figure 4. Figure 14 As shown in Figure D, the cDNA removal efficiency calculated after enrichment of MB / Clutch 5 was 80.26%, while the cDNA removal efficiency calculated after enrichment of MB@GO / Clutch 5 was 89.74%, indicating that the number of probe modification sites increased after graphene oxide coating, and the enrichment efficiency was improved by 1.12 times, which was used for subsequent actual sample pre-enrichment detection.

[0095] Figure 15 Then, a qPCR standard curve was established, as shown in Figure 15 As shown in AB, the concentration of ctDNA in the actual sample was subsequently analyzed and then compared with the detection results of the proposed fluorescence analysis method because of its high sensitivity (LOD of 0.062pM), which is better than the paper-based fluorescence analysis (LOD of 4.125pM). First, the HCC827 lung cancer cell sample was studied. The DNA extracted from the cells was diluted in different ratios, and then the concentration of the target EGFR 19del (1) mutation ctDNA was analyzed using qPCR and the proposed fluorescence biosensor method. The results are shown in Figure 15 As shown in C, the detection results of the two methods showed a high correlation (R 2 =0.991), indicating that the proposed method has certain potential for actual sample detection.

[0096] Figure 16-17 On this basis, we further detected ctDNA extracted from peripheral blood samples (n=10) of healthy mice, tumor-bearing mice and postoperative mice. Figure 17 A. The corresponding mouse model is shown in Figure 16 It is worth noting that Figure 17 On the left side of B, both qPCR and fluorescent biosensor failed to detect EGFR 19del(1) mutant ctDNA in the peripheral blood of healthy mice, and the response signal value corresponding to the calculated concentration was lower than the LOD (marked as not detected, ND). Figure 17 In the middle of Figure B, a significant increase in ctDNA levels was observed in the serum of tumor-bearing mice after 21 days. The results were statistically analyzed using IBM SPSS Statistics 29.0.1.0. Figure 17Gray and red in CD, both methods show that there are significant differences in the detection values ​​of ctDNA concentration between healthy mice and tumor-bearing mice (***p < 0.001), indicating that the proposed method has certain potential for the detection of ctDNA in blood samples of actual lung cancer patients.

[0097] In addition, monitoring ctDNA levels after treatment can be used as an effective indicator to help assess postoperative recurrence. To demonstrate this, tumor-bearing mice underwent surgery to remove the tumor and continued to monitor ctDNA levels two weeks after surgery. Figure 17 As shown on the right side of B, both methods detected that the ctDNA concentration was significantly reduced two weeks after surgery, but it was still higher than that of healthy mice. Figure 17 ***p<0.001 (gray and green) in CD indicate statistically significant differences. This change can be attributed to the possibility that ctDNA levels may increase again in advanced lung cancer and that residual lesions are associated with ctDNA levels, as evidenced by the regeneration of tumors in mice No. 5 and No. 8 after surgery ( Figure 16 ), and their ctDNA levels reflected this trend. Previous studies have shown this correlation between tumor volume and ctDNA concentration. In order to evaluate the diagnostic accuracy of the proposed biosensor, the area under the receiver operating characteristic (ROC) curve (AUC) was calculated, as shown in Figure 2. Figure 17 The AUC value should be greater than 0.8 for clinical diagnosis. Notably, the AUCs of both the qPCR method and the proposed biosensor reached 1, clearly distinguishing cancer mice from healthy controls, indicating the reliability and potential practical applicability of the MBene NSs-based fluorescence sensing method for ctDNA detection.

[0098] During the above detection process, the relevant nucleotide sequences are shown in Table 1 below.

[0099] Table 1 Different nucleotide sequences

[0100]

[0101] In summary, the present invention synthesized MBene nanosheets (MBene NSs) by etching and ultrasonic aqueous phase exfoliation. Density functional theory (DFT) calculations of adsorption energy and fluorescence kinetic adsorption experiments demonstrated the excellent DNA adsorption capacity of MBene NSs and its ability to distinguish between single-stranded (ssDNA) and double-stranded DNA (dsDNA). Compared with MXene, it has higher adsorption properties for bases A, G, and C. Subsequently, a highly sensitive ctDNA fluorescence sensing method was established using Exo III-assisted fluorescence signal cyclic amplification, with a detection range of 0.1 to 100 pM and a LOD as low as 0.062 pM, showing excellent detection performance. In order to extend this sensing method to point-of-care (POCT) applications, a paper-based microfluidic chip fluorescence analysis was designed, and the configuration optimization analysis of the mixing channel was carried out through numerical simulation and experiments. Finally, a cross-shaped structure mixing channel was selected. At the same time, the paper-based chip also contains fast / slow channels in the delay area to control the sequential flow of fluids. On this paper-based chip, ctDNA detection was successfully achieved, and the test results were collected using a smartphone and read out through the designed WeChat applet "ctDNADetection". MB@GO was then used to magnetically pre-enrich single-stranded ctDNA in actual samples. The detection results of this method and the standard qPCR method for cell and animal samples showed high consistency, indicating the accuracy of the proposed method (AUC = 1). At the same time, the successful detection of ctDNA in the peripheral blood of postoperative mice by this method further implies its potential in cancer diagnosis and postoperative monitoring. In addition, this work has opened up a path for the study of MBene-DNA interface effects and inspired further exploration, such as more precise adsorption regulation through metal coordination. In addition to biosensing, it is also expected to be applied to bioimaging and nanopore-based nucleic acid sequencing, in which the differential adsorption between MBene nanosheets and various DNA bases is used as a digital electrical signal output to achieve intelligent detection.

[0102] 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 can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions, which should all be included in the scope of the claims of the present invention.

Claims

1. A microfluidic detection system based on MBene nanosheets and functional partitioning, characterized by: The microfluidic detection system includes a microfluidic chip and a detection reagent. The microfluidic chip includes two layers, the upper layer is used for the reaction zone, and the lower layer contains a mixing channel and a delay zone, with a fast channel and a slow channel; the detection reagent includes MBene nanosheets, fluorescently labeled ssDNA, and nuclease exonuclease III.

2. The microfluidic detection system based on MBene nanosheets and functional partitioning according to claim 1, characterized in that: The preparation method of the MBene nanosheets is as follows: S1: Precursor etching treatment: lithium salt and concentrated hydrochloric acid solution are mixed to form an etching system, MoAlB powder is added and stirred at 37-40°C for 20-24 hours. The concentration of the concentrated hydrochloric acid is 8-10M, and the mass ratio of lithium salt to MoAlB is (1.5-1.7):

1. A precipitate is obtained by centrifugation; S2: Purification treatment: The precipitate obtained in step S1 was washed with deionized water until the pH value was 7, the precipitate was collected by centrifugation at 5000 rpm for 5 min, and dried at 60-80°C for 8-12 hours to obtain MBene powder; S3: Nanosheet exfoliation: The purified MBene powder was dispersed in deionized water to prepare a 1-3 mg / mL aqueous solution, which was then treated in a water bath with ultrasonic waves for 20-24 hours at a power of 70-90% of the total instrument power. S4: Gradient centrifugation: The ultrasonic product was centrifuged at 3000 rpm to collect the supernatant, and then the supernatant was centrifuged at 12000 rpm to collect the precipitated product, which was then freeze-dried in vacuum to obtain the MBene nanosheets.

3. The detection method of the microfluidic detection system based on MBene nanosheets and functional partitioning according to claim 1 or 2, characterized in that: The detection steps are as follows: S1: Mix the fluorescently labeled ssDNA probe with the target to be detected, then add exonuclease III and incubate at 37°C in the dark for 30 minutes; S2: Then, 25 μL of 0.05 mg / mL MBene nanosheets was added and reacted for 10 min. After the reaction was completed, the fluorescence intensity was measured at an excitation wavelength of 480 nm and an emission wavelength of 520 nm.

4. The detection method according to claim 3, wherein: The molar ratio of the fluorescently labeled ssDNA probe to the target to be detected and the exonuclease III is 1:1:

1.

5. Use of the microfluidic detection system based on MBene nanosheets and functional partitioning according to claim 1 or 2 in the preparation of a biosensor.

6. Application of the microfluidic detection system based on MBene nanosheets and functional partitioning according to claim 1 or 2 in ctDNA detection.

7. The use according to claim 6, characterized in that The detection steps are as follows: S1: 25 μL of 0.5 μM FAM-labeled ssDNA probe and 25 μL of ctDNA to be tested were added dropwise to the mixing channel on the microfluidic chip respectively; S2: Add 25 μL of 40 U exonuclease III to the fast channel and 25 μL of 0.05 mg / mL MBene nanosheets to the slow channel; S3: Incubate the paper-based chip at 37°C in the dark for 40 minutes, then place the paper-based chip in a dark box. Use a blue LED excitation light with a 480 nm filter to illuminate the microfluidic chip, and take a picture through a 520 nm filter under the lens. S4: Use Image J software to obtain the RGB values ​​of the reaction area photos and calculate the ctDNA concentration.

8. The use according to claim 7, characterized in that: The nucleic acid sequence of the ssDNA is shown in SEQ ID NO:

1.

9. The use according to claim 7, characterized in that: The ctDNA is lung cancer ctDNA.

Images