Rare earth nano probe, preparation method and application

Abstract

The invention discloses a rare earth nanoprobe, a preparation method and application thereof, and belongs to the technical field of biomedical detection.According to the technology, the rare earth nanoprobe with the high fluorescence quantum yield is synthesized, specific recognition of tumor markers is achieved through the aptamer technology, and the detection sensitivity and selectivity are remarkably improved. The fluorescence signal amplification effect of the rare earth nanoprobe and the modern fluorescence detection technology are innovatively combined, so that simultaneous detection of multiple tumor markers such as carcino-embryonic antigen (CEA), prostate specific antigen (PSA) and alpha fetoprotein (AFP) becomes possible. According to the method, the detection limit reaches the pg / mL level, the operation is simple and convenient, the cost is low, and the feasibility of clinical application is greatly improved. Besides, through multiple detection design, the detection efficiency and accuracy are further improved, an effective tool is provided for early diagnosis, curative effect monitoring and prognosis evaluation of tumors, and wide clinical application prospects and market potential are shown.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical detection, and particularly relates to a rare earth nanoprobe, a preparation method and an application thereof. Background Art

[0002] Tumor markers, as important indicators for early cancer diagnosis, play a crucial role in clinical detection. Existing tumor marker detection technologies mainly include methods such as enzyme-linked immunosorbent assay (ELISA) and chemiluminescence immunoassay (CLIA). These technologies can, to a certain extent, achieve the detection of tumor markers, but generally suffer from problems such as low sensitivity, limited detection range, complex operation, and high cost, which limit their application in early cancer screening and clinical diagnosis.

[0003] With the rapid development of nanoscience and biotechnology, bio-detection technologies based on nanomaterials have gradually become a research hotspot. Rare earth nanomaterials are widely regarded as having great application potential in the field of bio-detection due to their unique optical properties, such as high fluorescence quantum yield, tunable emission wavelength, and good biocompatibility. In particular, rare earth nanoprobes have shown their unique advantages in aspects such as bio-labeling and imaging-guided surgery.

[0004] Applying rare earth nanoprobes to the detection of tumor markers still faces some challenges. For example, how to improve the stability and biocompatibility of the probes, how to achieve the simultaneous detection of multiple tumor markers, and how to improve the sensitivity and accuracy of detection. In addition, existing detection technologies often require complex sample pretreatment and professional operators, which to a certain extent limits their popularization in clinical applications. Summary of the Invention

[0005] In view of the above problems, the present invention proposes a rare earth nanoprobe, a preparation method and an application thereof, aiming to achieve highly sensitive, highly specific, low-cost and simple-operation detection of tumor markers through innovative probe design and fluorescence detection methods, so as to play an important role in early cancer diagnosis and treatment monitoring.

[0006] In a first aspect, the present invention provides a preparation method of a rare earth nanoprobe, comprising the following steps:

[0007] Step 1: Select rare earth elements;

[0008] Step 2: Use nanomaterial synthesis technology to achieve uniform synthesis of rare earth nanoparticles;

[0009] Step 3: Perform surface modification on the rare earth nanoparticles to obtain a probe-aptamer complex.

[0010] In some embodiments, in step 1, according to the requirements of fluorescence characteristics, rare earth elements such as europium (Eu), terbium (Tb), or neodymium (Nd) are selected. These elements exhibit excellent fluorescence properties due to their unique 4f electron transitions and are suitable as fluorescence labels for biological detection.

[0011] In some embodiments, in step 2, after the element selection, nanomaterial synthesis techniques such as chemical co-precipitation method, thermal decomposition method, or solvothermal synthesis method are used to precisely control the reaction conditions, including reactant concentration, temperature, pH value, and reaction time, etc., to achieve the uniform synthesis of rare earth nanoparticles. These methods can ensure the consistency of the size and morphology of the nanoparticles, laying a foundation for the subsequent optimization of fluorescence characteristics. The synthesized rare earth nanoparticles are size-selected by a series of physical methods, such as ultracentrifugation technology, to ensure the uniformity of particle size. In addition, by adjusting the parameters in the synthesis process, such as the reaction solvent, the type and concentration of surfactants, precise control of the shape of the nanoparticles is achieved, and their fluorescence characteristics are optimized, such as spherical, rod-shaped, or polyhedral, etc. Finally, the fluorescence characteristics of the rare earth nanoparticles are optimized. By finely adjusting the doping ratio of rare earth elements, the thickness and composition of the surface modification layer, precise control of parameters such as the fluorescence spectrum, quantum yield, and fluorescence lifetime of the particles is achieved. This step ensures that the rare earth nanoprobe has a high fluorescence quantum yield and good photostability, meeting the requirements of high-sensitivity detection.

[0012] In some embodiments, the surface modification of rare earth nanoparticles in step 3 is specifically operated as follows:

[0013] Step 301: Disperse the rare earth nanoparticles in an ethanol solution with a pH value of 4.0, and add tetraethoxysilane as a silicon source at 60 °C. A SiO2 layer is formed through an acid-base catalyzed reaction. The thickness of the SiO2 layer is 5 - 10 nm, which can ensure that it does not significantly affect the fluorescence characteristics of the rare earth nanoparticles and at the same time provides good biological stability;

[0014] Step 302: On the basis of SiO2 coating, use atom transfer radical polymerization (ATRP) technology to graft biocompatible polymers such as polyethylene glycol (PEG) or poly(lactic-co-glycolic acid) (PLGA) on the surface of the rare earth nanoparticles. These polymers not only increase the water solubility of the particles but also reduce non-specific protein adsorption by forming a hydrophilic outer shell;

[0015] Step 303: Graft lipoic acid or oleic acid on the surface of the SiO2 / PEG-modified rare earth nanoparticles through an amidation reaction.

[0016] In some embodiments, oleic acid is mixed with rare earth nanoparticles in a phosphate buffer solution (PBS) with a pH value of 7.4, and oleic acid is stably grafted onto the SiO2 coating layer by forming amide bonds or ester bonds. This grafting improves the dispersibility of the particles and provides protection against aggregation and biological oxidation. After the surface modification is completed, techniques such as Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and transmission electron microscopy (TEM) are used to characterize the modification effect. These characterization results verify the successful coating of the SiO2 layer and the polymer layer, as well as the successful grafting of the surfactant, ensuring the performance of the rare earth nanoprobes in subsequent biological applications.

[0017] In some embodiments, the specific operation of step 302: Using 0.1 M CuBr2 / Me6TREN as the catalyst system, the polymerization reaction is carried out at 80 °C, and the molecular weight of PEG is controlled within the range of 5000 to 20000 g / mol to achieve the best water solubility and biocompatibility.

[0018] In some embodiments, the specific operation of grafting oleic acid onto the surface of SiO2 / PEG-modified rare earth nanoparticles in step 303: In a phosphate buffer solution with a pH value of 7.4, the concentration of oleic acid is controlled at 1 mM, and the reaction is carried out at room temperature for 24 hours to ensure that oleic acid molecules are stably grafted onto the SiO2 / PEG layer through amide bonds or ester bonds.

[0019] In a second aspect, the present invention also provides a rare earth nanoprobe, which is prepared by using a preparation method of a rare earth nanoprobe.

[0020] In a third aspect, the present invention also provides a rare earth nanoprobe as a probe reagent for detecting anti-tumor markers in the body.

[0021] In some embodiments, the use steps of the probe reagent are as follows:

[0022] Step 1-1: Synthesize the aptamer and fix the synthesized aptamer on the surface of the rare earth nanoparticles through chemical bonds and / or non-covalent interactions.

[0023] Step 1-2: Mix the probe-aptamer complex with the sample to be tested, and allow the aptamer to specifically bind to the tumor marker in the sample to form a complex-tumor marker conjugate.

[0024] Step 1-3: Use fluorescence detection technology to collect and analyze the fluorescence signal of the complex-tumor marker conjugate.

[0025] Step 1-4: Quantitatively analyze the concentration of the tumor marker according to the change in the intensity of the fluorescence signal.

[0026] Steps 1-5: Distinguish the fluorescence signals of different probe-aptamer complexes through multi-color fluorescence detection technology to achieve the simultaneous detection of at least two tumor markers.

[0027] In some embodiments, the synthesis of the aptamer in Step 1-1 starts from a carefully designed single-stranded DNA or RNA sequence, which is optimized for the active site of a specific tumor marker through computational simulation and literature research. The synthesis of the aptamer adopts solid-phase synthesis technology, which gradually adds nucleotides on an insulated solid-phase carrier. Using phosphate-buffered saline (PBS) with controlled pH value as the reaction medium, through an automatic DNA synthesizer, phosphodiester bonds are gradually added according to the designed sequence to accurately synthesize the aptamer. During the solid-phase synthesis process, the formation efficiency of phosphodiester bonds in each step is controlled above 98% to ensure the accuracy of aptamer synthesis. In addition to solid-phase synthesis, liquid-phase synthesis methods are also used to synthesize aptamers, especially when synthesizing longer or more complex-structured aptamers. Liquid-phase synthesis is carried out in solution and purified by high-performance liquid chromatography (HPLC) to ensure that the purity of the synthesized aptamer is higher than 90% and avoid non-specific binding. The synthesized aptamer is fixed on the surface of the rare-earth nanosensor through chemical bonding or non-covalent interactions. Using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as activators, the carboxyl group of the aptamer forms a stable amide bond with the amino group modified on the surface of the rare-earth nanosensor. The reaction is carried out at room temperature, and the pH value is controlled between 7.0 and 7.4 to optimize the bonding efficiency. For aptamers containing sulfhydryl groups, stable covalent bonds are formed with lipoic acid or cysteine residues on the surface of the rare-earth nanosensor through disulfide bond fixation technology. In PBS containing 10 mM 2-mercaptoethanol, the pH value is adjusted to 8.0 to promote the formation of disulfide bonds and enhance the binding stability between the aptamer and the sensor. In addition to covalent bonding, non-covalent interactions such as hydrogen bonds, π-π stacking, and van der Waals forces are also used for the fixation of aptamers. By adjusting solution conditions such as ionic strength and polarity, the adsorption and orientation of aptamers on the sensor surface are optimized to achieve non-covalent fixation. After the aptamer is fixed, techniques such as enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), or atomic force microscopy (AFM) are used to evaluate the fixation effect. By measuring the immobilization amount, orientation, and activity of the aptamer, the fixation conditions are optimized to ensure the uniform distribution and biological activity of the aptamer on the sensor surface. Through this series of fine steps of aptamer synthesis and fixation, this technical solution realizes the effective binding of the aptamer and the rare-earth nanosensor, providing a key biorecognition unit for the highly sensitive and highly specific detection of tumor markers.

[0028] In some embodiments, specific recognition and complex formation in Step 1-2: In the stage of specific recognition and complex formation, first, the aptamer-modified rare earth nanoprobes are mixed with the sample to be tested. The mixing process is carried out under precisely controlled temperature and pH conditions. Generally, the pH value is maintained at 7.2 to 7.4, and the temperature is controlled at 25 to 37 °C. These conditions are conducive to the maximum binding affinity between the aptamer and the tumor marker. To ensure the specific binding between the aptamer and the tumor marker, the incubation time of the mixing system is strictly controlled. Generally, the incubation time is set to 30 minutes to 2 hours to ensure sufficient time to reach the binding equilibrium. At the same time, by optimizing the concentration of the rare earth nanoprobes, it is ensured that there are sufficient probe molecules around each tumor marker molecule to improve the detection sensitivity. After specific binding, the stability of the formed probe-aptamer-tumor marker complex is evaluated by techniques such as dynamic light scattering (DLS) and ultraviolet-visible spectroscopy (UV-Vis). DLS is used to monitor the particle size change of the complex, and UV-Vis is used to monitor the absorption spectrum change caused by the complex formation to ensure the stability of the complex during subsequent detection. To avoid the influence of non-specific binding on the detection results, an appropriate blocker, such as bovine serum albumin (BSA) or gelatin, is added to the buffer to cover the surface of the unreacted probes. In addition, the unbound probes and background molecules are removed by multiple washing steps to further improve the detection specificity. After forming a stable probe-aptamer-tumor marker complex, an appropriate separation technique, such as magnetic bead separation or ultracentrifugation, is used to separate and enrich the complex from the sample. For example, using the ultracentrifugation technique, centrifuging at a speed of 100,000 g for 30 minutes effectively precipitates the complex. Finally, the fluorescence characteristics of the complex, including parameters such as fluorescence intensity, peak wavelength, and fluorescence lifetime, are analyzed using a fluorescence spectrometer. By comparing the fluorescence changes before and after adding the sample, the concentration of the tumor marker is quantitatively analyzed.

[0029] In some embodiments, the detection limit of the probe reagent reaches the picogram / milliliter level.

[0030] To achieve multi-target detection, rare earth nanoprobes with different fluorescence characteristics are designed and synthesized. These probes absorb light of different wavelengths and emit fluorescence of different colors by doping different rare earth elements, such as europium (Eu), terbium (Tb), neodymium (Nd), etc. For example, Eu 3+ ions produce red light at 612 nm, Tb 3+ ions produce green light at 545 nm, and Nd 3+Ions generate near-infrared light at 880 nm. Specific aptamers are designed for each rare-earth nanosensor, and these aptamers have high affinity and specificity for specific tumor markers through sequence optimization. The diversity of aptamers ensures specific recognition of different tumor markers without interfering with each other. Using multi-color fluorescence detection technology, the fluorescence signals of different probe-aptamer complexes can be simultaneously excited and detected. By setting different excitation wavelengths and emission filters, the fluorescence signals of different probes can be distinguished and measured. For example, using a three-color fluorescence microscope or flow cytometer, the fluorescence signals of Eu3+, Tb3+ and Nd3+ probes can be simultaneously detected. In multi-target detection, different probe-aptamer complexes are distinguished by the peak wavelength differences of the fluorescence signals. By quantitatively analyzing the intensity of each fluorescence signal, the concentration of the corresponding tumor marker can be determined. For example, by measuring the fluorescence intensities at 612 nm, 545 nm and 880 nm, the concentrations of three tumor markers, CEA, PSA and AFP, can be quantitatively analyzed. To ensure the accuracy and repeatability of multi-target detection, the experimental conditions are precisely controlled. This includes pH value, temperature, reaction time, and the concentrations of the probes and aptamers. For example, maintaining the pH value at 7.2, the temperature at 37 °C, the reaction time controlled at 60 minutes, and the concentrations of the probes and aptamers within the optimized range. Finally, the multi-color fluorescence data collected are analyzed by professional software to establish a standard curve for each tumor marker to achieve quantitative analysis. By comparing the fluorescence signal intensity of the sample with the standard curve, the concentration of the tumor marker is calculated and clinical diagnosis or biomedical research is carried out based on the results.

[0031] The detection process is extremely simple and easy to perform, avoiding the complex sample pretreatment steps required in traditional detection methods and not relying on expensive or professional detection equipment, making the whole operation process more economical and efficient. This simplicity greatly reduces the technical threshold, making the detection process applicable not only to well-resourced clinical settings, but also to research laboratories, and even can be extended to point-of-care testing (POCT) and areas with limited resources, greatly expanding the accessibility and application scope of tumor marker detection.

[0032] By optimizing each step, the goal of rapid detection is achieved. From the sample collection to the final fluorescence signal detection, the whole process is designed to be completed within two hours. This rapidity is achieved through careful planning and experimental verification to ensure that each step can be executed in the most efficient way. The sample processing steps are simplified and accelerated to reduce the overall detection time. For example, by using ready-to-use buffers and pre-treated reagents, the sample preparation time is significantly shortened. In addition, the aptamer-modified rare earth nanoprobes have the characteristic of rapid binding, enabling the specific recognition process with tumor markers to be completed within 30 minutes. By selecting aptamers with high affinity and optimizing the concentration of rare earth nanoprobes, the formation process of the complex is accelerated. Under suitable conditions, the formation of the probe-aptamer-tumor marker complex can be completed within 1 hour, providing the possibility for rapid detection. The fluorescence signal detection step is also designed to be rapid and efficient. By using highly sensitive detection devices such as photomultiplier tubes (PMT) or charge-coupled devices (CCD), the fluorescence signal acquisition can be completed within a few minutes. In addition, through automated software control, the signal acquisition, processing, and data analysis are automated, further improving the detection speed. Each step in the entire detection process is coordinated to ensure rapid and accurate results. From the addition of the sample to the reading of the fluorescence signal, each step is precisely timed and controlled to avoid unnecessary waiting and delays, ensuring the continuity and efficiency of the detection.

[0033] In a fourth aspect, the present invention also provides a detection kit, which includes rare earth nanoprobes, reaction buffers, standards, and control samples.

[0034] Compared with the prior art, the present invention has the following advantages:

[0035] 1. Significantly improved detection sensitivity: The rare earth nanoprobes used in the technical solution of the present invention, through fine-tuning of their core-shell structure and surface modification, achieve ultrasensitive in vitro detection of tumor markers such as carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), and alpha-fetoprotein (AFP), with detection limits reaching 0.15, 0.62, and 70 pg / mL respectively, which is 2-3 orders of magnitude higher than that of traditional DELFIA kits. An extremely high fluorescence quantum yield is achieved, and with the precise recognition of aptamers, the detection sensitivity is as high as 91.36% to 94.33%. The realization of this ultra-high sensitivity gives this technical solution significant advantages in the field of early tumor diagnosis, enabling the detection of trace tumor markers that are difficult to detect by traditional methods, providing strong technical support for the early detection of cancer.

[0036] 2. Highly simplified operation process and rapid detection capability: This technical solution optimizes the detection process, highly integrates and simplifies the steps of sample processing, probe-aptamer complex binding, fluorescence signal collection and analysis, etc., making the entire detection process not only easy to operate, but also fast and efficient. From sample collection to result output, the entire process can be completed within two hours, greatly shortening the detection cycle and improving detection efficiency. This rapid response capability is of great significance for clinical situations where rapid diagnosis is urgently needed, and can provide doctors with timely diagnostic information and speed up treatment decisions.

[0037] 3. Significant cost-effectiveness and wide application range: This technical solution reduces the detection cost by reducing the dependence on professional equipment, making high-sensitivity tumor marker detection more economical and practical. At the same time, due to the diversity and designability of aptamers, this technical solution has good versatility and can be customized for different tumor markers to achieve simultaneous detection of multiple tumor markers. This cost-effectiveness and wide application range make this technical solution not only suitable for clinical and testing in medical institutions, but also for large-scale cancer screening and health examinations, and has broad market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a step diagram of the tumor marker detection technology method of the present invention. DETAILED DESCRIPTION

[0039] The preferred embodiments of the present invention are described in detail below so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0040] Example 1

[0041] This embodiment provides a method for preparing a rare earth nanoprobe, such as Figure 1 As shown, the specific implementation is as follows:

[0042] Preparation process of rare earth nanoprobes: In the preparation process of rare earth nanoprobes, first, rare earth elements are carefully selected according to specific requirements of fluorescence applications. In this technical solution, rare earth elements such as europium (Eu), terbium (Tb), or neodymium (Nd) are selected. Due to the transition characteristics of their 4f electrons, these elements exhibit excellent fluorescence properties. For example, europium (Eu) is commonly used in bioimaging due to its strong red light emission, while terbium (Tb) is favored for its green fluorescence characteristics. After the rare earth elements are selected, synthesis is carried out at 90 °C by chemical coprecipitation method, and the pH value of the solution is precisely controlled to 10.5 to ensure the uniform precipitation of rare earth ions. During the synthesis process, the growth of nanoparticles is precisely controlled by adjusting the concentration ratio of reactants and reaction time to achieve size consistency. For example, by controlling the concentration of europium nitrate in the range of 0.01 M to 0.1 M, uniform Eu2O3 nanoparticles with a diameter of 5 to 10 nanometers can be synthesized. The synthesized rare earth nanoparticles are size-selected by ultracentrifugation technology to ensure the uniformity of particle size. The ultracentrifugation speed is set to 100,000 g, and the centrifugation time is 1 hour to separate the nanoparticles within the required size range. In addition, by adjusting the concentration of surfactants such as oleic acid to 0.1% during the synthesis process, the shape of nanoparticles can be controlled, such as the transformation from spherical to rod-shaped. Based on size and shape control, the fluorescence characteristics of rare earth nanoparticles are further optimized. By adjusting the doping ratio of rare earth elements, for example, controlling the doping ratio of europium (Eu) between 1% and 5%, the fluorescence quantum yield of nanoparticles can be significantly improved. At the same time, by coating a 5- to 10-nanometer-thick silica (SiO2) protective layer on the surface of nanoparticles, not only the biological stability of the particles is improved, but also the fluorescence efficiency is further enhanced by restricting the non-radiative relaxation of fluorescence quanta.

[0043] Surface modification of rare earth nanoprobes: To enhance the stability of rare earth nanoparticles in vivo and reduce non-specific adsorption, this technical solution takes a series of precisely controlled surface modification steps. These steps include coating with silica (SiO2), grafting of polymer layers, and chemical modification of surfactants.

[0044] The rare earth nanoparticles were coated with SiO2 by the sol-gel method. In this process, the rare earth nanoparticles were dispersed in an ethanol solution with a pH value of 4.0, and 0.05 M tetraethyl orthosilicate (TEOS) was added as the silicon source at 60 °C. The SiO2 layer was formed through an acid-base catalyzed reaction. The thickness of this layer was precisely controlled by adjusting the concentration of TEOS and the reaction time. The specific method was as follows: The rare earth nanoparticles were dispersed in an ethanol solution with a pH value of 4.0. At 60 °C, different concentrations of TEOS solutions were added dropwise and the timing was started. A series of TEOS solutions with different concentrations were prepared, such as 0.01 M, 0.05 M, 0.1 M, etc. For each concentration, the required reaction time to achieve the expected SiO2 layer thickness was recorded. For each TEOS concentration, the growth of the SiO2 layer at different reaction times was observed. Generally, the longer the reaction time, the thicker the SiO2 layer, but it may also affect the fluorescence properties of the rare earth nanoparticles. Techniques such as Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and transmission electron microscopy (TEM) were used to characterize the thickness and uniformity of the SiO2 layer. The effects of different TEOS concentrations and reaction times on the thickness of the SiO2 layer were analyzed. According to the characterization results, the SiO2 layer thickness in the range of 5 - 10 nm was selected. The optimal ratio of TEOS concentration and reaction time was determined to ensure that the thickness of the SiO2 layer was uniform and did not affect the fluorescence properties of the rare earth nanoparticles. The fluorescence properties of the rare earth nanoparticles modified with the optimized SiO2 layer were tested. Ensure that the fluorescence quantum yield and stability meet the detection requirements. Repeat the above experiment at least three times and take the average value. Record all experimental conditions and results, and analyze the data to determine the optimal ratio of TEOS concentration and reaction time. Generally, it is controlled within 5 - 10 nm to ensure that it does not affect the fluorescence properties of the rare earth nanoparticles.

[0045] On the basis of the SiO2 coating, polyethylene glycol (PEG) was further grafted through atom transfer radical polymerization (ATRP) technology. In this step, 0.1 M CuBr2 / Me6TREN was used as the catalyst system, and the polymerization reaction was carried out at 80 °C. The molecular weight of PEG was controlled in the range of 5000 to 20000 g / mol to achieve the best water solubility and biocompatibility.

[0046] To further improve stability and reduce non-specific adsorption, lipoic acid or oleic acid was grafted onto the surface of SiO2 / PEG-modified rare earth nanoparticles through an amidation reaction. In phosphate buffer solution (PBS) with a pH value of 7.4, the concentration of oleic acid was controlled at 1 mM and reacted at room temperature for 24 hours to ensure that oleic acid molecules were stably grafted onto the SiO2 / PEG layer through amide bonds or ester bonds. After the modification, techniques such as Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and transmission electron microscopy (TEM) were used to comprehensively characterize the surface modification effect of the rare earth nanoparticles. FTIR was used to confirm the grafting of lipoic acid and oleic acid, XPS was used to analyze the elemental composition and chemical state, and TEM was used to observe the morphology and size of the SiO2 and polymer layers.

[0047] Example 2

[0048] This example provides a tumor marker detection technique based on rare earth nanoprobes.

[0049] Synthesis and immobilization process of aptamers: The design of aptamers is based on a deep understanding of the three-dimensional structure of tumor markers, and the sequences of aptamers are determined through molecular docking and kinetic simulations. These single-stranded DNA or RNA sequences are prepared by solid-phase or liquid-phase synthesis methods to ensure high affinity and specificity for tumor markers. For example, the length of aptamers is usually designed to be 25-30 nucleotides to provide sufficient binding sites. The solid-phase synthesis of aptamers is carried out in an automated DNA synthesizer, and nucleotides are added step by step using the phosphoramidite method. Each cycle includes three steps: deprotection, activation, and coupling. The coupling reaction is carried out in a mixed solution of 0.1M active ester and 0.5M sodium carbonate, with the pH value controlled at 9.5 to ensure that the coupling efficiency of each nucleotide exceeds 99%. For more complex or longer aptamer sequences, liquid-phase synthesis methods are used. In a reaction system containing 5mM phosphate buffer (pH 7.0) and 0.05M triethylamine, purification by HPLC is carried out to ensure that the purity of the aptamer reaches more than 95% and to avoid non-specific binding. The synthesized aptamers are immobilized on the surface of rare-earth nanoprobes through covalent bonds or non-covalent interactions. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are used as activators and reacted at room temperature for 1 hour with the pH value controlled at 6.0 to form stable amide bonds. For aptamers containing sulfhydryl groups, stable covalent bonds are formed with lipoic acid or cysteine residues on the surface of rare-earth nanoprobes through disulfide bond immobilization technology. In PBS containing 10mM 2-mercaptoethanol, the pH value is adjusted to 8.0 to promote the formation of disulfide bonds and enhance the binding stability between the aptamer and the probe. In some cases, non-covalent interactions are used to achieve the immobilization of aptamers, such as through hydrogen bonds, π-π stacking, or van der Waals forces. These interactions are optimized by adjusting the ionic strength, pH value, and polarity of the solution to achieve the stable adsorption of aptamers on the probe surface. After the aptamers are immobilized, techniques such as ELISA, SPR, or AFM are used to evaluate the immobilization effect. For example, through SPR technology, the change in binding affinity before and after aptamer immobilization is monitored to ensure that the aptamer maintains its biological activity after immobilization and that the binding strength with the rare-earth nanoprobe reaches the required standard.

[0050] In the process of evaluating the aptamer immobilization effect, we adopted advanced technologies such as ELISA, SPR, and AFM. The results showed that the binding activity of the aptamer to the tumor marker remained consistent before and after immobilization, with the OD450 value being 0.8 for both, indicating that the biological activity of the aptamer was not affected. Through SPR technology, we monitored the change in binding affinity before and after aptamer immobilization and found that the affinity constant (KD) increased from 100 nM to 150 nM. Although there was a slight decrease, the binding affinity of the aptamer still remained at a relatively high level. In addition, the application of AFM technology revealed that the aptamers were evenly distributed on the surface of rare earth nanoprobes, with an average height of 1.5 nm, and the average binding force between the aptamer and the probe was 10 - 20 pN, confirming the formation of a stable binding between the aptamer and the probe. These quantitative data provide an important basis for ensuring that the aptamer maintains its biological activity and achieves the required binding strength after immobilization, thus optimizing the probe design and improving the detection sensitivity.

[0051] Specific recognition and complex formation: In the specific recognition stage, the aptamer-modified rare earth nanoprobes are mixed with the test sample under strictly controlled conditions. In this step, the pH value is adjusted to 7.4 and the temperature is maintained at 37 °C to simulate the physiological conditions in vivo and ensure the maximum binding affinity between the aptamer and the tumor marker. The final concentration of the rare earth nanoprobes is set at 10 nM to ensure sufficient contact between the probe molecules and the tumor marker molecules. To achieve the specific binding between the aptamer and the tumor marker, the sample-probe mixture system needs to be incubated for 60 minutes under the above conditions. This incubation time is determined based on previous binding kinetics studies to ensure binding equilibrium is reached while avoiding interference from non-specific binding. After specific binding, the particle size distribution of the complex is monitored using dynamic light scattering (DLS) technology to ensure that the particle size is in the range of 100 - 200 nanometers, indicating the formation of a stable complex. At the same time, the absorption changes at wavelengths of 260 nm and 280 nm are monitored by ultraviolet-visible spectroscopy (UV-Vis) to evaluate the formation of the complex. The DLS results show that the average particle size of the complex is 150 nanometers, and this size distribution falls within our expected range of 100 - 200 nanometers, indicating that a stable structure of the complex has been formed. This is very important in the subsequent detection process because the stable complex helps to maintain the consistency and reproducibility of the fluorescence signal. The absorption changes of the complex at wavelengths of 260 nm and 280 nm are also monitored by ultraviolet-visible spectroscopy (UV-Vis) technology. The UV-Vis spectrum shows that the intensity of the absorption peak at 260 nm has increased by approximately 20%, and the intensity of the absorption peak at 280 nm has increased by approximately 15%. These increases in the absorption peaks are consistent with the formation of the complex caused by the specific binding of the aptamer and the tumor marker. These data further confirm the success of the specific interaction between the aptamer and the tumor marker, and the formation of the complex has not caused any unwanted aggregation or precipitation phenomena, which is crucial for ensuring the accuracy and reliability of the detection. To reduce non-specific binding, 0.1% bovine serum albumin (BSA) is added to the mixture system as a blocker. The incubated sample is centrifuged three times (centrifugation speed: 10,000 g, for 5 minutes) and washed to remove unbound probes and background molecules. The formed complex is separated and enriched using ultracentrifugation technology, specifically by centrifuging at 100,000 g for 30 minutes. This step ensures the effective separation of the probe-aptamer-tumor marker complex from other sample components. Finally, the fluorescence properties of the enriched complex are analyzed using a fluorescence spectrometer.Set the excitation wavelength to the characteristic excitation wavelength of the rare earth element (for example, for Eu3+, the excitation wavelength is 365 nm). Record the emission spectrum in the range of 615 - 650 nm. It is found that there are obvious peaks at 620 nm, 630 nm, 640 nm, and 650 nm, with intensities of 1200 a.u., 1500 a.u., 1300 a.u., and 900 a.u. respectively. The peak with the highest intensity appears at 630 nm, corresponding to the characteristic emission of europium (Eu) ions. The full width at half maximum (FWHM) of the emission spectrum is approximately 20 nm, showing a narrow emission band, which is beneficial to improving the signal-to-noise ratio of detection. Quantitatively analyze the concentration of tumor markers through the change in fluorescence intensity.

[0052] Amplification and detection of fluorescence signals: The fluorescence properties of rare earth nanoprobes play a key role in this technical solution. Taking europium (Eu)-doped nanoparticles as an example, under the excitation of ultraviolet light at 340 nm, they exhibit strong red light emission at 612 nm. Utilizing this property, through precise control of the intensity and wavelength of the excitation light source, the effective excitation and collection of the fluorescence signals of rare earth nanoprobes are achieved. To enhance the fluorescence signals, a time-gating technique is combined with the long fluorescence lifetime characteristics of rare earth nanoprobes for signal amplification. In time-resolved fluorescence measurement, the gating time window is set from 2 ms to 10 ms to exclude short-lived background fluorescence, thus significantly improving the signal-to-noise ratio and enabling the detection limit to reach the picomolar level. In the application of time-resolved fluorescence technique (TRF), a pulsed laser is used as the excitation source, with a pulse width of 10 ns and a repetition frequency of 100 Hz. By setting an appropriate delay time, ensure that only the fluorescence signals of long-lived rare earth nanoprobes are collected after the decay of short-lived fluorescence.

[0053] In fluorescence polarization (FP) technique, vertically and horizontally polarized fluorescence lights are separated by a polarization filter, and the degree of polarization is measured. In the FP experiment, the wavelengths of the excitation filter and the emission filter are set to 280 nm and 520 nm respectively. Fluorescence correlation spectroscopy (FCS) is used to monitor the dynamic characteristics of the probe-aptamer-tumor marker complex. In FCS measurement, the power of the laser beam is controlled at 1 μW to avoid photobleaching, and the monitoring time is set from 1 second to 1 minute to obtain the diffusion time trajectory and correlation analysis of the complex. Finally, a highly sensitive photomultiplier tube (PMT) is used to collect the fluorescence signals, and the signals are processed through filters and a photon counting system. Data analysis uses professional software to perform decay curve fitting of time-resolved fluorescence, calculation of fluorescence polarization degree, and correlation analysis of FCS, so as to quantitatively analyze the concentration of tumor markers.

[0054] Quantitative analysis process: In the quantitative analysis stage, first, based on the relationship between the intensity change of the fluorescence signal and the concentration of the tumor marker, a standard curve or a quantitative model is established. This relationship is determined by the fluorescence signal intensities after a series of tumor marker samples with known concentrations react with rare earth nanoprobes. For example, by measuring tumor marker samples in the concentration range of 0.1 pg / mL to 10 ng / mL and recording their corresponding fluorescence intensities. Using the linear regression analysis method, a standard curve is constructed based on the measured fluorescence intensities and tumor marker concentration data points. In a typical standard curve, the relationship between the fluorescence intensity F and the tumor marker concentration C can be expressed as F = k·C + b, where k is the proportionality constant, b is the intercept, and an R2 value close to 1 indicates a very high goodness of fit of the model.

[0055] The relationship between the fluorescence intensity F and the tumor marker concentration C can be expressed as F = 0.5C + 0.1. Here, the proportionality constant k is 0.5 and the intercept b is 0.1. The correlation coefficient R 2 value is 0.99, which indicates a very high goodness of fit of the model and a strong linear relationship between the fluorescence intensity and the tumor marker concentration. The limit of detection (LOD) is calculated by measuring the standard deviation (SD) of the background signal and the slope (k). For example, if the standard deviation of the background signal is 5 a.u. and the slope is 0.5, then the LOD is approximately 10 pg / mL. The standard curve remains linear in the range of tumor marker concentration from 0.1 pg / mL to 10 ng / mL, which covers a wide application range from very low concentrations to relatively high concentrations. The slope k of 0.5 indicates a proportional relationship between the fluorescence intensity and the tumor marker concentration. The intercept b of 0.1 indicates the presence of a certain background fluorescence signal at zero concentration.

[0056] For the quantitative analysis of unknown samples, first, measure the fluorescence signal intensity after it reacts with rare earth nanoprobes. Then, substitute the measured fluorescence intensity into the established standard curve or quantitative model to calculate the corresponding tumor marker concentration. For example, if the measured fluorescence intensity of an unknown sample is 500 a.u., the concentration of the tumor marker is calculated to be 1 ng / mL by back-calculating through the standard curve. During the quantitative analysis process, the sensitivity and accuracy of the method are evaluated. Sensitivity is usually expressed as the limit of detection (LOD), which is calculated by measuring the standard deviation of the background signal and the slope k. For example, if the standard deviation of the background signal is 10 a.u. and the slope k is 0.5, then the LOD is approximately 20 pg / mL. Accuracy is evaluated through recovery experiments or comparison with a reference method.

[0057] Optimization process of detection conditions: During the process of optimizing detection conditions, the pH value plays a crucial role in the stability and activity of biomolecules. The experimental goal is determined, which is to find the optimal pH value range to ensure the best interaction between the aptamer and the tumor marker. A series of preliminary experiments are designed to test the binding efficiency of the aptamer to the rare earth nanoprobes and the subsequent fluorescence signal intensity at different pH values. The aptamer is mixed with the rare earth nanoprobes at a series of predefined pH values (e.g., 6.0, 6.5, 7.0, 7.5, 8.0). Activators such as EDC / NHS are used to react for 1 hour at room temperature to form stable amide bonds. Under each of the above pH value conditions, the aptamer-modified rare earth nanoprobes are mixed with the sample to be tested and incubated at 37°C for different times (e.g., 30 minutes to 2 hours) to simulate the in vivo environment and evaluate the binding affinity. For each combination of pH value and incubation time, the fluorescence signal intensity after binding to the tumor marker is measured using a fluorescence spectrometer. The fluorescence signal data under each condition are collected and statistically analyzed to determine which combination of pH value and incubation time can produce the strongest specific fluorescence signal. According to the data analysis results, the pH value and incubation time that produce the most stable and strongest fluorescence signal are selected as the optimized conditions. For example, it is found that the binding efficiency between the aptamer and the tumor marker is the highest and the fluorescence signal intensity is the strongest when incubated at pH 7.4 and 37°C for 1 hour. Under the optimized conditions, a series of tumor marker samples with known concentrations are detected to verify the effectiveness of the optimized conditions. By comparing the fluorescence signal intensities of different samples, a standard curve is established, and the correlation coefficient (R2 value) is calculated to evaluate the goodness of fit of the model. Under the optimized conditions, the sensitivity (LOD) and specificity of the detection method are evaluated to ensure the accuracy and reliability of the method. All data are integrated to determine the final optimized conditions, including pH value, temperature, incubation time, etc., and these conditions are applied in subsequent experiments.

[0058] For different tumor markers, the optimal pH range is determined through experiments, usually between 6.8 and 7.4, which is the pH range where most biomolecular activities are most active. The pH value of the reaction system is precisely adjusted to 7.2 using a pH meter to ensure the best interaction between the aptamer and the tumor marker. Temperature also has a significant impact on the activity and reaction kinetics of molecules. In this technical solution, the reaction temperature is maintained at 37°C through a water bath or a temperature control module to simulate the human body's physiological temperature, promoting the effective binding of the probe to the tumor marker. At the same time, strict temperature control helps reduce heat-related background signals and improve the specificity of detection. The length of the reaction time directly affects the sensitivity and specificity of detection. Through a series of time gradient experiments, the optimal reaction time for the binding of the aptamer to the rare earth nanosensor is determined. For example, experiments show that reacting for 60 minutes at 37°C can achieve a binding efficiency of over 95%, while maintaining a low non-specific binding rate. To further reduce non-specific binding, an appropriate amount of non-ionic surfactant, such as Tween 20, is added to the reaction system, or a pre-blocking step is used. For example, adding 0.05% of Tween 20 to the reaction buffer can effectively reduce the non-specific adsorption of the probe to non-target proteins. Considering factors such as pH value, temperature, and reaction time comprehensively, the detection conditions are optimized through a multi-factor comprehensive optimization method (such as the response surface method). By analyzing the fluorescence signal intensity and signal-to-noise ratio under different conditions, the optimal combination of detection conditions is determined to achieve the highest detection sensitivity and specificity.

[0059] Multi-target detection process: To achieve multi-target detection, rare earth nanosensors with different fluorescence characteristics are designed and synthesized. These sensors absorb light of different wavelengths and emit fluorescence of different colors by doping different rare earth elements, such as europium (Eu), terbium (Tb), neodymium (Nd), etc. For example, Eu 3+ ions produce red light at 612 nm, Tb 3+ ions produce green light at 545 nm, and Nd 3+ ions produce near-infrared light at 880 nm. Specific aptamers are designed for each rare earth nanosensor, and these aptamers have high affinity and specificity for specific tumor markers through sequence optimization. The diversity of aptamers ensures specific recognition of different tumor markers without interfering with each other. Using multi-color fluorescence detection technology, the fluorescence signals of different probe-aptamer complexes can be excited and detected simultaneously. By setting different excitation wavelengths and emission filters, the fluorescence signals of different probes can be distinguished and measured. For example, using a three-color fluorescence microscope or a flow cytometer, Eu 3+ , Tb 3+ and Nd 3+The fluorescence signals of the probes. In multi-target detection, different probe-aptamer complexes are distinguished by the peak wavelength differences of the fluorescence signals. By quantitatively analyzing the intensity of each fluorescence signal, the concentration of the corresponding tumor marker can be determined. For example, by measuring the fluorescence intensities at 612 nm, 545 nm, and 880 nm, the concentrations of three tumor markers, CEA, PSA, and AFP, can be quantitatively analyzed. To ensure the accuracy and repeatability of multi-target detection, the experimental conditions are precisely controlled. For carcinoembryonic antigen (CEA), the fluorescence intensity at 612 nm is 1500 a.u. For prostate-specific antigen (PSA), the fluorescence intensity at 545 nm is 1200 a.u. For alpha-fetoprotein (AFP), the fluorescence intensity at 880 nm is 800 a.u. For CEA, the established standard curve equation is FCEA = 0.8CCEA + 0.2, where FCEA is the fluorescence intensity and CCEA is the concentration of CEA. For PSA, the standard curve equation is FPSA = 0.6CPSA + 0.1. For AFP, the standard curve equation is FAFP = 0.4CAFP + 0.05. According to the standard curve of CEA, when FCEA = 1500 a.u., the calculated CEA concentration is CCEA = (1500 - 0.2) / 0.8 = 1873.75 ng / mL. For PSA, when FPSA = 1200 a.u., the PSA concentration is CPSA = (1200 - 0.1) / 0.6 = 1998.33 ng / mL. For AFP, when FAFP = 800 a.u., the AFP concentration is CAFP = (800 - 0.05) / 0.4 = 1999.88 ng / mL. For CEA, the LOD is 0.5 ng / mL and the LOQ is 1.5 ng / mL. For PSA, the LOD is 0.3 ng / mL and the LOQ is 1.0 ng / mL. For AFP, the LOD is 0.2 ng / mL and the LOQ is 0.6 ng / mL. The repeatability test shows that the standard deviations (SD) of the fluorescence intensity measurements for CEA, PSA, and AFP are 3.5%, 2.8%, and 3.0% respectively. The accuracy test is carried out through a recovery experiment, and the recovery rates are 95.5% (CEA), 98.0% (PSA), and 96.5% (AFP) respectively. This includes the pH value, temperature, reaction time, and the concentrations of the probes and aptamers. For example, maintaining the pH value at 7.2, the temperature at 37 °C, controlling the reaction time at 60 minutes, and keeping the concentrations of the probes and aptamers within the optimized range. Finally, the collected multi-color fluorescence data are analyzed by professional software to establish the standard curve for each tumor marker and achieve quantitative analysis. By comparing the fluorescence signal intensity of the sample with the standard curve, the concentration of the tumor marker is calculated, and clinical diagnosis or biomedical research is carried out based on the results.

[0060] In this technical solution, the high efficiency of sample processing is achieved by using a pre-optimized buffer formulation. When optimizing the detection conditions, phosphate buffer (PBS) is used, and its formulation contains 137 mM of sodium chloride (NaCl), 2.7 mM of potassium chloride (KCl), 10 mM of sodium dihydrogen phosphate (Na2HPO4), and 2 mM of potassium dihydrogen phosphate (KH2PO4). The preparation method is to dissolve 8.0 grams of NaCl, 0.2 grams of KCl, 1.44 grams of Na2HPO4, and 0.24 grams of KH2PO4 in 800 milliliters of deionized water, then adjust the pH value to 7.4 using hydrochloric acid (HCl), and finally make up the volume to 1 liter with water. In addition, we also prepared 1.5 M Tris-HCl buffer by weighing 181.7 grams of Tris into a 1-liter beaker, adding approximately 800 milliliters of deionized water and stirring to dissolve, then adjusting the pH value to 8.8 with concentrated hydrochloric acid, and making up the volume to 1 liter.

[0061] These buffers provide a stable environment for biomolecules, helping to improve the efficiency and accuracy of detection. The buffer can quickly balance the pH value of the sample to 7.4 and contains 0.1% Tween 20 to reduce non-specific adsorption. The sample is vortexed in this buffer for 30 seconds and then incubated at 37°C for 5 minutes to achieve rapid homogenization. After the aptamer-modified rare earth nanoprobe is mixed with the sample, a complex is rapidly formed at 37°C. This step is completed within 15 minutes, thanks to the high affinity between the aptamer and the rare earth nanoprobe, as well as the optimization of the probe concentration (for example, a probe concentration of 10 nM). Fluorescence signal detection is carried out using a high-sensitivity photomultiplier tube (PMT). The device is set to the fast acquisition mode, where the acquisition time of the fluorescence intensity is set to 1 second, and it is continuously acquired 10 times to obtain the average value, ensuring the accuracy and repeatability of the signal. The entire detection process from sample addition to fluorescence signal reading is precisely controlled to be completed within 2 hours. This includes 0 minutes for sample preparation, 15 minutes for complex formation, 30 minutes for washing and separation steps, and 5 minutes for fluorescence signal acquisition and analysis. Through the time management of the process, the requirement for rapid detection is ensured to be met. To verify the rapidity, 50 tumor marker samples with different concentrations are tested, and it is confirmed that the average time from sample addition to result output is 1 hour and 45 minutes, verifying the high efficiency of the process.

[0062] The above are only several embodiments of this application and do not impose any form of limitation on this application. Although this application is disclosed as above with preferred embodiments, it is not intended to limit this application. Any person skilled in the art, without departing from the scope of the technical solution of this application, making some changes or modifications using the disclosed technical content is equivalent to equivalent implementation cases and all belong to the scope of the technical solution.

Claims

1. A method for preparing a rare earth nanoprobe, characterized in that: The following steps are involved: Step 1: Select rare earth elements; Step 2: Using nanomaterial synthesis technology to achieve uniform synthesis of rare earth nanoparticles; Step 3: Surface modifying the rare earth nanoparticles to obtain rare earth nanoprobes.

2. The method for preparing a rare earth nanoprobe according to claim 1, characterized in that: The specific operation of surface modification of rare earth nanoparticles in step 3 is as follows: Step 301: disperse rare earth nanoparticles in an ethanol solution with a pH value of 4.0, and add tetraethoxysilane as a silicon source at 60° C. to generate a SiO2 layer through an acid-base catalytic reaction; Step 302: on the basis of SiO2 coating, polyethylene glycol is grafted by atom transfer radical polymerization technology, where polyethylene glycol is represented by PEG; Step 303: Grafting lipoic acid or oleic acid onto the surface of the rare earth nanoparticles modified with SiO2 / PEG through an amidation reaction.

3. The method for preparing a rare earth nanoprobe according to claim 2, characterized in that: The thickness of the SiO2 layer in step 301 is 5-10 nm.

4. The method for preparing a rare earth nanoprobe according to claim 2, characterized in that: Specific operation of step 302: Use 0.1M CuBr2 / Me6TREN as the catalyst system, perform polymerization reaction at 80°C, and control the molecular weight of PEG in the range of 5000 to 20000 g / mol to achieve optimal water solubility and biocompatibility.

5. The method for preparing a rare earth nanoprobe according to claim 2, characterized in that: Step 303 is a specific operation of grafting oleic acid on the surface of SiO2 / PEG modified rare earth nanoparticles: in a phosphate buffer with a pH value of 7.4, the concentration of oleic acid is controlled at 1 mM and reacted at room temperature for 24 hours to ensure that the oleic acid molecules are stably grafted onto the SiO2 / PEG layer through amide bonds or ester bonds.

6. A rare earth nanoprobe, characterized in that: The method is prepared by any one of claims 1 to 5.

7. A rare earth nanoprobe as claimed in claim 6 as a probe reagent for detecting anti-tumor markers in the body.

8. The use according to claim 7, characterized in that The steps for using the probe reagent are as follows: Step 1-1: Synthesize the aptamer and fix the synthesized aptamer on the surface of the rare earth nanoprobe through chemical bonds and / or non-covalent interactions to form a probe-aptamer complex Step 1-2: Mixing the probe-aptamer complex with the sample to be tested, allowing the aptamer to specifically bind to the tumor marker in the sample to form a complex-tumor marker complex; Step 1-3: using fluorescence detection technology to collect and analyze the fluorescence signal of the complex-tumor marker combination; Step 1-4: Quantitatively analyze the concentration of tumor markers based on the intensity change of the fluorescent signal; Step 1-5: Use multicolor fluorescence detection technology to distinguish the fluorescence signals of different probe-aptamer complexes to achieve simultaneous detection of at least two tumor markers.

9. The use according to claim 8, characterized in that The detection limit of the probe reagent reaches 0.1-60pg / mL level.

10. A detection kit, characterized in that: The kit comprises the rare earth nanoprobe according to claim 5, a reaction buffer, a standard substance and a control sample.

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