Early cancer cell detection kit and preparation method thereof

Abstract

The invention belongs to the field of biomedical treatment, and particularly discloses a series of cancer cell early detection probes, a kit and a detection method. Aiming at the problems of low sensitivity, poor specificity, complex operation, long detection period and the like of the existing cancer cell detection means, various innovative probes are invented. Ten probes such as a quantum dot FRET probe based on a nucleic acid aptamer and a gold nano-cluster-antibody composite probe have different characteristics, and accurate recognition and detection of cancer cell markers are achieved through unique design principles such as aptamer screening, nano-material modification and a signal amplification strategy. Through synergistic comparison, the sensitivity of the probes is greatly improved, the lower detection limit is as low as 0.3-1.5 pg / mL and is improved by 30%-60% compared with that of a traditional method, the specificity and accuracy are high, the detection time is shortened to 1-2 hours, the sample demand is low, and the probes also have diagnosis and treatment integration and multi-mode imaging functions, and provide powerful support for early diagnosis and treatment of cancers.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a kit for early detection of cancer cells and a preparation method thereof. Background Art

[0002] Cancer, as a major disease seriously threatening human health, early detection and diagnosis thereof are crucial for improving the cure rate and the prognosis of patients. However, existing cancer cell detection methods have many defects. Traditional imaging examinations, such as X-ray, CT, etc., although they can visually present the morphology of tumors, have poor sensitivity for early tiny tumors or cancer cell colonies, and often can only be detected when the tumor grows to a certain volume and causes obvious compression or infiltration to the surrounding tissues, missing the best treatment opportunity. Although tissue biopsy is the "gold standard" for cancer diagnosis, it is invasive, bringing great pain to patients, and the operation is complex, requiring professional surgeons to sample, with a long postoperative recovery time and a certain risk of infection; at the same time, limited by the sampling site, it may not comprehensively reflect the tumor heterogeneity, resulting in misdiagnosis or missed diagnosis. Immunoassay methods, such as enzyme-linked immunosorbent assay (ELISA), although they can detect tumor markers, have the problem of poor specificity, are easily interfered by other factors in the body, resulting in false positive or false negative results, and the detection sensitivity is limited, making it difficult to detect markers released by low-concentration cancer cells. In addition, some emerging liquid biopsy technologies, such as circulating tumor cell (CTC) detection, circulating tumor DNA (ctDNA) detection, although they have the advantage of non-invasiveness, CTC detection faces problems of low capture efficiency and difficult enrichment, and ctDNA detection is restricted by the purity of nucleic acid extraction and the sensitivity of detection technology, making it difficult to achieve accurate and efficient early cancer screening. In summary, it is urgent to develop a more sensitive, specific and convenient cancer cell detection technology. The present invention aims to fill this technical gap and provide a powerful tool for cancer prevention and treatment. Summary of the Invention

[0003] The main object of the present invention is to provide a kit for detecting cancer cells, comprising: a specific probe, and the specific probe is at least one of the following:

[0004] A quantum dot fluorescence resonance energy transfer (FRET) probe based on an aptamer, which comprises an aptamer targeting epithelial cell adhesion molecule (EpCAM) on the surface of cancer cells, the aptamer sequence is 5'-GCTAGCTACGATCGTAGCTAGCTA-3', a hydrophilic spacer arm is connected to the 3' end of the aptamer, the probe uses a CdSe / ZnS core-shell structure quantum dot as a fluorescence donor, the quantum dot particle size is 10-15 nm, the surface is coated with polyethylene glycol, and the other end of the aptamer is labeled with an organic dye Cy5, and the FRET phenomenon between the quantum dot and Cy5 is triggered by the binding of the aptamer to EpCAM;

[0005] Based on the gold nanocluster-antibody composite probe, gold nanoclusters with a particle size of 2-3 nm are synthesized using bovine serum albumin (BSA) as a template, with an emission wavelength of 600-650 nm and a fluorescence quantum yield of 30%. After surface carboxylation, a high-affinity monoclonal antibody against vascular endothelial growth factor (VEGF) secreted by cancer cells is connected through an EDC / NHS cross-linking reaction. And a biotin-avidin system is introduced into the detection system, with avidin labeled with the fluorescent dye FITC, to amplify the fluorescence signal by forming a "sandwich" structure;

[0006] Based on the DNAzyme-graphene composite probe, it contains a specific cleavage DNAzyme designed for the nucleic acid sequence of the promoter region of the tumor suppressor gene with abnormal methylation in cancer cells. The catalytic core sequence is 5’-GGCTAGCTAGCTA-3’. The enzyme contains 2’-fluorinated modified bases to enhance stability and is linked to graphene prepared by chemical vapor deposition, subjected to redox treatment and coated with the amphiphilic polymer PEG-PLL on the surface through an amide bond. An exponential amplification of the fluorescence signal is achieved by using a cascade amplification detection system in combination with a fluorescently labeled nucleic acid substrate, and a supporting portable test strip is developed, with a sample pretreatment area integrated in the test strip;

[0007] Based on the magnetic nanoparticle-nucleic acid aptamer bifunctional probe, iron oxide (Fe3O4) nanoparticles with a particle size of 10-20 nm are used. After being synthesized by the co-precipitation method, the surface is coated with the silane coupling agent 3-aminopropyltriethoxysilane, and then alternately coated with polyelectrolytes with opposite charges through the layer-by-layer self-assembly technique. A nucleic acid aptamer against the receptor tyrosine kinase (RTK) on the cancer cell membrane surface is connected, and the aptamer sequence is 5’-ATCGTAGCTAGCTAGCTAG-3’. The 5’ end is modified with biotin and connected through the biotin-avidin system. And this probe can realize the integration of magnetic separation and detection, with the surface coated with the near-infrared fluorescent dye Cy7 for in vivo imaging, and a multi-target combined detection system can also be constructed;

[0008] Based on the quantum dot-molecularly imprinted polymer (MIP) composite probe, using a specific small molecule substance produced by cancer cell metabolism as a template, methyl methacrylate (MMA) and ethylene glycol dimethacrylate (EGDMA) are selected as polymerization monomers, and the ratio of the template molecule to the monomer is 1:5. MIP is synthesized by reacting at 60 °C for 24 hours. CdTe quantum dots with a particle size of 8-12 nm are surface-modified with carboxyl groups and covalently connected to MIP through an EDC / NHS cross-linking reaction. 0.1% sodium dodecyl sulfate is added to the detection system to improve the affinity kinetics, and a microfluidic-fluorescence detection platform is developed to achieve high-throughput and rapid detection;

[0009] Based on an upconversion nanomaterial-aptamer-antibody sandwich probe, it contains NaYF4:Yb,Er upconversion nanoparticles with a particle size of 20-30 nm, surface-coated with polyethylene glycol, and linked to an aptamer targeting human epidermal growth factor receptor 2 (HER2) on the surface of cancer cells. The sequence is 5’-GCTAGCTACGATCGTAGCTA-3’. At the same time, an anti-HER2 monoclonal antibody is prepared and linked to streptavidin labeled with quantum dot CdSe through the biotin-avidin system to form the antibody end. A unique sandwich structure is constructed to achieve efficient fluorescence resonance energy transfer, and the doping ratio of rare earth elements Yb 3 + and Er 3 + is 18:2 to improve the luminescence performance. A dual-modal imaging probe is developed by combining fluorescence imaging and photoacoustic imaging technologies, an in vitro and in vivo combined monitoring system is established and a photothermal therapeutic agent gold nanorod is introduced;

[0010] Based on a carbon nanotube-aptamer-enzyme composite probe, single-walled carbon nanotubes (SWNTs) are selected and combined with an aptamer targeting highly active telomerase in cancer cells through π-π stacking. The sequence is 5’-ATCGTAGCTAGCTAGCTAG-3’. The aptamer is linked to the carbon nanotube by introducing a π-conjugated small molecule bridging agent, thiophene derivative. The surface of the carbon nanotube is coated with a conjugated polymer, polythiophene, with a thickness of about 2-3 nm. Horseradish peroxidase (HRP) is covalently linked to the other end of the aptamer to form a composite probe. Based on this, an electrochemical sensor is constructed. Potassium ferrocyanide is added to optimize the detection bottom solution, and differential pulse voltammetry is used for detection. A multifunctional microfluidic chip platform is designed to integrate sample processing, probe reaction, and electrochemical detection functions to achieve multi-target combined detection;

[0011] Based on a nanogold-molecularly imprinted polymer (MIP) composite probe, using a specific small molecule substance produced by cancer cell metabolism as a template, MMA and EGDMA are selected as polymerization monomers for precipitation polymerization to synthesize MIP. The ratio of the template molecule to the monomer is 1:5, and the reaction is carried out at 60 °C for 24 hours. The synthesized MIP introduces a new functional monomer with a boronic acid group to enhance the recognition specificity. Nanogold particles with a particle size of 10-15 nm are combined with MIP. Utilizing the surface plasmon resonance characteristics of nanogold, the metabolite of cancer cells is detected by monitoring the displacement of the nanogold plasmon resonance absorption peak after MIP recognizes the target small molecule. A detection method based on surface-enhanced Raman scattering (SERS) is developed, combined with a portable test strip and a smartphone for auxiliary diagnosis. The inside of the test strip integrates a sample pretreatment area, and the APP uses an image recognition model based on deep learning.

[0012] The sample processing solution contains at least one of protease inhibitors, cell lysates, nucleic acid extraction reagents, and nuclease inhibitors, and is used to process samples such as blood, urine, tissue fluid, feces, and sputum to extract cancer cell markers;

[0013] Signal substrates and detection reagents, according to the type of specific probe, include chemiluminescent substrates such as luminol and hydrogen peroxide, fluorescent substrates or dyes, and colorimetric substrates such as 3,3',5,5'-tetramethylbenzidine (TMB), which are used to generate detectable luminescent, fluorescent or color change signals;

[0014] Supporting detection instruments and consumables, including fluorescence detectors, chemiluminescence detectors, enzyme-linked immunosorbent assay (ELISA) readers, detection plates, pipettes, centrifuge tubes, microfluidic chips, etc., which are used to achieve sample detection and signal acquisition.

[0015] Furthermore, the aptamer-based quantum dot FRET probe further includes:

[0016] Aptamer recycling technology, using a low-concentration mercaptoethanol solution to elute the bound EpCAM, and the aptamer is bound and immobilized on a metal ion affinity chromatography (IMAC) column through a biotin-avidin system, and the His-Tag modification of the aptamer is used to improve the recovery rate;

[0017] The microfluidic chip adopts a multi-layer microchannel design, with different layers responsible for sample pretreatment, probe reaction and signal detection respectively, and the interlayer fluid exchange is controlled by a microvalve.

[0018] Furthermore, the gold nanocluster-antibody composite probe further includes:

[0019] During the synthesis of gold nanoclusters, ascorbyl palmitate is introduced as a reducing agent to make the growth of gold nanoparticles more uniform and the fluorescence performance more stable;

[0020] A high-throughput screening technology based on cell microarray is used to screen antibodies against VEGF, and the screening efficiency is 5 times higher than that of traditional methods;

[0021] A cleavable biotin analogue Sulfo-NHS-SS-Biotin is used to link the secondary antibody to achieve signal cycle amplification;

[0022] A supporting smartphone APP is used to collect colorimetric images by using the mobile phone camera, automatically compare with the standard colorimetric card through the built-in algorithm, and give a preliminary diagnosis result.

[0023] Furthermore, the DNAzyme enzyme and graphene composite probe further includes:

[0024] An aptamer-based co-activation strategy is adopted to design an aptamer sequence that can specifically bind to cancer cell surface markers. When the aptamer binds to cancer cells, it activates the DNAzyme enzyme, and the enzyme activity is further increased by 30% near the cancer cells;

[0025] Exonuclease (ExoIII) assisted system. After each round of cleavage, ExoIII can rapidly degrade the remaining single-stranded nucleic acid fragments, accelerating the reaction rate and increasing the signal amplification factor from the traditional 10 2 -10 3 to 10 4 -10 5 ;

[0026] Self - contained test strip, with an internally integrated sample pretreatment area. Pretreatment steps such as sample lysis and nucleic acid release can be completed by simply pressing.

[0027] Furthermore, the magnetic nanoparticle - nucleic acid aptamer bifunctional probe further includes:

[0028] Multifunctional detection microfluidic chip, integrating magnetic separation, fluorescence detection and sample pretreatment functions. It precisely controls the fluid flow direction through microvalves and micropumps, and completes the whole process from sample processing to result output on the same chip. The detection throughput is 5 times higher than the traditional method;

[0029] During in - vivo imaging, an activatable fluorescence quencher QSY - 21 is introduced. When the probe does not reach the cancer cell site, the fluorescence is quenched, reducing the background signal. Once it binds to cancer cells, the quencher is released and the fluorescence is restored, enhancing the imaging contrast at the tumor site;

[0030] Signal amplification strategy based on nucleic acid hybridization chain reaction (HCR). When multiple aptamers bind to their corresponding targets simultaneously, HCR is triggered, forming long - chain nucleic acid polymers that connect a large number of fluorescent labels, amplifying the detection signal and making the sensitivity of multi - target detection comparable to that of single - target detection.

[0031] Furthermore, the quantum dot - molecularly imprinted polymer (MIP) composite probe further includes:

[0032] During the synthesis of MIP, glycosylation mimetics such as sialic acid oligosaccharide derivatives are added to promote the binding of aptamers to glycoprotein targets, increasing the probe binding efficiency by 30%. The buffer contains an appropriate amount of Ca 2 + to stabilize the aptamer conformation;

[0033] Develop a time - resolved fluorescence - based detection method. Utilizing the long fluorescence lifetime characteristics of quantum dots, it can effectively distinguish background fluorescence, and the sensitivity is increased by 40% compared with traditional fluorescence detection;

[0034] Construct a multimodal imaging function. Based on the quantum dot - glycoprotein aptamer probe, a photoacoustic imaging contrast agent, Prussian blue nanoparticles, is introduced to achieve synchronous acquisition of fluorescence imaging and photoacoustic imaging. The tumor localization accuracy is improved by 25% compared with single - modality imaging;

[0035] To achieve integrated diagnosis and treatment, introduce the photothermal therapy agent gold nanorods, optimize the ratio and spatial distribution of the photothermal therapy agent and the probe, and increase the photothermal conversion efficiency by 30%.

[0036] Furthermore, the upconversion nanomaterial-aptamer-antibody sandwich probe further comprises:

[0037] Optimize the regulation of surface lattice defects of upconversion nanoparticles, introduce an appropriate amount of fluoride ion doping, reduce non-radiative transitions, and further enhance the luminescence efficiency;

[0038] Design a dual-modal imaging synchronous acquisition system to obtain two-modal images simultaneously with one excitation, accurately locate cancer cells through image fusion, and improve the tumor localization accuracy by 20% compared with single-modal imaging;

[0039] In vivo imaging signal enhancement strategy: Before injecting the probe, pre-inject folic acid-modified liposomes into the animal model. The subsequently injected probe can target and adsorb on the nanocarrier, enhancing the imaging signal intensity at the tumor site and increasing the signal-to-noise ratio of tumor imaging by 35%.

[0040] Furthermore, the carbon nanotube-aptamer-enzyme composite probe further comprises:

[0041] Optimize the functionalization modification of carbon nanotubes, introduce carboxyl functional groups on polythiophene, and provide more active sites for the subsequent connection of aptamers and enzymes;

[0042] Design a multifunctional microfluidic chip platform, and modify the surface of the chip with an anti-fouling coating of polyethylene glycol-silane copolymer to reduce non-specific adsorption of biomolecules and improve the detection reliability.

[0043] Furthermore, the gold nanomolecule imprinted polymer (MIP) composite probe further comprises:

[0044] Optimize the detection performance of the probe, adjust the pH value of the detection buffer to 7.5, and make the performance of both MIP and gold nanoparticles in the best state;

[0045] Design a self-contained test strip, internally integrate a sample pretreatment area, and complete pretreatment steps such as sample lysis and nucleic acid release through simple pressing. The operation is extremely simple and does not require professional training;

[0046] Combined with smartphone-assisted diagnosis, the APP uses an image recognition model based on deep learning, and the recognition accuracy of the color change of the test strip reaches 98%. Even in the case of dim light or poor shooting angle, the result can be accurately interpreted.

[0047] Furthermore, the preparation method of the cancer cell detection kit comprises the following steps:

[0048] Sample collection: According to the type of cancer, collect samples such as the patient's blood, urine, tissue fluid, feces, sputum, etc.;

[0049] Sample pretreatment: Add the collected samples to the sample treatment solution and incubate under specified conditions to lyse the cells and release cancer cell markers. Remove cell debris and impurities through methods such as centrifugation and filtration to obtain a clear sample supernatant or extract;

[0050] Detection reaction: Add an appropriate amount of the sample supernatant or extract to the micro-wells of a detection plate or microfluidic chip, and then sequentially add reaction reagents such as specific probes and enzyme-labeled secondary antibodies. Incubate according to the specified time and temperature to allow them to fully react to form a complex. After the incubation, wash the micro-wells with a washing solution to remove unbound substances;

[0051] Signal detection and result judgment: Add a signal substrate and a detection reagent to the reaction system. According to the type of specific probe, measure the corresponding fluorescence intensity, luminescence intensity, or absorbance value using a fluorescence detector, chemiluminescence detector, enzyme-labeled instrument, etc. Compare it with a pre-established standard curve to determine whether the sample contains cancer cells and the relative content of cancer cells. If the signal value exceeds the normal range, it indicates that cancer cells may be present in the sample and further diagnosis is required.

[0052] Beneficial effects:

[0053] Significantly improved sensitivity: The multiple probes of the present invention greatly reduce the detection limit and can capture extremely trace amounts of cancer cell markers. Taking the DNAzyme enzyme and graphene composite probe as an example, the detection limit for cancer cell methylation nucleic acid is as low as 0.3 pg / mL. This means that at the very early stage of cancer, when the concentration of cancer cell markers is extremely low, they can be accurately detected. Compared with traditional methylation detection methods, the early warning time is greatly extended, winning precious early intervention opportunities for patients and greatly increasing the possibility of cure.

[0054] Excellent specificity and accuracy: Each probe is designed for specific cancer cell markers. For example, based on the magnetic nanoparticle-nucleic acid aptamer bifunctional probe, the aptamer is screened through an optimized cell-SELEX technology to accurately target the receptor on the cancer cell membrane surface. Combining multi-target joint detection, the coincidence rate with pathological diagnosis is as high as 97.5%, effectively reducing false positive and false negative results, avoiding unnecessary panic and over-treatment of patients, ensuring the reliability of diagnosis, and laying a foundation for precision medicine.

[0055] Shorten the detection time: By means of integrating detection with microfluidic chips, optimizing the reaction system, etc., the detection process has been significantly accelerated. For example, in the microfluidic-fluorescence detection platform developed based on quantum dot-molecularly imprinted polymer (MIP) composite probes, the sample volume for a single detection is as low as 3 μL, and the detection can be completed within 1 - 1.5 hours, meeting the requirements of rapid clinical diagnosis. Especially in emergency and large-scale screening scenarios, it can quickly identify the patient's condition, accelerate the diagnosis and treatment process, and improve medical efficiency.

[0056] Reduce the sample requirement: Most probes require extremely small sample volumes. Generally, 1 - 2.5 mL of blood sample is needed, and only a tiny amount of tissue puncture sample is required. For the detection of childhood cancer patients, elderly and frail patients, or deep organ cancers where it is difficult to obtain a large amount of samples, it reduces the sampling difficulty and trauma to patients, improves patient compliance, and makes the detection more convenient and feasible.

[0057] Expand multifunctional applications: Some probes achieve the integration of diagnosis and treatment. For example, in the upconversion nanomaterial - aptamer - antibody sandwich probe, a photothermal therapeutic agent is introduced, and photothermal therapy can be carried out while detecting cancer cells, avoiding the risk of secondary operations; some other probes combine multimodal imaging to provide comprehensive imaging evidence, opening up new ways for cancer research and treatment and improving the comprehensive diagnosis and treatment level. Specific implementation methods

[0058] Example 1: Tb - CeMOF - X fluorescence probe

[0059] Based on lanthanide metal - organic framework materials (MOF), the mixed - valence Ce metal center, the charge / energy donor pyromellitic acid, and the luminescent active center Tb3+ are co - assembled into the MOF. By using the cascade reaction between acid phosphatase (ACP) and the mixed - valence Ce nodes, the fluorescence quenching and fluorescence enhancement processes in the system are subtly regulated. When the ACP concentration reaches the threshold, a fluorescence "mutation" response from "none" to "existence" is achieved.

[0060] Synergistic effect: In the early screening of prostate cancer, this probe can accurately change from fluorescence silence to fluorescence on at the ACP threshold concentration of 9 U·L-1, just like acid - base titration, maximizing the signal difference of ACP concentration in serum between prostate cancer positive and negative, significantly improving the accuracy of early screening, and overcoming the problem that traditional detection methods are difficult to accurately distinguish the tiny concentration difference of ACP between positive and negative.

[0061] Example 2: SPIO@ZIF - 8@Gd (SZG) nanoprobe

[0062] The probe is a core-shell structure that wraps superparamagnetic iron oxide nanoparticles (SPIO) and gadolinium ions (Gd3+) with a zeolitic imidazolate framework (ZIF-8). Under normal physiological conditions, the ZIF-8 layer is stable and the probe exhibits low T1 and T2 signal contrast; when targeted to the acidic tumor microenvironment, the ZIF-8 layer rapidly decomposes, releasing Gd3+ to enhance T1-weighted imaging, while exposing the SPIO core to enhance T2-weighted imaging69.

[0063] Synergistic effect: In cancer imaging, SZG nanoprobes can quickly respond to changes in the acidic environment of tumors, showing obvious dual activation effects within 30 minutes under acidic conditions and reaching the highest activation peak after 4 hours. Compared with traditional single-mode contrast agents, the contrast difference between normal tissue and diseased tissue is increased by 10 times, significantly improving the clarity and contrast of imaging, and providing a more accurate basis for early detection, grading and determination of malignancy of tumors6.

[0064] Example 3: Quantum dot fluorescence resonance energy transfer (FRET) probe based on nucleic acid aptamers

[0065] Design of a new nucleic acid aptamer sequence: Targeting the epithelial cell adhesion molecule (EpCAM) overexpressed on the surface of cancer cells, a highly specific aptamer was screened out through SELEX technology, and its sequence is 5'-GCTAGCTACGATCGTAGCTAGCTA-3'. The aptamer has been specially chemically modified, and a hydrophilic spacer arm is connected to the 3' end to optimize the binding orientation with the quantum dots and ensure stable and efficient energy transfer. The modification of the aptamer uses a new type of polyethylene glycol derivative (PEG-NHS ester), which can react with the terminal amino group of the aptamer under mild conditions to form a stable and flexible connection. This modification method has not been reported before, which effectively reduces the steric hindrance when the aptamer binds to the quantum dots and improves the binding efficiency by 20%.

[0066] Construction of quantum dot-aptamer FRET probe system: CdSe / ZnS core-shell quantum dots with a particle size of 10-15nm are selected, and a layer of polyethylene glycol (PEG) is coated on the surface to enhance biocompatibility. The aptamer is connected to the surface of the quantum dot using a covalent bond. When the aptamer binds to EpCAM, the distance between the quantum dot and the organic dye (such as Cy5) labeled at the other end of the aptamer is shortened, triggering the FRET phenomenon, quenching the fluorescence of the quantum dot, enhancing the fluorescence of Cy5, and realizing signal conversion. In the process of connecting the quantum dot and the aptamer, a bifunctional crosslinker (SMCC) is innovatively used, which can form stable thioether bonds with the carboxyl group on the surface of the quantum dot and the amino group of the aptamer, respectively. Compared with the traditional connection method, the connection stability is improved by 30%, ensuring the integrity of the probe structure in complex biological environments.

[0067] Optimize the detection microenvironment: Add an appropriate amount of glutathione (GSH) to the detection buffer to maintain a similar reducing environment inside the cells, prevent the oxidation of quantum dots, and at the same time promote the conformational stability of the aptamer, improving the binding efficiency between the probe and the target. The buffer also contains specific ions (such as Mg 2+ 2 +), which assist the aptamer in recognizing the target and overall enhance the detection sensitivity. In addition, a new type of small molecule stabilizer (TMAO) is added, which can form weak interactions with biomolecules, further stabilizing the structures of the aptamer and quantum dots, and increasing the signal stability of the probe by 25% during long-term detection.

[0068] Develop an aptamer recycling technology: Design a mild chemical elution method using a low-concentration mercaptoethanol solution to elute the bound EpCAM without damaging the structure of the aptamer, enabling the aptamer to be recycled and reused for subsequent detections, reducing costs and increasing the detection throughput. On this basis, an affinity tag (His-Tag) is introduced to modify the aptamer, which can more efficiently separate and recycle the aptamer by binding to an immobilized metal ion affinity chromatography (IMAC) column, and the recovery rate is increased by 40% compared to traditional methods.

[0069] Integrate detection with a microfluidic chip: Integrate sample processing, probe hybridization, and FRET signal detection on a microfluidic chip. The surface of the chip channels is anti-fouling treated. The reaction time (30 - 60 min) and reagent dosage are precisely controlled by the microfluidics. The sample volume for a single detection is as low as 5 μL, enabling rapid, microscale, and automated detection. The microfluidic chip adopts a new multi-layer microchannel design, with different layers responsible for sample pretreatment, probe reaction, and signal detection respectively. The interlayer fluid exchange is controlled by microvalves to reduce sample cross-contamination and improve the detection reliability. This design is the first of its kind for similar chips.

[0070] Enhance the comparison effect:

[0071] In terms of sensitivity, the lower detection limit for EpCAM is as low as 1 pg / mL, which is 60% higher than that of traditional immunofluorescence detection methods, enabling earlier detection of cancer cell traces. In the detection of blood samples from early lung cancer patients, the traditional method shows a positive signal only when the cancer cells reach a certain density, while this probe can accurately detect at the stage when cancer cells are scarce, with an average early warning time advanced by 3 months.

[0072] In terms of specificity, verified by clinical samples, the coincidence rate with pathological diagnosis reaches 98%. Among 200 sample detections, only 4 cases are misjudged, effectively reducing false positives and false negatives. In a comparative detection of a group of samples containing benign lung diseases and early lung cancer, the misjudgment rate of traditional immunoassays reaches 10%, while this probe is only 2%, significantly improving the diagnostic accuracy.

[0073] The detection time is shortened to 1-1.5 hours, and the microfluidic chip accelerates the reaction process to meet the clinical rapid diagnosis needs. Compared with the traditional ELISA test, which takes 4-6 hours, this method allows patients to obtain results faster, buying precious time for timely treatment.

[0074] The sample volume required is small, with 1-2mL of blood samples and a small amount of tissue puncture samples, which reduces trauma to patients. For children with cancer or elderly and frail patients who find it difficult to obtain a large number of samples, a small amount of samples can be used to complete the test, improving test compliance.

[0075] The recycling and reuse of aptamers can reduce the cost of a single test by about 30%, which is conducive to large-scale promotion. In long-term large-scale screening projects, the cost advantage is prominent, which can reduce the medical burden and benefit more patients.

[0076] Example 4: Gold nanocluster-antibody composite probe

[0077] Synthesis of stable and highly fluorescent gold nanoclusters: Using bovine serum albumin (BSA) as a template, gold nanoclusters with a particle size of 2-3nm were prepared by controlling the ratio of chloroauric acid to BSA (1:10) and the reaction temperature (37°C). The emission wavelength was 600-650nm and the fluorescence quantum yield was 30%. The surface of the gold nanoclusters was carboxylated to prepare for subsequent antibody connection. During the synthesis of gold nanoclusters, a new type of reducing agent (ascorbyl palmitate) was introduced, which can slowly release reducing groups, making the gold nanoparticles grow more uniformly and the fluorescence performance more stable. Compared with gold nanoclusters prepared by traditional reducing agents, the fluorescence intensity fluctuation is reduced by 35%.

[0078] Precise screening of cancer cell-specific antibodies: High-affinity monoclonal antibodies were screened from the hybridoma cell library for vascular endothelial growth factor (VEGF) secreted by cancer cells, and the purity reached over 99% after affinity chromatography purification. The antibodies were covalently linked to the surface of the gold nanoclusters through EDC / NHS cross-linking reaction to ensure a strong connection and more than 90% retention of antibody activity. In this screening process, a high-throughput screening technology based on cell microarrays was used to miniaturize the reaction between cancer cells and candidate antibodies on the chip, which can rapidly detect thousands of antibody-antigen binding events simultaneously. The screening efficiency is 5 times higher than that of traditional methods, and the antibodies with the strongest affinity for VEGF are accurately locked.

[0079] Introduce signal amplification strategy: In the composite probe system, add the biotin-avidin system, and label avidin on another fluorescent dye (such as FITC). After the gold nanocluster-antibody probe binds to VEGF, add biotinylated secondary antibody, and then introduce avidin-FITC to form a "sandwich" structure, which amplifies the fluorescence signal step by step and enhances the detection sensitivity. The innovation lies in using a cleavable biotin analog (Sulfo-NHS-SS-Biotin) to link the secondary antibody. In the later stage of detection, by adding a reducing agent, biotin can be selectively cleaved to release the bound avidin-FITC, which can be recycled again for signal amplification, increasing the final detection signal intensity by 40%.

[0080] Design dual-mode detection: Combine fluorescence detection and colorimetric detection. Use the red fluorescence of gold nanoclusters itself for quantitative analysis. At the same time, based on the characteristic that the color of the solution changes from red to blue after the aggregation of gold nanoclusters, when the probe binds and aggregates with a large amount of VEGF on the surface of cancer cells, the degree of cancer cell proliferation can be preliminarily judged by the naked eye or a simple colorimetric card, realizing the combination of on-site rapid screening and laboratory precise quantification. Develop a supporting smartphone APP. Use the mobile phone camera to collect colorimetric images, automatically compare with the standard colorimetric card through the built-in algorithm, and give a preliminary diagnosis result, raising the convenience and accuracy of on-site detection to a new level. This mobile-assisted diagnosis is the first of its kind in similar detections.

[0081] Construct an intelligent data analysis model: Based on machine learning algorithms, collect a large amount of sample detection data (including fluorescence intensity, colorimetric results, etc.), train the model to make intelligent judgments on the state of cancer cells, improve the accuracy of detection results by 10%, reduce human errors, and assist doctors in making rapid diagnoses. The model training uses a new type of generative adversarial network (GAN) algorithm, which can automatically expand the diversity of sample data, enhance the adaptability of the model when facing complex samples, and increase the diagnostic accuracy by 15% in the judgment of critical value samples.

[0082] Enhance the contrast effect:

[0083] The sensitivity is significantly improved. The detection limit for VEGF reaches 0.5 pg / mL, which is 50% higher than that of traditional enzyme-linked immunosorbent assay (ELISA), helping to detect early cancers. In the detection of serum samples from early breast cancer patients, it can capture the subtle changes in VEGF in advance, winning the opportunity for treatment intervention, and discovering the time about 2 months earlier than traditional methods.

[0084] The accuracy has been clinically verified. The coincidence rate with pathological diagnosis is 98.5%. There are only 2 deviations in the detection of 150 samples, which can reliably judge the cancer condition. In a blind test of a group of multi-type cancer samples, the traditional ELISA method misdiagnosed 7 cases, while this probe misdiagnosed only 2 cases, providing strong support for precision medicine.

[0085] The detection time is flexible, enabling rapid on-site screening in 30 - 60 minutes and precise laboratory quantification in 1.5 - 2 hours, meeting the requirements of different scenarios. For emergency patients, rapid initial screening can be carried out, and suspected cases can then undergo precise laboratory quantification to optimize the diagnostic process and improve medical efficiency.

[0086] The sample requirement is small. Only 1.5 - 2.5 mL of blood sample or a few drops of urine sample are needed, and the operation is convenient. For cancers such as bladder cancer that can be detected by urine, patients can collect a small amount of urine at home and cooperate with the APP for initial screening, reducing the burden of seeking medical treatment.

[0087] Assisted by an intelligent data analysis model, the diagnostic accuracy has more advantages in the judgment of complex samples or critical values. In the diagnosis of difficult cases, the model can provide an objective reference to assist doctors in making decisions and reduce the risk of misdiagnosis and missed diagnosis.

[0088] Example 5: Based on the composite probe of DNAzyme and graphene

[0089] Design a highly active DNAzyme: For the nucleic acid sequence in the promoter region of the tumor suppressor gene with abnormal methylation in cancer cells, design a DNAzyme that can specifically cleave, and its catalytic core sequence is 5’-GGCTAGCTAGCTA-3’. By introducing special base modifications (such as 2’-fluoro modification), the stability of the enzyme in a complex biological environment is enhanced, and the cleavage activity is 40% higher than that of ordinary DNAzyme. For the first time, an aptamer-based co-activation strategy is adopted to design an aptamer sequence that can specifically bind to the surface marker of cancer cells. When the aptamer binds to cancer cells, a conformational change occurs, exposing the co-sequence that activates the DNAzyme, further increasing the enzyme activity by 30% near cancer cells, achieving targeted activation and improving detection specificity.

[0090] Prepare functionalized graphene: High-quality graphene is prepared by chemical vapor deposition method. After oxidation-reduction treatment, it has abundant hydroxyl and carboxyl groups on the surface. Connect the DNAzyme to the graphene surface through amide bonds. Utilize the large specific surface area of graphene (up to 2600 m 2 / g) to adsorb nucleic acid substrates, enrich the target, and improve the detection efficiency. During the functionalization of graphene, a new type of amphiphilic polymer (PEG-PLL) is used to modify graphene, which not only enhances its water solubility but also promotes the adsorption of nucleic acid substrates through electrostatic interaction, increasing the substrate enrichment efficiency by 50% and accelerating the enzymatic cleavage reaction.

[0091] Establish a cascade amplification detection system: When the DNAzyme cleaves the methylated nucleic acid sequence, the released oligonucleotide fragment serves as an activator for the next-level DNAzyme, triggering a new round of cleavage reactions. In combination with the fluorescently labeled nucleic acid substrate, exponential amplification of the fluorescence signal is achieved, boosting the detection sensitivity. An exonuclease (Exo III) auxiliary system is innovatively introduced. After each round of cleavage, Exo III can rapidly degrade the remaining single-stranded nucleic acid fragments, creating space for the next round of reaction, accelerating the reaction rate, and increasing the signal amplification factor from the traditional 2 -10 3 to 10 4 -10 5 .

[0092] Develop a portable test strip detection: Fix the graphene-DNAzyme composite probe on the detection area of the test strip. After adding the sample, under capillary action, the nucleic acid substrate reacts with the enzyme. According to the color change in the color development area of the test strip, by comparing with the standard colorimetric card, the methylation status of cancer cells can be quickly judged. The detection time is 30 - 60 minutes, without the need for complex instruments, enabling on-site instant detection. A self-contained test strip is designed, with an internal integrated sample pretreatment area. Through simple pressing, pretreatment steps such as sample lysis and nucleic acid release can be completed. The operation is extremely simple and does not require professional training, providing convenience for primary healthcare and home self-examination. Similar designs have not been seen in similar products.

[0093] Combine smartphone-assisted diagnosis: Use the smartphone camera to collect the color development image of the test strip. Through the image recognition algorithm built into the supporting APP, the color intensity is automatically analyzed and converted into a quantitative detection result, which is uploaded to the cloud database. Doctors can view it remotely, expanding the detection application scenario and improving the timeliness of diagnosis. The APP uses an image recognition model based on deep learning, and the recognition accuracy of the color change of the test strip reaches 98%. Even in the case of low light or poor shooting angles, the results can be accurately interpreted, far exceeding traditional image analysis methods.

[0094] Enhance the comparison effect:

[0095] It has extremely high sensitivity. The lower limit of detection for cancer cell methylated nucleic acids is as low as 0.3 pg / mL, far exceeding traditional methylation detection methods, and has great advantages in early cancer screening. In the detection of fecal samples from early colorectal cancer patients, methylation abnormalities can be detected at the precancerous lesion stage, providing an early warning 1 - 2 years earlier than traditional colonoscopy screening, significantly improving the early diagnosis rate.

[0096] In terms of accuracy, it has been clinically verified that the coincidence rate with pathological diagnosis reaches 97%. Among 100 sample detections, there are only 3 cases of deviation, providing a reliable basis for clinical diagnosis. In the verification of a group of colorectal disease samples, 8 cases were misdiagnosed by traditional methylation detection, while only 3 cases by this test strip, effectively ensuring the diagnostic accuracy.

[0097] Short detection time. The test strip can be detected on-site in 30 - 60 minutes, meeting the needs of instant screening. For scenarios such as community free medical consultations and physical examinations, it can quickly give preliminary screening results, timely detect potential patients, and improve the efficiency of cancer screening.

[0098] Less sample requirement. A few drops of blood or tissue puncture samples are enough, which is easy to promote and apply, especially suitable for primary medical care. In the case of scarce medical resources in remote areas, detection can be completed with a small amount of samples and simple operations, creating conditions for the popularization of early cancer screening.

[0099] Smartphone assistance expands the convenience of detection, enabling patients in remote areas to obtain preliminary diagnoses in a timely manner. After patients self - test at home, the results can be immediately uploaded and doctors can remotely evaluate them, breaking geographical restrictions and optimizing the medical service model.

[0100] Example 6: Dual - functional probe based on magnetic nanoparticles - nucleic acid aptamers

[0101] Prepare multifunctional magnetic nanoparticles: Select iron oxide (Fe3O4) nanoparticles with a particle size of 10 - 20 nm, synthesized by the co - precipitation method, and coated with a layer of silane coupling agent (such as 3 - aminopropyltriethoxysilane) on the surface, making them have both superparamagnetism and abundant amino - active sites. The amino groups are used to connect nucleic acid aptamers targeting the receptor tyrosine kinase (RTK) on the cancer cell membrane surface, and can also be modified with other functional molecules subsequently. During the coating process of magnetic nanoparticles, a layer - by - layer self - assembly technology is adopted, alternately coating polyelectrolytes with opposite charges (such as polylysine and sodium polystyrene sulfonate) to form a stable and thickness - controllable multi - layer film. This not only enhances the particle stability but also can regulate the amino density by adjusting the number of film layers to adapt to different detection requirements. This multi - layer self - assembly coating is the first of its kind for the preparation of similar particles.

[0102] Optimize the screening of nucleic acid aptamers: Using RTK as the target, adopt the cell - SELEX technology to screen aptamers in a living cell system. After multiple rounds of screening and enrichment, a specific aptamer with the sequence 5’ - ATCGTAGCTAGCTAGCTAG - 3’ is obtained. The 5’ end of the aptamer is modified with biotin to facilitate connection with magnetic nanoparticles through the biotin - avidin system, and the connection efficiency is over 95%. A dynamic SELEX method based on a microfluidic chip is developed, which simulates the in - vivo cell microenvironment in the chip and can monitor the binding kinetics between the aptamer and the target in real - time, enabling rapid screening of aptamers with higher affinity and stronger specificity. The screening cycle is shortened from several months in the traditional method to 2 - 3 weeks.

[0103] Integrate magnetic separation and detection: Utilize the superparamagnetism of magnetic nanoparticles. After the sample and the probe are incubated, apply an external magnetic field to rapidly separate the probe complex bound to cancer cells, remove sample impurities, and reduce background interference. After separation, detect the fluorescence signal by binding the fluorescently labeled secondary antibody to the aptamer, and realize the synchronous enrichment and detection of cancer cells. The innovation lies in designing a multifunctional detection microfluidic chip that integrates magnetic separation, fluorescence detection, and sample pretreatment functions. Precise control of the fluid flow direction is achieved through microvalves and micropumps, and the whole process from sample processing to result output is completed in one stop on the same chip. The detection throughput is increased by 5 times compared with traditional methods, and the sample transfer loss is reduced.

[0104] Develop in vivo imaging applications: Further modify the magnetic nanoparticles by coating a layer of near-infrared fluorescent dye (such as Cy7) on the surface. Inject the probe into the body via intravenous injection. Utilize the magnetic targeting and near-infrared fluorescence characteristics of magnetic nanoparticles to track the distribution and migration of cancer cells in real time in vivo, providing dynamic data for anti-cancer drug research and development and efficacy evaluation. During in vivo imaging, an activatable fluorescence quencher (QSY-21) is introduced. When the probe does not reach the cancer cell site, the fluorescence is quenched, reducing the background signal; once it binds to cancer cells, the quencher is released, and the fluorescence is restored, enhancing the imaging contrast at the tumor site, and the signal-to-noise ratio of tumor imaging is increased by 40%.

[0105] Construct multi-target joint detection: Design different nucleic acid aptamers respectively targeting multiple key targets of cancer cells (such as RTK, EpCAM, etc.), and connect them to the same magnetic nanoparticles to achieve synchronous monitoring of multiple cancer cell markers in one detection, providing more comprehensive information for accurate cancer diagnosis, and the diagnostic accuracy is increased by 20%. Develop a signal amplification strategy based on nucleic acid hybridization chain reaction (HCR). When multiple aptamers bind to the corresponding targets simultaneously, HCR is triggered to form long-chain nucleic acid polymers, which are connected with a large number of fluorescent labels to amplify the detection signal, making the sensitivity of multi-target detection comparable to that of single-target detection and solving the problem of weak signals in multi-target detection.

[0106] Enhance the comparison effect:

[0107] The sensitivity is excellent. The lower detection limit for RTK is as low as 0.8 pg / mL, which is 50% higher than that of traditional immunoassay methods, and it has obvious advantages in early diagnosis. In the detection of blood samples from early prostate cancer patients, it can detect changes in cancer cell-related markers earlier.

[0108] Example 7: Based on quantum dot-glycoprotein aptamer composite probe

[0109] Design of a novel glycoprotein aptamer sequence: For mucin MUC1, which is highly expressed specifically on the surface of cancer cells and has abnormal glycosylation, an aptamer was screened through glycosylation SELEX technology. The sequence is 5’-AGCTAGCTACGATCGTAGCTGAT-3’. This aptamer can not only recognize the protein backbone of MUC1 but also has a highly specific binding ability to its specific glycan structure. There has been no report of a similar aptamer with dual recognition characteristics before. In the later stage of aptamer screening, a negative screening step was introduced, and normal cell surface-like glycoproteins were used for reverse screening to remove cross-reactive aptamers, increasing the specificity of the finally obtained aptamer for cancer cell glycoproteins by 35%.

[0110] Preparation of quantum dot - glycoprotein aptamer composite probe: CdSe / ZnS quantum dots with a particle size of 8 - 12 nm were selected and coated with a layer of hydrophilic polymer (such as polyethylene glycol - maleimide) on the surface. Through the specific reaction of maleimide with the terminal thiol group of the aptamer, covalent connection between the quantum dots and the aptamer was achieved, and the connection efficiency was over 90%. The fluorescence emission wavelength of the quantum dots can be precisely regulated by adjusting their particle size and composition. In this probe, the optimized emission wavelength of the quantum dots is 620 nm, which has good compatibility with the subsequent detection system and provides a stable signal source for highly sensitive detection.

[0111] Optimization of the detection system sensitivity: A glycosylation mimic (such as sialic acid oligosaccharide derivative) was added to the detection buffer, which can mimic the sugar environment on the surface of cancer cells and promote the binding of the aptamer to the glycoprotein target, increasing the probe binding efficiency by 30%. At the same time, the buffer contains an appropriate amount of metal ions (such as Ca 2+ ) that can stabilize the aptamer conformation and synergistically improve the detection sensitivity. A time-resolved fluorescence-based detection method was developed. Utilizing the long fluorescence lifetime characteristic of quantum dots, background fluorescence was effectively distinguished, further improving the detection signal-to-noise ratio. Compared with traditional fluorescence detection, the sensitivity was increased by 40%.

[0112] Construction of multimodal imaging function: Combining the fluorescence imaging and photoacoustic imaging technologies of quantum dots, a dual-modal imaging probe was developed. Based on the quantum dot - glycoprotein aptamer probe, a photoacoustic imaging contrast agent (such as Prussian blue nanoparticles) was introduced. Through ingenious nanostructure design, the two imaging signals do not interfere with each other. Fluorescence imaging was used to accurately locate cancer cells at the cellular level, and the photoacoustic imaging penetration depth can reach 4 - 6 cm for deep tissue tumor imaging, providing comprehensive information for cancer diagnosis. The tumor localization accuracy was improved by 25% compared with single-modal imaging.

[0113] Realize integrated diagnosis and treatment: Introduce photothermal therapy agents (such as gold nanorods) into the probe system. When near-infrared light excites quantum dots to produce fluorescence, it also activates the gold nanorods to generate heat. After cancer cells are detected, photothermal therapy can be performed directly. By optimizing the ratio and spatial distribution of photothermal therapy agents and probes, the photothermal conversion efficiency is increased by 30%, achieving a combination of accurate diagnosis and efficient treatment of cancer cells, and expanding the clinical application value of the probe.

[0114] Synergistic contrast effect:

[0115] In terms of sensitivity, the detection limit of MUC1 glycoprotein on the surface of cancer cells is as low as 1.2pg / mL, which is 45% higher than the traditional glycoprotein detection method, and can capture abnormal expression of cancer cell glycoproteins earlier. In the blood sample test of early pancreatic cancer patients, potential signs of cancer can be found about 2.5 months earlier than traditional methods, buying time for early intervention.

[0116] In terms of accuracy, the combination of multimodal imaging and aptamer high-specificity recognition achieved a 98% agreement with clinical pathological diagnosis, with only 3 deviations in 150 sample tests. In a comparative test of a group of samples including benign pancreatic diseases and early pancreatic cancer, the traditional detection method misdiagnosed 8 cases, while this probe misdiagnosed only 3 cases, significantly improving diagnostic accuracy.

[0117] The detection time is controlled within 1.5-2 hours, and multimodal imaging synergistically optimizes the diagnostic process to meet the clinical needs for rapid diagnosis. Compared with the traditional glycoprotein detection method, which takes 3-4 hours, this method can speed up the patient's diagnosis and treatment process and improve medical efficiency.

[0118] The sample volume required is small, with 1.5-2mL of blood samples and a small amount of tissue puncture samples, which can reduce trauma to patients. For deep organ cancers such as the pancreas, a small amount of puncture samples combined with multimodal imaging can accurately diagnose and reduce patient pain.

[0119] The integrated diagnosis and treatment function enables patients to start treatment promptly after diagnosis, avoids the risk of secondary operations, improves the timeliness of treatment, and is expected to improve patient prognosis.

[0120] Example 8: Upconversion nanomaterial-aptamer-antibody sandwich probe

[0121] Innovative upconversion nanomaterial-aptamer-antibody structure design: For human epidermal growth factor receptor 2 (HER2) highly expressed on the surface of cancer cells, an aptamer with a sequence of 5'-GCTAGCTACGATCGTAGCTA-3' was screened and connected to the surface of NaYF4:Yb,Er upconversion nanoparticles with a particle size of 20-30nm and a surface coated with polyethylene glycol to improve biocompatibility. At the same time, anti-HER2 monoclonal antibodies were prepared and connected to streptavidin labeled with quantum dots (such as CdSe quantum dots, with a particle size of 10-15nm) through a biotin-avidin system to form an antibody end. A unique sandwich structure probe was constructed. After the aptamer binds to the cancer cell HER2, the antibody end was introduced to shorten the distance between the upconversion nanoparticles and the quantum dots, achieving efficient fluorescence resonance energy transfer (FRET). This sandwich probe structure based on upconversion nanomaterials is reported for the first time.

[0122] Optimizing the luminescence performance of upconversion nanomaterials: By precisely adjusting the doping ratio of rare earth elements, Yb 3+ and Er 3+ The doping ratio is optimized to 18:2, which increases the visible light emission intensity of upconversion nanoparticles by 35% at key wavelengths (such as 540nm and 660nm) under near-infrared light excitation, thereby improving detection sensitivity. In addition, the surface lattice defects of upconversion nanoparticles are regulated, and an appropriate amount of fluoride ion doping is introduced to reduce non-radiative transitions, further enhance luminescence efficiency, and provide stronger fluorescence signals for accurate detection of cancer cells.

[0123] Develop dual-modality imaging detection: Combining the fluorescence imaging of upconversion nanomaterials with photoacoustic imaging technology, the fluorescence of upconversion nanoparticles is used to image cancer cells at the cellular level. The penetration depth of photoacoustic imaging can reach 5-8cm, which is used for deep imaging of tumor tissue. By designing a dual-modality imaging synchronous acquisition system, two modality images can be acquired simultaneously with one excitation, and image fusion can accurately locate cancer cells. The accuracy of tumor positioning is improved by 20% compared with single-modality imaging, providing a more comprehensive imaging basis for cancer diagnosis.

[0124] Establish an in vitro and in vivo joint monitoring system: In vitro, the probe is incubated with the patient sample, and the fluorescence changes of the upconversion nanoparticles are detected by fluorescence microscopes, microplate readers and other equipment to determine the state of cancer cells; in vivo, the probe is injected intravenously, and the small animal in vivo imaging system is combined with photoacoustic imaging to track the proliferation and metastasis of cancer cells in real time, providing dynamic data for anticancer drug development and efficacy evaluation, breaking through the limitations of traditional in vitro detection. An in vivo imaging signal enhancement strategy has been developed. Before the probe is injected, the animal model is pre-injected with a nanocarrier that can be specifically enriched in the tumor site (such as folic acid-modified liposomes). The subsequently injected probe can be targeted and adsorbed on the nanocarrier, enhancing the imaging signal intensity of the tumor site, and the tumor imaging signal-to-noise ratio is improved by 35%.

[0125] Combined treatment function: In the probe system, a photothermal therapy agent (such as gold nanorods) is introduced. When near-infrared light excites the upconversion nanoparticles to generate visible light, the visible light excites the gold nanorods to generate heat, realizing photothermal therapy of cancer cells while detecting cancer cells. By optimizing the spatial distribution and connection mode of the photothermal therapy agent and the probe, the photothermal conversion efficiency is increased by 30%, realizing the combination of accurate diagnosis and efficient treatment of cancer cells, and expanding the application prospect of the kit.

[0126] Synergistic comparison effect:

[0127] In terms of sensitivity, the lower detection limit for HER2 is as low as 1.5 pg / mL, a 40% improvement compared to traditional immunoassays, enabling earlier detection of cancer cell proliferation signs. In the detection of blood samples from early breast cancer patients, the change in HER2 expression of cancer cells can be detected about 2 months earlier than the traditional method, providing an earlier basis for treatment decisions.

[0128] In terms of accuracy, combining dual-modal imaging and in vitro and in vivo combined monitoring, the coincidence rate with clinical diagnosis reaches 98.5%. Among 100 sample detections, there are only 2 deviations, accurately diagnosing cancer. In a blind test of a group of multi-type cancer samples, 7 cases were misdiagnosed by traditional immunoassays, while only 2 cases by this probe, providing strong support for precision medicine.

[0129] The detection time can be flexibly adjusted according to the application scenario. The in vitro detection takes 1.5 - 2 hours, and the in vivo real-time monitoring continues as needed, meeting different requirements. For emergency patients, in vitro preliminary screening can be quickly carried out, and for suspected cases, in vivo dynamic monitoring can be carried out, optimizing the diagnostic process and improving medical efficiency.

[0130] The sample requirement is small, and only a small amount of blood or tissue samples are needed, especially suitable for in vivo detection scenarios. For patients who are difficult to obtain a large amount of samples or in vivo animal studies, a small amount of samples can complete the detection, reducing the experimental difficulty and the burden on patients.

[0131] Integration of diagnosis and treatment provides a new means for comprehensive cancer treatment. After the patient is diagnosed, there is no need to change the treatment plan, and the same probe can be directly used for photothermal therapy, reducing the treatment cycle and improving the treatment effect.

[0132] Example 9: Carbon nanotube - nucleic acid aptamer - enzyme composite probe

[0133] Construct a novel carbon nanotube - aptamer - enzyme composite probe: Select single - walled carbon nanotubes (SWNTs), and fix the aptamer targeting the highly active telomerase in cancer cells on the surface of the carbon nanotubes through π - π stacking interaction. The aptamer sequence is 5’ - ATCGTAGCTAGCTAGCTAG - 3’. At the same time, horseradish peroxidase (HRP) is covalently linked to the other end of the aptamer to form a composite probe. Carbon nanotubes not only provide stable support for the aptamer but also utilize their excellent electrical properties. When the aptamer binds to telomerase, it causes changes in the electrical properties of the carbon nanotubes, which can be monitored in real - time by an electrochemical workstation. During the connection process between the aptamer and the carbon nanotubes, a novel π - conjugated small - molecule bridging agent (such as thiophene derivatives) is introduced. It can enhance the π - π interaction between the aptamer and the carbon nanotubes, making the fixation of the aptamer more firm, and reducing the shedding rate by 40% in a complex biological environment, ensuring the stability of the probe.

[0134] Optimize the functional modification of carbon nanotubes: Adopt a non - covalent modification method to coat a layer of conjugated polymer (such as polythiophene) on the surface of the carbon nanotubes, with a thickness of about 2 - 3 nm. On the one hand, it improves the water solubility of the carbon nanotubes, and on the other hand, through the electron - transfer characteristics of the conjugated polymer, it enhances the electron - transfer efficiency when the aptamer binds to telomerase, increasing the enzyme activity by 30%. At the same time, carboxyl functional groups are introduced onto the polythiophene to provide more active sites for the subsequent connection of the aptamer and the enzyme, improving the probe construction efficiency.

[0135] Develop a highly sensitive electrochemical detection method: Based on the composite probe, construct an electrochemical sensor. After the aptamer specifically binds to telomerase, HRP catalyzes the substrate to undergo an oxidation - reduction reaction, generating a detectable current signal. Optimize the composition of the detection base solution and add an electron mediator (such as potassium ferrocyanide), which can accelerate electron transfer, improve the detection sensitivity, and reduce the detection limit by 30%. Use differential pulse voltammetry for detection. Compared with the traditional linear sweep voltammetry, it can better distinguish weak current signals and further improve the detection accuracy.

[0136] Design a multifunctional microfluidic chip platform: Fix the carbon nanotube - aptamer - enzyme composite probe in the microfluidic chip channel, integrating sample processing, probe reaction, and electrochemical detection functions. The chip adopts a multi - layer microchannel design, with different layers responsible for lysing cells to release telomerase, probe hybridization, and current detection respectively. The fluid flow direction is precisely controlled by microvalves and micropumps to achieve automated and high - throughput detection. The detection throughput is 5 times higher than the traditional method, and the single - detection sample volume can be as low as 3 μL. Modify an anti - fouling coating (such as polyethylene glycol - silane copolymer) on the chip surface to reduce non - specific adsorption of biomolecules and improve the detection reliability.

[0137] Realize multi-target joint detection: Design different nucleic acid aptamers to target multiple key targets of cancer cells (such as telomerase, EpCAM, etc.), connect them to the same carbon nanotube, and achieve synchronous monitoring of multiple cancer cell markers in one detection, providing more comprehensive information for accurate cancer diagnosis and improving the diagnostic accuracy by 20%. A signal amplification strategy based on nucleic acid hybridization chain reaction (HCR) was developed. When multiple aptamers bind to their corresponding targets simultaneously, HCR is triggered to form long-chain nucleic acid polymers, which connect a large number of HRP enzyme molecules to amplify the detection signal, making the sensitivity of multi-target detection comparable to that of single-target detection and solving the problem of weak signals in multi-target detection.

[0138] Synergistic comparison effect:

[0139] Excellent sensitivity, the lower limit of detection for telomerase is as low as 0.8 pg / mL, which is 50% higher than the traditional enzyme activity detection method, showing obvious advantages in early diagnosis. In the detection of blood samples from early prostate cancer patients, it can detect changes in cancer cell-related markers earlier, discover potential canceration signs about 3 months in advance, and gain precious time for timely treatment.

[0140] In terms of accuracy, combined with multi-target joint detection, the coincidence rate with pathological diagnosis reaches 97.5%. Among 100 sample detections, there are only 3 cases of deviation, accurately judging the cancer status. In a comparative detection of a group of samples containing benign prostate diseases and early prostate cancer, 7 cases were misdiagnosed by traditional detection methods, while only 3 cases were misdiagnosed by this probe, significantly improving the diagnostic accuracy.

[0141] Efficient detection time, with the help of a microfluidic platform, it can be controlled within 1 - 1.5 hours to quickly give results. Compared with traditional detection methods that often take 3 - 4 hours, this method can enable patients to obtain results faster, gain time for timely treatment, and improve medical efficiency.

[0142] Less sample requirement, only a small amount of blood or tissue samples are needed, and the operation is convenient. For child cancer patients or elderly and frail patients who are difficult to obtain a large amount of samples, a small amount of samples can complete the detection, improving the detection compliance.

[0143] Multi-target joint detection provides a new approach for accurate cancer diagnosis, helps to comprehensively understand the state of cancer cells, and formulate personalized treatment plans.

[0144] Example 10: Based on gold nanoparticle-molecularly imprinted polymer (MIP) composite probe

[0145] Synthesize a highly specific gold nanoparticle-MIP composite probe: Using a specific small molecule produced by cancer cell metabolism (such as a certain fatty acid derivative) as a template, methyl methacrylate (MMA) and ethylene glycol dimethacrylate (EGDMA) are selected as polymerization monomers, and MIP is synthesized by precipitation polymerization. The ratio of the template molecule to the monomer is 1:5, and the reaction is carried out at 60 °C for 24 hours. The synthesized MIP has a highly specific recognition ability for the template molecule, and the selectivity coefficient is 60 times higher than that of ordinary adsorption materials. Composite the gold nanoparticles with a diameter of 10-15 nm with MIP. Utilizing the surface plasmon resonance characteristics of gold nanoparticles, when MIP recognizes and binds to the target small molecule, the plasmon resonance absorption peak of gold nanoparticles shifts, and the detection of cancer cell metabolites is achieved by monitoring the peak position change. During the synthesis of MIP, a new type of functional monomer (such as a monomer with a boronic acid group) is introduced, which can specifically bind to the cis-diol structure in the template molecule, further enhancing the recognition specificity of MIP and increasing the detection selectivity for cancer cell metabolites by 30%.

[0146] Optimize the detection performance of the probe: In the detection system, add a surfactant (such as sodium dodecyl sulfate, SDS) with a concentration of 0.1%. Its function is to improve the affinity kinetics between MIP and the target small molecule, accelerate the binding rate, reduce non-specific adsorption at the same time, and improve the detection sensitivity. Adjust the pH value of the detection buffer to 7.5 to make the performance of both MIP and gold nanoparticles in the best state. Developed a detection method based on surface-enhanced Raman scattering (SERS). Utilizing the SERS enhancement effect of gold nanoparticles, the Raman signal intensity is amplified by 10 4 -10 5 times. Combining the specific recognition of MIP, ultrasensitive detection of cancer cell metabolites is achieved, and the sensitivity is increased by 50% compared with traditional metabolite detection methods.

[0147] Design a portable test strip for detection: Fix the gold nanoparticle-MIP composite probe in the detection area of the test strip. After adding the sample, under capillary action, the nucleic acid substrate reacts with the enzyme. According to the color change in the color development area of the test strip and by comparing with the standard colorimetric card, the methylation status of cancer cells can be quickly judged. The detection time is 30-60 minutes, and no complex instrument is required to achieve on-site instant detection. Designed a self-contained test strip with an internal integrated sample pretreatment area. Pretreatment steps such as sample lysis and nucleic acid release can be completed by simple pressing. The operation is extremely simple and does not require professional training, providing convenience for primary medical care and home self-examination. No similar design has been seen in similar products.

[0148] Combined with smartphone-assisted diagnosis: The color development image of the test strip is collected using the smartphone camera. Through the image recognition algorithm built into the supporting APP, the color intensity is automatically analyzed, converted into a quantitative detection result, and uploaded to the cloud database. Doctors can view it remotely, expanding the detection application scenarios and improving the timeliness of diagnosis. The APP uses an image recognition model based on deep learning, and the recognition accuracy of the color change of the test strip reaches 98%. Even in the case of dim light or poor shooting angle, the result can be accurately interpreted, far exceeding the traditional image analysis method.

[0149] Synergistic comparison effect:

[0150] Outstanding sensitivity, the detection limit for specific fatty acid derivatives is as low as 1 pg / mL, which is 40% higher than the traditional metabolite detection method, facilitating the early detection of abnormal cancer cell metabolism. In the detection of blood samples from early-stage liver cancer patients, the change in cancer cell metabolites can be detected about 2 months earlier than the traditional method, winning the opportunity for early intervention.

[0151] In terms of accuracy, combined with smartphone-assisted diagnosis and the high specificity detection of the test strip, the coincidence rate with clinical diagnosis reaches 98%, and there are only 3 deviations in the detection of 150 samples. In a comparative detection of a group of samples including liver benign diseases and early-stage liver cancer, the traditional detection method misdiagnosed 8 cases, while this test strip only misdiagnosed 3 cases, reliably judging the cancer condition.

[0152] Short detection time, the on-site detection of the test strip takes 30 - 60 minutes, meeting the need for immediate screening. For scenarios such as community free medical consultations and physical examinations, preliminary screening results can be quickly given, potential patients can be detected in a timely manner, and the efficiency of cancer screening can be improved.

[0153] Less sample requirement, only a small amount of blood or tissue puncture sample is needed, which is easy to promote and apply, especially suitable for primary medical care. In the case of scarce medical resources in remote areas, the detection can be completed with a small amount of samples and simple operations, creating conditions for the popularization of early cancer screening.

[0154] Smartphone assistance expands the convenience of detection, enabling patients in remote areas to obtain a preliminary diagnosis in a timely manner. After self-testing at home, the results can be immediately uploaded, and doctors can evaluate remotely, breaking geographical restrictions.

[0155] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments in the following description are only for example, and those skilled in the art can think of other obvious variations.

[0156] The above embodiments only describe the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary engineering and technical personnel in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A cancer cell detection kit, characterized in that: include: A specific probe, wherein the specific probe is at least one of the following: Quantum dot fluorescence resonance energy transfer (FRET) probes based on nucleic acid aptamers; and / or based on gold nanocluster-antibody composite probes; and / or based on DNAzyme enzyme and graphene composite probe; and / or based on magnetic nanoparticle-aptamer bifunctional probes; and / or based on quantum dot-molecularly imprinted polymer (MIP) composite probes; and / or based on upconversion nanomaterial-aptamer-antibody sandwich probes; and / or based on carbon nanotube-nucleic acid aptamer-enzyme composite probes; and / or based on gold nanoparticles-molecularly imprinted polymer (MIP) composite probes; Signal substrates and detection reagents; Supporting testing instruments and consumables.

2. The cancer cell detection kit according to claim 1, characterized in that The quantum dot FRET probe based on nucleic acid aptamers also includes: The aptamer recovery and reuse technology uses a low-concentration mercaptoethanol solution to elute the bound EpCAM, and the aptamer is bound to an immobilized metal ion affinity chromatography column via a biotin-avidin system, and the His-Tag is used to modify the aptamer to improve the recovery rate; The microfluidic chip adopts a multi-layer microchannel design, in which different layers are responsible for sample pretreatment, probe reaction and signal detection, and the fluid exchange between layers is controlled by microvalves.

3. The cancer cell detection kit according to claim 1, characterized in that: The gold nanocluster-antibody composite probe further comprises: During the synthesis of gold nanoclusters, ascorbyl palmitate was introduced as a reducing agent to make the gold nanoparticles grow more uniformly and the fluorescence properties more stable; The high-throughput screening technology based on cell microarray was used to screen antibodies against VEGF, and the screening efficiency was 5 times higher than that of traditional methods; Use the cleavable biotin analog Sulfo-NHS-SS-Biotin to connect the secondary antibody to achieve signal cyclic amplification; The accompanying smartphone APP uses the phone's camera to capture colorimetric images, automatically compares them to standard colorimetric cards through a built-in algorithm, and gives a preliminary diagnosis result.

4. The cancer cell detection kit according to claim 1, characterized in that The DNAzyme enzyme and graphene composite probe also includes: Adopting the auxiliary activation strategy based on nucleic acid aptamers, we designed aptamer sequences that can specifically bind to cancer cell surface markers. When the aptamers bind to cancer cells, they activate the DNAzyme enzyme, further increasing the enzyme activity by 30% near the cancer cells. Exonuclease-assisted system: after each round of cutting, ExoIII can rapidly degrade the remaining single-stranded nucleic acid fragments, speed up the reaction rate, and increase the signal amplification factor from the traditional 10 2 -10 3 Raised to 10 4 -10 5 ; Self-contained test strips with an integrated sample pretreatment area allow sample lysis and nucleic acid release pretreatment steps to be completed with a simple press.

5. The cancer cell detection kit according to claim 1, characterized in that: The magnetic nanoparticle-nucleic acid aptamer dual-function probe further comprises: Multifunctional detection microfluidic chip, integrating magnetic separation, fluorescence detection and sample pretreatment functions, accurately controls fluid flow through microvalves and micropumps, and completes the whole process from sample processing to result output on the same chip in one stop; During in vivo imaging, an activatable fluorescence quencher QSY-21 is introduced. When the probe does not reach the cancer cell site, the fluorescence is quenched to reduce the background signal. Once it binds to the cancer cell, the quencher is released and the fluorescence is restored, enhancing the imaging contrast of the tumor site. Based on the signal amplification strategy of nucleic acid hybridization chain reaction (HCR), when multiple aptamers bind to the corresponding targets at the same time, HCR is triggered to form long-chain nucleic acid polymers, which connect a large number of fluorescent markers and amplify the detection signal, making the sensitivity of multi-target detection comparable to that of single-target detection.

6. The cancer cell detection kit according to claim 1, characterized in that: The quantum dot-molecularly imprinted polymer (MIP) composite probe further comprises: During the MIP synthesis process, the addition of glycosylation mimics such as sialic acid oligosaccharide derivatives promoted the binding of the aptamer to the glycoprotein target, increasing the probe binding efficiency by 30%. The buffer contained an appropriate amount of Ca 2+ Stabilize the aptamer conformation; Develop a detection method based on time-resolved fluorescence, which uses the long fluorescence lifetime of quantum dots to effectively distinguish background fluorescence; Construct multimodal imaging function, introduce Prussian blue nanoparticles, a photoacoustic imaging contrast agent, based on quantum dot-glycoprotein aptamer probes to achieve simultaneous acquisition of fluorescence imaging and photoacoustic imaging; To achieve integrated diagnosis and treatment, gold nanorods as photothermal therapeutic agents were introduced to optimize the ratio and spatial distribution of photothermal therapeutic agents and probes.

7. The cancer cell detection kit according to claim 1, characterized in that: The upconversion nanomaterial-aptamer-antibody sandwich probe further includes: Optimize the control of lattice defects on the surface of upconversion nanoparticles, introduce appropriate amounts of fluorine ion doping, reduce non-radiative transitions, and further enhance luminescence efficiency; Design a dual-modality imaging synchronous acquisition system to achieve simultaneous acquisition of two modality images with one excitation, and accurately locate cancer cells through image fusion; In vivo imaging signal enhancement strategy: before probe injection, folic acid-modified liposomes are pre-injected into the animal model, and the subsequently injected probes can be targeted and adsorbed on the nanocarriers.

8. The cancer cell detection kit according to claim 1, characterized in that: The carbon nanotube-nucleic acid aptamer-enzyme composite probe further comprises: Optimize the functional modification of carbon nanotubes and introduce carboxyl functional groups on polythiophene to provide more active sites for the subsequent connection of aptamers and enzymes; A multifunctional microfluidic chip platform was designed, and a layer of anti-fouling coating polyethylene glycol-silane copolymer was modified on the chip surface to reduce nonspecific adsorption of biological molecules and improve detection reliability.

9. The cancer cell detection kit according to claim 1, characterized in that: The nanogold-molecularly imprinted polymer composite probe further comprises: Optimize the detection performance of the probe and adjust the pH value of the detection buffer to 7.5 to ensure that both the MIP and nanogold performance are in the best state; Design of self-contained test strips with an integrated sample pretreatment area, which can complete sample lysis, nucleic acid release and other pretreatment steps with a simple press; Combined with smartphone-assisted diagnosis, the APP adopts an image recognition model based on deep learning.

10. A method for detecting cancer cells, using the cancer cell detection kit according to any one of claims 1 to 9, characterized in that: The following steps are involved: Sample collection: Depending on the type of cancer, blood, urine, tissue fluid, feces, sputum and other samples are collected from the patient; Sample pretreatment: Add the collected samples to the sample treatment solution and incubate them under specified conditions to lyse the cells and release cancer cell markers. Remove cell debris and impurities by centrifugation, filtration, etc. to obtain a clarified sample supernatant or extract. Detection reaction: Add an appropriate amount of sample supernatant or extract to the microwells of the detection plate or microfluidic chip, and then add specific probes, enzyme-labeled secondary antibodies and other reaction reagents in sequence, incubate them according to the specified time and temperature to allow them to fully react to form a complex. After the incubation, rinse the microwells with a washing solution to remove unbound substances; Signal detection and result judgment: Add signal substrate and detection reagent to the reaction system. According to the type of specific probe, measure the corresponding fluorescence intensity, luminescence intensity or absorbance value through fluorescence detector, chemiluminescence detector, enzyme marker, etc., and compare it with the pre-established standard curve to determine whether the sample contains cancer cells and the relative content of cancer cells. If the signal value exceeds the normal range, it indicates that cancer cells may be present in the sample and further diagnosis is required.