Preparation method of CoB NP nanozyme and detection kit and detection system

Abstract

The application relates to the technical field of biological detection, in particular to a preparation method of CoB NP nanometer enzyme, a detection kit and a detection system. The preparation method is as follows: mixing ethylene glycol, polyvinylpyrrolidone and cobalt boride powder, adding a H2O2 and glacial acetic acid mixture to seal the reaction, collecting and washing the precipitate through centrifugation, and finally redissolving, ultrasonic treatment and centrifugation to obtain the CoB NP nanometer enzyme. The kit comprises a biochip and a detection reagent, wherein the detection reagent contains a SP-CoB NP nanometer enzyme signal probe with a specific DNA sequence, and a corresponding capture probe is connected on the biochip. The biochip has a three-layer structure and is used for sample adding, reaction and waste liquid collecting, so that the convenience and efficiency of detection are improved. The application has the beneficial effect that the CoB NP nanometer enzyme is connected with the signal probe, a special biochip is combined, high sensitivity and high specificity detection of the target are realized, the detection process is more simple and fast, and the application is suitable for on-site rapid detection.

Description

Technical Field

[0001] This invention belongs to the field of biodetection technology, and particularly relates to a method for preparing CoB NP nanozymes and a detection kit and detection system based on CoB NP nanozymes. Background Technology

[0002] Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer death worldwide. Numerous reports indicate that NSCLC patients with epidermal growth factor receptor (EGFR) gene alterations are eligible for targeted therapy, and EGFR gene testing can provide personalized treatment plans and better prognoses for NSCLC patients. Since circulating tumor DNA (ctDNA) is a fragmented gene derived from tumors and present in the blood of cancer patients, liquid biopsy of ctDNA from peripheral blood has become a promising new non-invasive method for cancer diagnosis and treatment. Although many polymerase chain reaction (PCR)-based methods or biosensors based on fluorescence, electrochemistry, and chemiluminescence technologies have been applied to detect ctDNA, several drawbacks, including high cost and the need for advanced equipment, limit their widespread use.

[0003] Nanozymes, as nanomaterials that mimic the activity of natural enzymes, have shown great potential in fields such as biosensing, environmental monitoring, and medical diagnostics due to their unique physicochemical properties and stability. Colorimetric sensing systems, as a simple, intuitive detection method that requires no complex instruments, have greatly expanded their application scope by combining with nanozymes. Specifically, nanozyme colorimetric sensing systems can achieve qualitative and quantitative detection of target analytes by catalyzing color changes in specific substrates. Nanozyme colorimetric sensing systems have been widely used in the detection of biomarkers, pathogens, toxins, etc. For example, platinum nanoparticle-based colorimetric sensors can sensitively detect glucose levels in blood; gold nanoparticles are widely used to detect biomolecules such as proteins and DNA. However, existing nanozyme colorimetric sensing systems still face some challenges in practical applications, such as the need for improvement in detection sensitivity, selectivity, stability, and portability. Furthermore, for the detection of low-abundance target analytes in complex biological samples, especially circulating tumor DNA (ctDNA), traditional methods suffer from problems such as complex operation, long processing times, and high equipment requirements.

[0004] Cobalt boride nanomaterials exhibit excellent catalytic activity in certain chemical reactions, and can replace or partially replace traditional noble metal catalysts, thus reducing costs. Cobalt boride possesses good electrical conductivity, which is particularly important for electrochemical applications (such as batteries and supercapacitors). Compared to some noble metal catalysts, cobalt boride nanomaterials show better stability under harsh environments, making them more suitable for industrial production processes. By controlling the synthesis conditions, the morphology, size, and surface properties of cobalt boride nanomaterials can be tuned, thereby optimizing their catalytic performance. However, cobalt boride nanomaterials also face several disadvantages and challenges. Although they show great potential in catalysis, their biocompatibility has not been fully studied, limiting their application in the biomedical field. Currently, there is relatively little research on the toxicity of cobalt boride nanomaterials. While the cost of cobalt boride nanomaterials is lower than that of noble metal catalysts, their synthesis process may still require high energy and raw material inputs. The environmental stability (such as corrosion resistance and aging resistance) of cobalt boride nanomaterials in practical applications needs further verification and optimization.

[0005] Cobalt boride nanomaterials have shown great potential in fields such as catalysis, but they still face many challenges in practical applications. Further research and development are needed to address these issues and achieve the widespread application of cobalt boride nanomaterials.

[0006] In point-of-care testing (POCT) methods, nanozyme-based colorimetric sensing systems exhibit unique advantages. By designing nanozyme probes that specifically recognize ctDNA and combining them with portable colorimetric detection devices, rapid and sensitive detection of ctDNA can be achieved. However, existing technologies still have some shortcomings, such as insufficient specificity, the need to improve sensitivity, and inadequate ease of operation. Summary of the Invention

[0007] In view of this, one of the objectives of the present invention is to provide a method for preparing CoB NP nanozymes.

[0008] The preparation method of CoB NP nanozyme includes the following steps:

[0009] S1: In a polytetrafluoroethylene reactor containing ethylene glycol, add polyvinylpyrrolidone and cobalt boride powder as raw material. The ratio of each raw material is as follows: ethylene glycol:polyvinylpyrrolidone:cobalt boride powder volume mass ratio is 4:45:15 (ml:mg:mg). Mix well and set aside.

[0010] S2: Add a mixture of H2O2 and glacial acetic acid to the reactor in S1. The volume ratio of H2O2 to glacial acetic acid is 1:100 (ml:ul). Seal the reactor and place it in an oven. React at 160°C for 120 minutes.

[0011] S3: Centrifuge at 12000 rpm for 20 minutes to collect the precipitate, and wash it three times with anhydrous ethanol and deionized water respectively.

[0012] S4: The collected precipitate was redissolved in deionized water and treated with an ultrasonic processor. After centrifugation at 5000 rpm for 1 minute, the supernatant was collected. The supernatant was then centrifuged at 12000 rpm for 10 minutes. The collected precipitate was CoB NP nanozyme.

[0013] The second objective of this invention is to provide a kit for CoB NP nanozymes;

[0014] To achieve the above objectives, the present invention provides the following technical solution:

[0015] A bioassay kit based on CoB NP nanozymes includes a biochip and a detection reagent. The detection reagent contains an SP-CoB NP nanozyme signal probe prepared from the CoB NP nanozyme. The DNA sequence of the SP-CoB NP nanozyme signal probe (SP) is shown in SEQ ID NO: 3. The 5′ end of the signal probe (SP) is modified with an amino group. A capture probe is linked on the biochip. The DNA sequence of the capture probe DNA (CP) is shown in SEQ ID NO: 1. The 3′ end of the capture probe (CP) is modified with an amino group.

[0016] Furthermore, the biochip has a three-layer structure: a top dividing layer containing channels and a sample application area; a bottom collection layer for collecting reaction waste liquid; and a middle detection layer, wherein the reaction regions on the detection layer are respectively connected by amino covalent linkages to capture probe DNA (CP).

[0017] Furthermore, the preparation method of the SP-CoB NP nanozyme signal probe includes the following steps:

[0018] S1: A 5 μM amino-modified signal probe (SP) and a 1 mg / ml CoB NP nanozyme were mixed in a 1:1 volume ratio in 0.01 M phosphate buffer solution.

[0019] S2: The reaction is carried out at 4°C for approximately 12 hours, during which the amino groups on the signal probe (SP) are covalently linked to the hydroxyl groups on the surface of the CoB NP nanozyme.

[0020] S3: The sediment collected by centrifugation of the product from step S2 is the SP-CoB NP nanozyme signal probe. The SP-CoB NP nanozyme signal probe is redissolved in 0.01M phosphate buffer solution for later use.

[0021] Furthermore, the method for preparing the biochip includes the following steps:

[0022] S1: Drop 2.5 μL of chitosan (0.25 mg / mL) onto the reaction area of ​​the paper-based chip detection layer;

[0023] S2: After drying the detection layer treated in step S1 at room temperature, add 2.5 μL of glutaraldehyde (2.5%, v / v). The aldehyde group of glutaraldehyde is covalently linked to the amino group of chitosan.

[0024] After step S3:S2 is completed, react for 2 hours, then add 2.5 μL of 5 μM amino-modified capture probe (CP) and covalently link for half an hour. Then add 1% BSA to block unreacted sites for half an hour to obtain the biochip.

[0025] Furthermore, the biochip has two reaction zones: one is a colorimetric reaction zone, and the other is a pressure reaction zone, with each reaction zone connected to the captured DNA.

[0026] Furthermore, the biochip is a dual-mode pop-up paper chip, and the biochip can specifically bind to lung cancer target ctDNA.

[0027] The third objective of this invention is a detection system based on a CoB NP nanozyme bioassay kit, which includes the aforementioned bioassay kit and further includes an intelligent colorimetric processing terminal and a portable barometer, wherein the intelligent colorimetric processing terminal can be a mobile terminal.

[0028] The steps for detecting the target using the above detection system are as follows:

[0029] S1: Drop the mixture of target and SP-CoB NP onto the sample layer of the split region of the constructed chip, incubate, and wash;

[0030] S2: Cut out the two reaction areas in the detection layer respectively. For colorimetric mode, drop sodium acetate buffer containing a mixture of TMB and H2O2 into the chip, then place it in a 3D-printed dark box, and then take a picture with a smartphone after 3 minutes and detect it through a mobile terminal with a colorimetric application. For pressure mode, put the cut paper into an EP tube containing H2O2 (30%, wt%) and immediately seal it with a cap, then insert a saline infusion needle and connect it to a portable barometer for detection, and record the pressure after 6 minutes.

[0031] Furthermore, the target detected was lung cancer ctDNA.

[0032] Compared with existing technologies, the present invention has the following advantages:

[0033] This invention provides a simple, rapid, and cost-effective method for detecting ctDNA, which is particularly suitable for detecting ctDNA in lung cancer.

[0034] The system integrates both colorimetric and barometric detection modes, improving the sensitivity and specificity of the detection.

[0035] The pop-up paper chip design makes operation more convenient and is suitable for on-site testing and instant diagnosis.

[0036] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0037] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0038] Figure 1 (A) shows the synthesis process of CoB NP, (B) shows the SEM image of the original CoB powder, (C) shows the SEM image and DSL analysis of CoB NP, (D) shows the SEM image and element point scan of CoB NP, and (E) shows the high-angle annular dark field scanning TEM image of CoB NP and the corresponding EDS elemental mapping.

[0039] Figure 2 (A) is the FT-IR spectrum of the original CoB powder and CoB NP, (B) is the XRD pattern of the original CoB powder and CoB NP, (C) is the geometric structure diagram of CoB NP, (D) is the charge density diagram of CoB NP, (E) is the density of states diagram of CoB NP, and (F) is the band structure diagram of CoB NP.

[0040] Figure 3 (A) shows the peroxidase and catalase reaction process of CoB NP; (B) shows the UV-Vis absorbance spectra of different reactions based on the peroxidase activity of CoB NP; (C) shows the UV-Vis absorbance spectra of the catalase activity of CoB NP and the reaction of natural catalase with H2O2; (D) and (E) show the steady-state kinetics of CoB NP with different concentrations of TMB and H2O2 based on peroxidase activity; (F) shows the effect of various scavengers on the oxidation of TMB by CoB NP; (G) shows the ESR spectrum of ·OH; (H) shows the change in fluorescence intensity of terephthalic acid with the concentration of CoB NP; and (I) shows the cyclic voltammetry curves of CoB NP in sodium acetate buffer with or without H2O2.

[0041] Figure 4In the diagram, (A) and (B) are the pathway free energy diagrams of the CoB NP catalytic reaction based on the activities of peroxidase and catalase, respectively. (C) and (D) are the corresponding intermediates (purple circles) and their charge density differences (pink circles) at each step of the pathway, respectively.

[0042] Figure 5 This section describes the manufacturing process of paper-based chips and introduces the chips themselves.

[0043] Figure 6 (A) shows the sequencing of PCR amplification products of ctDNA extracted from different cells; (B) shows hematoxylin and eosin staining of mouse tumors; and (C) shows the results of qPCR detection of ctDNA in mouse peripheral blood.

[0044] Figure 7 (i) describes the properties of the CoB NP nanozyme, (ii) shows the sensor construction process, and (iii) shows the sensor's dual-mode detection process for ctDNA.

[0045] Figure 8 The interface of the smartphone application "ctDNAAssay" is described as follows: (i) Login; (ii) Obtaining images from CAMERA or PICTURES; (iii) Binarization of images; (iv) Detection results after clicking DETECTION; (v) Recording of detection results.

[0046] Figure 9 In the diagram, (A) and (B) represent the grayscale values ​​and gas pressures of different modifications on the paper substrate, respectively; (C) represents the Zeta potentials of different substances; (D) shows the FT-IR spectra of filter paper (a) and filter paper immobilized with DNA probes (b); and (E) shows the AFM images and surface roughness of the paper substrates with different modifications. (i: filter paper, ii: P / chitosan, iii: P / C / CP, ​​iv: P / C / CP / TP / SP-CoB NP, P: filter paper, C: chitosan, CP: capture probe, SP: signal probe)

[0047] Figure 10 In the middle, (A) and (B) are optimizations for capture concentration, and (C) and (D) are optimizations for incubation time.

[0048] Figure 11 In the middle, (A) and (B) show the gray value and air pressure of the sensor when measuring different concentrations of ctDNA, respectively. (C) is a comparison of the LOD and detection linear range of various methods for nucleic acid analytes. (D) shows the gray value and air pressure readings of the sensor when detecting different analytes. (E) shows the reproducibility of the sensor in dual-mode detection. (F) shows the storage stability of the sensor in dual-mode detection. (G) is a schematic diagram of actual samples in dual-mode analysis. (H) and (I) show the detection results of ctDNA in cell samples and mouse peripheral blood samples by the proposed method and qPCR method, respectively. Detailed Implementation

[0049] Example 1

[0050] Preparation of a biological kit for CoB NP nanozymes:

[0051] I. Preparation of CoB NP nanozyme signal probes, the specific method is as follows:

[0052] (1) Synthesis of CoB NP nanozymes (e.g.) Figure 1 In a polytetrafluoroethylene reactor containing 40 mL of ethylene glycol, 450 mg of polyvinylpyrrolidone and 150 mg of cobalt boride powder were first added. Then, 4 mL of H₂O₂ and 400 μL of glacial acetic acid were added. These solutions and reagents were mixed thoroughly and then placed in an oven at 160 °C for 120 minutes. After cooling to room temperature, the precipitate was collected by centrifugation at 12000 rpm for 20 minutes and washed three times each with anhydrous ethanol and deionized water. The collected precipitate was then redissolved in deionized water and treated with a cell sonicator for half an hour. The supernatant was collected by centrifugation at 5000 rpm for 1 minute, followed by centrifugation at 12000 rpm for 10 minutes. The collected supernatant and precipitate were then collected as the final product, yielding 0.2 mg of CoB NP nanozyme.

[0053] (2) Synthesis of CoB NP nanozyme signal probe. 250 μL of 5 μM amino-modified DNA signal probe was added to 250 μL of 1 mg / ml CoB NP solution (CoB NP solution was prepared with 0.01 M phosphate buffer). In this reaction, the amino groups on the DNA are covalently linked to the hydroxyl groups on the surface of CoB NP. After mixing at 4 °C for about 12 h, the CoB NP-DNA signal probe in the sediment was collected by centrifugation and redissolved in 0.01 M phosphate buffer solution for further use.

[0054] II. Chip Fabrication: First, design the channels to be used on the paper chip using AutoCAD. Then, create a mesh from these channel patterns to serve as a mask for screen printing. During fabrication, a heating plate is placed at the bottom and heated to approximately 60°C. Filter paper is then placed on the heating plate, and the mask is placed on the filter paper. Molten wax is then applied to the mask using a crayon. Finally, a squeegee is used to evenly spread the molten wax over the channel patterns. Some wax will drip through the screen onto the filter paper below, forming a hydrophobic barrier. The paper-based chip fabrication is now complete.

[0055] The chip has a three-layer structure. The two regions in the middle layer are used to construct the reaction zones for colorimetric and pressure modes, respectively. Figure 5This design enables dual detection. A top splitting layer and a bottom collection layer are incorporated to simultaneously clean and collect waste liquid during sensor construction. In use, the paper base unfolds like a greeting card, popping out three layers that connect by pressing. Once the sensor construction is complete, the three layers separate. For large-scale paper base manufacturing, this design allows for better preservation when fully pressed shut, similar to a closed greeting card, keeping the reaction area clean.

[0056] III. Using the chip fabricated in step II above to construct the sensor: First, drop 2.5 μL of chitosan (0.25 mg / mL) onto the two reaction zones of the paper-based chip detection layer, and allow it to dry at room temperature. Then, drop 2.5 μL of glutaraldehyde (2.5%, v / v), where the aldehyde group of glutaraldehyde will covalently link to the amino group of chitosan. After reacting for 2 hours, drop 2.5 μL of 5 μM amino-modified capture probe DNA (CP), and covalently link it for half an hour. Then, add 1% BSA to block unreacted sites for half an hour. After each modification, wash away unreacted reagents twice with 20 μL of washing buffer. Prepare the washing buffer using 20 mM PBS (pH 7.5), 50 mM NaCl, and 0.5% Tween 20.

[0057] Example 2

[0058] CoB NP Nanozyme Bioassay Kit for Detecting ctDNA:

[0059] I. ctDNA Extraction: Mice were first modeled with tumor cells, and then 150 μL of H1975 tumor cells were injected into each mouse. The mice were fed for approximately 2 weeks. Peripheral blood was then collected from the tumor-bearing mice after enucleation, centrifuged at 4°C (3000 rpm, 10 min) to obtain serum, and stored at -20°C. ctDNA was then extracted from the serum using a cell-free plasma DNA extraction kit (DP339, Tiangen Biotech, Beijing, China). The DNA product was then amplified by PCR under the following conditions: 2 μL extracted DNA, 0.3 μL forward primer (F), 0.3 μL reverse primer (R), 10 μL 2×T5 Super mix, 7.4 μL nuclease-free water, 98°C, 5 min, followed by 42 cycles (95°C-20s → 60°C-30s). The standard qPCR method used the same procedure, replacing the 2×T5 Super mix with 2×SYBR MIX. The observation procedure for tumor tissue is as follows: the tumor tissue is cut off, fixed in paraformaldehyde solution, dehydrated, immersed in paraffin, heated to allow the paraffin to penetrate into the tumor tissue, cooled and fixed, then the tumor tissue is sliced, one slice of tumor tissue is dewaxed, and stained with hematoxylin-eosin.

[0060] Sequencing of PCR amplification products of ctDNA extracted from different cells ( Figure 6 A), staining of mouse tumors with hematoxylin and eosin ( Figure 6 B), Results of qPCR detection of ctDNA in mouse peripheral blood ( Figure 6 C). The extracted cellular DNA was subjected to PCR amplification, and the products were sequenced by Tsingke Biotech Co., Ltd. (Beijing, China). The results showed that the mutations in the experimental group were correct. Figure 6 A). Animal samples were used to model tumors in mice using H1975 tumor cells. A stained section of tumor tissue from a mouse ( Figure 6 B) shows that tumor cells grow densely in clusters, are tightly packed, and are in the mitotic phase. They are spherical, polygonal, and irregular in shape and size, with less cytoplasm and larger nuclei. The results of qPCR detection of ctDNA in mouse peripheral blood show ( Figure 6 C) Normal mice do not contain ctDNA, while tumor-bearing mice used for modeling contain ctDNA.

[0061] The relevant nucleotide sequences are shown in Table 1 below.

[0062] Table 1 Nucleotide sequence list

[0063]

[0064] II. Dual-mode detection of target ctDNA ( Figure 7 Add 2.5 μL of a mixture of ctDNA and SP-CoB NP to the sensor constructed above, incubate for 60 minutes, and then wash. The detection process is as follows: Figure 7 As shown in ii, firstly, the CP is anchored to the paper-based chip. Then, SP is prepared in advance and linked to the nanozyme to obtain SP-CoB NP (see Example 1, Preparation of CoB NP Nanozyme Signal Probe). Finally, a mixture of the target ctDNA and SP-CoB NP is added, where ctDNA is paired with half of the SP and half of the CP, forming a sandwich structure to capture the target ctDNA. Different signal probes (SP) and capture probes (CP) can be designed to detect ctDNA from different tumors. Then, the two reaction regions in the detection layer are cut out for dual-mode detection. For colorimetric mode, 2.5 μL of 0.2 M sodium acetate buffer containing a TMB / H2O2 mixture is dropped into the chip, then placed in a 3D-printed dark box. A photo is taken after 3 minutes using a Huawei (nova 9 Pro) smartphone, and the result is detected via a designed smartphone application. For pressure mode, the cut paper was placed into an EP tube containing 100 μL H2O2 (30%, wt%) and immediately capped and sealed. Then, a saline infusion needle was inserted and connected to a handheld gas pressure gauge for testing, and the pressure was recorded after 6 minutes.

[0065] The smartphone application has the following usage steps ( Figure 8 First, log in to the APP and take a picture of the paper-based chip after the reaction by taking a photo or using the album. Then, click BINARIZATION to lock the reaction area on the paper chip, and then click DETECT to extract the gray value. The standard curve equation of the relationship between ctDNA concentration and gray value is obtained through experiments. The gray value is converted into ctDNA concentration, and then the test results can be saved and checked.

[0066] Example 3

[0067] CoB NP Nanozyme Bioassay Kit System Performance Testing:

[0068] To test the peroxidase-like activity of CoB NP, spectrophotometry (UV-vis) was used to detect it at 652 nm. The solution contained 100 μL CoB NP (0.1 mg / mL), 100 μL 0.5 mM TMB, 100 μL 2 mM H₂O₂, and 200 μL sodium acetate buffer (pH 5.0). To test the catalase-like activity of CoB NP, 500 μL CoB NP (0.1 mg / mL) was added to 500 μL H₂O₂ (30%, wt%), and the decomposition of H₂O₂ at 240 nm was recorded.

[0069] Steady-state dynamic parameters are calculated using the Michaelis-Menten equation: 1 / V = (K m / V max (1 / [S])1 / V max Where V max [S] is the maximum reaction rate, [S] is the substrate concentration, and K is the maximum reaction rate. m This is the Michaelis constant. In the enzyme reaction system, 100 μL of tryptophan (Trp, 1 mM), p-benzoquinone (p-BQ, 5 mM), and thiourea (TH, 5 mM) were used as singlet oxygen (…). 1 O2), superoxide anion (O2) ·- The types of reactive oxygen species (ROS) generated were explored by using scavengers of CoB NP and hydroxyl radicals (·OH), and their UV-Vis spectra were then measured. Additionally, fluorescent probes (terephthalic acid, TA, 5 mM) were added to enzyme reaction systems with different concentrations of CoB NP to test for generated ·OH. The fluorescence spectra of the mixtures were then recorded. In ESR experiments, 5,5-dimethyl-1-pyrrolomorpholine N-oxide (DMPO), CoB NP (2 mg / ml), H₂O₂ (10 mM), and DMPO were mixed for 10 minutes, dispersed in sodium acetate buffer, and then used to record the ESR signal.

[0070] DFT was performed using the CASTEP package in Materials Studio. Exchange-correlation interactions were studied using Perdew-Burke-Ernzerhof (PBE) parameterization and the generalized gradient approximation (GGA). The cutoff energy for the ultrasoft pseudopotential was 600 eV. The Hellmann-Feynman force was maintained using a (2×2×2) k-point grid. Below this, the total energy difference is less than 2 × 10⁻⁶ eV / atom, and the interionic displacement is less than... The board model has 5 layers, with the bottom 3 layers restricted and... A vacuum layer was established there. Interactions between consecutive cells can be neglected. The Gibbs free energy is calculated using the following formula: G = E DFT +ZPE-TShereE DFT It is the energy calculated by DFT, ZPE is the zero-point energy, and TS is the entropy correction.

[0071] The original CoB powder was layered and in large lumps. Figure 1 B). In contrast, the synthesized CoB NPs are smaller in size, exhibiting square or round shapes, and the embedded particle size distribution DSL plot shows that their nanostructures are mainly around 50 nm. Figure 1 C). SEM images and element point scans of CoB NP ( Figure 1 D) and CoB NP high-angle annular dark-field scanning TEM images and corresponding EDS element mappings ( Figure 1 E) indicates the presence of Co and B.

[0072] Fourier transform infrared spectroscopy results show that ( Figure 2 A) After processing the original cobalt boride powder, the bending vibration of -OH appears at 1500 cm⁻¹. -1 ~1700cm -1 Within the range, B-OH appears at 900 cm⁻¹ -1 Left and right. X-ray diffraction (XRD) revealed a similar crystal phase structure between the original CoB powder and CoB NP. Figure 2 B). Further analysis of the CoB NP properties was conducted using density functional theory (DFT). The geometric structure diagram of the CoB NP shows that Co-B bonding occurs through covalent bonding and exhibits a layered structure. Figure 2 C). The presence of B and Co atoms in a planar plane endows them with high catalytic activity. The electron cloud density isosurface (top) and its associated cross-section (bottom) of the CoB NP three-dimensional model (…). Figure 2D), it can be seen that most of the contributed electrons come from metallic Co, indicating that metallic Co can act as an electron donor and as a catalytic active site center, promoting electron transfer between CoB NP and the adsorbed substrate. This will enhance electron transfer and change reaction kinetics. The density of states (DOS) and band structure of CoB NP (…) Figure 2 The results (EF) show the conductive metallic properties of CoB NP, indicating that all models have no band gap near the Fermi level. Since the higher the carrier density near the Fermi level (0 eV), the higher the conductivity of the material, the above results demonstrate the good catalytic activity of CoB NP.

[0073] CoBNP is a catalytic reaction based on the activities of peroxidase and catalase. TMB, TMB / H2O2, and TMB / CoBNP have low UV-Vis absorbance. Figure 3 B). At 652 nm, oxTMB shows a distinct characteristic peak at the TMB / CoB NP / H2O2 ratio, indicating that CoB NP exhibits peroxidase-like activity. It catalyzes the decomposition of H2O2 via a single-electron process to produce the oxTMB product. The catalase activity of CoB NP was assessed after the catalytic decomposition of H2O2, in which H2O2 is decomposed into dissolved O2 by CoB NP. The UV-Vis characteristic peak of H2O2 is located at 240 nm. Compared with natural catalase ( Figure 3 C) The absorption peak of H2O2 / CoB NP decreased further, and more O2 bubbles were observed in the inset, indicating the catalase-like activity of CoB NP. The catalytic curve of CoB NP substrate was well fitted by the Michaelis-Menten kinetic model. Figure 3 DE), CoB's K m (0.35 mM for TMB and 1.86 mM for the substrate) showed good substrate affinity. In the presence of thiourea (TH), the peroxidase activity of CoBNP was significantly inhibited, indicating that ·OH plays an important role in the catalytic process. Figure 3 F). DMPO is used to capture ·OH and form DMPO / ·OH adducts, with a typical ESR signal ratio of 1:2:2:1, indicating that ·OH generated in the CoB NP / H2O2 catalytic reaction system (F) Figure 3 G). When TA reacts with ·OH, the resulting hydroxyterephthalic acid exhibits a characteristic fluorescence emission peak around 420 nm under 315 nm excitation. Higher CoB NP concentrations result in stronger fluorescence intensity, indicating the generation of ·OH during the catalytic reaction. Figure 3H). To further understand the electron transfer occurring during the CoB NP catalysis process, CoB NP modified on a glassy carbon electrode exhibited a significant current response after H2O2 was added to PBS buffer, indicating that CoB NP possesses the ability to transfer electrons between the electron donor and the H2O2 substrate (electron acceptor). The inlay diagram shows that O2 bubbles were generated on the electrode surface during the reaction, further demonstrating the occurrence of catalysis. Figure 3 I).

[0074] CoB NP pathway free energy diagram based on peroxidase and catalase activity catalyzing the reaction ( Figure 4 A, 4B), the corresponding intermediates (purple circles) and their charge density differences (pink circles) at each step of the path (A, 4B). Figure 4 C, 4D). The results show that the optimized model indicates that transition metal elements act as electron donors, and H2O2 adsorbed on the surface of the nanozyme catalyst accepts electrons. The energy barrier of the rate-determining step (RDS) in either pathway I or II of the CoB NP peroxidase catalytic pathway indicates ( Figure 4 A), pathway I more readily generates ·OH. The adsorbed H₂O₂ is first heterogenized ( Figure 4 C), and forms *O and *OH on the metal Co element as active sites. In the reaction catalyzed by catalase CoB NP to decompose H2O2 into O2, pathway III is optimal. Figure 4 B), H2O2 gains electrons from metallic Co and tends to transfer hydrogen to form *OOH ( Figure 4 D).

[0075] Gray value and gas pressure tests on different modified paper bases: Compared with the target-free group, the gray value and gas pressure of the target group both showed significant changes. Figure 9 A and B) demonstrate the feasibility of constructing a biosensor for dual-mode real-time detection of ctDNA. Zeta potential analysis showed ( Figure 9 C) After modification with electronegative DNA, the negative potential of SP-CoB NP (-33.57 mV) was larger, indicating that the signal DNA successfully bound to CoB NP. After immobilizing the DNA probe on paper ( Figure 9 D) Due to the NH stretching vibration in nitrogenous DNA bases, the FT-IR peak is in the 3100-3400 cm⁻¹ range. -1 It widens at a point, reaching 1500-1600cm. -1 The peak at the specified location corresponds to the imine (C=N), indicating that a Schiff base is formed between the amino-modified DNA probe with glutaraldehyde as the cross-linking agent and the chitosan-modified paper substrate. Surface changes after paper substrate modification were observed using AFM. Figure 9 E). Comparison Figure 9In E i and ii, the surface roughness decreased after chitosan modification on the paper, indicating that chitosan smooths the paper surface, which helps in the uniform distribution of reagents and a more uniform reaction. Roughness increases with the modification of the probe and target on the paper chip, as shown by R. a =Increased from 43.73nm to 56.37nm, R q The increase in wavelength from 59.82 nm to 70.54 nm indicates the successful fabrication of the biosensor.

[0076] In dual-mode detection, a capture probe with a concentration of 5 μM was selected to obtain the optimal response. Figure 10 A, B). After 60 minutes of incubation, the signal response remained almost unchanged in both modes, therefore 60 minutes was chosen as the appropriate reaction time. Figure 9 C, D).

[0077] Measurement of grayscale values ​​and air pressure at different concentrations of ctDNA: As the ctDNA concentration increased from 200-1500 pM, the response signal in each mode showed a linear change. Figure 11 A, B). In colorimetric mode, the standard curve equation is fitted as Y = -0.032X + 188.992(R). 2 =0.994), detection limit LOD = 95.813 pM, under pressure mode, the standard curve equation is fitted as Y = 0.008X + 4.153(R) 2 =0.992), LOD = 79.875pM. Comparison with other nucleic acid target detection methods ( Figure 11 C) indicates that the proposed dual-mode ctDNA detection method has the potential to serve as an ideal sensing platform for other analytes. To evaluate the specificity of this method, non-complementary DNA (N-DNA), single-base mismatched DNA (S-DNA), double-base mismatched DNA (D-DNA), and wild-type EGFR were detected. Compared with other analytes ( Figure 11 D), the target signal response was significant in both modes, demonstrating good specificity. To examine specificity and reproducibility, dual-model detection showed no significant signal difference among the seven parallel sensors (D). Figure 11 The RSD was 2.05-2.82%. The constructed biosensor was then stored at 4°C for 15 days to study its stability. The results showed that after 15 days, the sensing response decreased to 90.37-91.24% of its original value. Figure 11 F). A schematic diagram illustrating the sensor's analytical capabilities in real samples, where a positive sample result is output based on an AND logic system. Figure 11G), normal samples are defined as "0", samples containing ctDNA are defined as "1", and only when both the colorimetric signal and pressure signal values ​​are 1 will the test result be evaluated as a positive sample. This is of great significance for improving the accuracy of the sensor in actual sample testing. Detection results of cell samples and mouse blood samples ( Figure 11 H), in both modes, the signal response of the experimental group was significantly different from that of the control group (p<0.01), and the results of standard qPCR detection of mouse blood samples ( Figure 11 I) The signal response of the experimental group was significantly different from that of the control group, indicating the potential for practical application of the sensor.

[0078] In summary, this invention presents a kit and detection system for detecting lung cancer ctDNA based on CoB NP nanozymes. The CoB NP nanozyme, utilizing the peroxidase and catalase properties, has been studied for the first time and successfully used as a signal marker for dual-mode ctDNA detection. The dual-mode signal generation is based on peroxidase activity, catalyzing the colorimetric reagent for color development, and reading the signal using a colorimetric program APP. It also utilizes catalase activity, catalyzing the decomposition of H₂O₂ into O₂, and reading the signal using a handheld barometer. This method provides new insights for ctDNA detection and the application of CoB NP in the biomedical field. Furthermore, the sensor is constructed on a pop-up paper chip, making the detection device economical and the construction process convenient and quick. This chip can also be used to construct other biosensors, demonstrating its versatility. Finally, the constructed sensor successfully detected cell and mouse serum samples, with results consistent with standard methods, indicating the reliability of this method.

[0079] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components. In the description of the present invention, it should be understood that if terms such as "upper," "lower," "left," "right," "front," and "rear" indicate the orientation or positional relationship based on the orientation or positional relationship 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 orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting the present invention. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A bioassay kit based on CoB NP nanozymes, characterized in that: The device includes a biochip and a detection reagent. The detection reagent contains an SP-CoB NP nanozyme signal probe prepared from CoB NP nanozyme. The 5′ end of the signal probe (SP) is modified with an amino group. A capture probe (CP) is linked to the biochip. The 3′ end of the capture probe is modified with an amino group. The DNA sequence of the SP-CoB NP nanozyme signal probe (SP) is shown in SEQ ID NO: 3, and the DNA sequence of the capture probe (CP) is shown in SEQ ID NO:

1. Both can specifically bind to lung cancer target ctDNA. The biochip has a three-layer structure: a top splitting layer containing channels and a sample application area; a bottom collection layer for collecting reaction waste liquid; and a middle detection layer, on which the capture probe (CP) is covalently linked to the reaction area via amino groups. The biochip has two reaction zones: one is a colorimetric reaction zone, and the other is a pressure reaction zone. The biochip is a dual-mode pop-up paper chip; The preparation method of the CoB NP nanozyme is as follows: S1: In a polytetrafluoroethylene reactor containing ethylene glycol, add polyvinylpyrrolidone and cobalt boride powder as raw material. The ratio of each raw material is as follows: ethylene glycol:polyvinylpyrrolidone:cobalt boride powder volume mass ratio is 4 ml:45 mg:15 mg. Mix well and set aside. S2: Add a mixture of H2O2 and glacial acetic acid to the reactor in S1. The volume ratio of H2O2 to glacial acetic acid is 1 ml: 100 μl. Seal the reactor and place it in an oven. React at 160 °C for 120 minutes. S3: Centrifuge at 12000 rpm for 20 minutes to collect the precipitate, and wash it three times with anhydrous ethanol and deionized water respectively. S4: The collected precipitate was redissolved in deionized water and treated with an ultrasonic processor. After centrifugation at 5000 rpm for 1 minute, the supernatant was collected. The supernatant was then centrifuged at 12000 rpm for 10 minutes. The collected precipitate was CoB NP nanozyme.

2. The bioassay kit based on CoB NP nanozyme according to claim 1, characterized in that: The preparation method of the SP-CoB NP nanozyme signal probe is as follows: S1: A 5 μM amino-modified signal probe (SP) and a 1 mg / ml CoB NP nanozyme were mixed in a 1:1 volume ratio in 0.01 M phosphate buffer solution. S2: The reaction is carried out at 4°C for 12 h, during which the amino groups on the signal probe (SP) are covalently linked to the hydroxyl groups on the surface of the CoB NP nanozyme. S3: The sediment collected by centrifugation of the product from step S2 is the SP-CoBNP nanozyme signal probe. The SP-CoBNP nanozyme signal probe is redissolved in 0.01 M phosphate buffer solution for later use.

3. The bioassay kit based on CoB NP nanozyme according to claim 1, characterized in that: The method for preparing the biochip includes the following steps: S1: Drop 2.5 μL of chitosan with a concentration of 0.25 mg / mL onto the reaction area of ​​the paper-based chip detection layer; S2: After drying the detection layer processed in step S1 at room temperature, 2.5 μL of glutaraldehyde with a volume ratio of 2.5% is dropped on it. The aldehyde group of glutaraldehyde is covalently linked to the amino group of chitosan. S3: After the S2 step is completed, react for 2 hours, then add 2.5 μL of 5 μM amino-modified capture probe (CP), covalently link for half an hour, then add 1% BSA to block unreacted sites for half an hour to obtain the biochip.

4. The detection system of the bioassay kit for CoB NP nanozymes according to any one of claims 1-3, characterized in that: The biological detection kit includes a smart colorimetric processing terminal and a portable barometer, wherein the smart colorimetric processing terminal is a mobile terminal.

5. The detection system according to claim 4, characterized in that: The steps for detecting the target are as follows: S1: Drop the mixture of target and SP-CoB NP onto the sample application area of ​​the split layer of the biochip, incubate, and wash; S2: Cut out the two reaction areas in the detection layer respectively. For colorimetric mode, drop sodium acetate buffer containing a mixture of TMB and H2O2 into the chip, then place it in a 3D-printed dark box. Take a picture with a smartphone after 3 minutes and detect it through a mobile terminal with a colorimetric application. For pressure mode, put the cut paper into an EP tube containing 30% H2O2 and immediately seal it with a cap. Then insert a saline infusion needle and connect it to a portable barometer for detection. Record the pressure after 6 minutes.

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