Rare earth tree-shaped probe and preparation method thereof

Abstract

The invention discloses a rare earth tree-shaped probe and a preparation method thereof, and relates to the technical field of biological analysis and detection.The probe takes a polyamide-amine tree-shaped polymer as a framework, the surface of the polyamide-amine tree-shaped polymer is grafted with a 1, 4, 7, 10-tetraaza dodecane-1, 4, 7, 10-tetraacetic acid or diethylenetriaminepentaacetic acid polydentate ligand, the ligand is chelated with rare earth ions, and the rare earth ions are adsorbed on the surface of the polyamide-amine tree-shaped polymer. On the basis of a unique highly branched structure and a clear three-dimensional structure of the dendrimer skeleton, metal ion bearing sites are remarkably increased, the metal loading capacity is effectively improved, and meanwhile, the dendrimer skeleton has the advantages that the metal loading capacity is greatly improved; the rigid structure greatly reduces non-specific adsorption through the steric hindrance effect, the detection specificity is enhanced, the key problems of contradiction between metal loading capacity and steric hindrance, non-specific adsorption and the like of an existing probe are successfully solved, and a high-sensitivity and high-precision detection tool is provided for single cell mass spectrum flow analysis.

Description

Technical Field

[0001] The present invention relates to the field of biological information technology, in particular to a rare earth tree probe and a preparation method thereof. Background Art

[0002] As a cutting-edge single-cell analysis method, single-cell mass cytometry has attracted widespread attention in the field of biomedical research in recent years. This technology uses metal element-labeled antibodies to achieve simultaneous detection of multiple components in single cells, providing a powerful tool for complex disease research and precision medicine. Its core principle is to use metal element-labeled antibodies to specifically bind to cell surface markers. After flow cytometric separation, high-sensitivity detection is performed by inductively coupled plasma mass spectrometry, and combined with bioinformatics analysis to reveal cell phenotypes and signaling networks. Compared with traditional flow cytometry, single-cell mass cytometry has higher detection throughput and multi-parameter analysis capabilities. It can simultaneously detect dozens of cell markers, providing a new perspective for in-depth understanding of cellular heterogeneity and functional status. However, the performance of this technology is highly dependent on the quality of the metal element-labeled probes. The sensitivity, specificity, and stability of the probes directly affect the accuracy and reliability of single-cell analysis.

[0003] At present, the metal element labeled probes used in single-cell mass spectrometry flow cytometry technology have the following main defects: although the rare earth ion chelates used in the early stage have the advantages of multiple isotopes and low biological background, each label usually contains only one detectable metal ion, resulting in limited detection sensitivity; although nanoparticles have a high atomic loading rate, they have problems such as severe nonspecific adsorption, limited types and large steric hindrance, which make it difficult to meet the needs of single-cell multi-component analysis; linear polymer element labels have the problems of cumbersome preparation, unstable product quality and the contradiction between the increase in the number of metal ions and the increase in steric hindrance, and cannot take into account the requirements of high specificity, high sensitivity and high precision detection. In addition, the metal loading capacity of existing reagents is limited and the sensitivity to low-expressed antigens is poor, which makes it difficult to meet the diverse needs of scientific research and clinical diagnosis. Therefore, the development of new, high-performance mass spectrometry flow cytometer supporting reagents is imminent. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide a rare earth dendrimer probe and its preparation method. It can use a polyamidoamine (PAMAM) dendrimer as the skeleton, and graft a multidentate ligand such as 1,4,7,10-tetraazadodecane-1,4,7,10-tetraacetic acid (DOTA) or diethylenetriaminepentaacetic acid (DTPA) on its surface. The ligand chelates with the rare earth ions, and the end of the dendrimer skeleton is connected to a specific antibody via a bifunctional coupling agent. This structure, thanks to the unique highly branched structure and clear three-dimensional framework of the dendrimer, increases the metal ion loading sites and improves the metal loading capacity. At the same time, the rigid structure effectively reduces nonspecific adsorption and enhances the detection specificity.

[0005] In order to solve the above-mentioned technical problems, the present invention provides the following technical solutions: On the one hand, a rare earth dendrimer probe is provided, which has a polyamide-amine dendrimer as a skeleton, and a multidentate ligand of 1,4,7,10-tetraazadodecane-1,4,7,10-tetraacetic acid or diethylenetriaminepentaacetic acid is grafted on its surface. The ligand chelates with the rare earth ion, and the end of the dendrimer skeleton is connected to a specific antibody via a bifunctional coupling agent.

[0006] Furthermore, the rare earth ions include one or more of Y, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu, and the bifunctional coupling agent is 1,2-bis(maleimidoethoxy)ethane.

[0007] In another aspect, a method for preparing a rare earth tree probe comprises the following specific steps:

[0008] Ligand grafting onto the dendrimer backbone: Dissolve the cystamine core G1-G5 generation PAMAM dendrimer in a buffer solution, add DOTA or DTPA ligands at a certain molar ratio, and add a condensing agent. Stir the reaction at a suitable temperature. After the reaction is completed, dialyze to remove impurities and freeze-dry to obtain the ligand-grafted dendrimer.

[0009] Polymer core reduction and coupling agent connection: The ligand-grafted dendrimer is dissolved in a solvent, and DTT or TCEP is added to reduce the cystamine core to a thiol under specific conditions. After the reduction is completed, a bifunctional coupling agent is added in proportion to react. After the reaction is completed, impurities are removed by ultrafiltration, freeze-dried, and stored at low temperature to obtain a functionalized polymer with a maleimide group at the end;

[0010] Rare earth ion chelation: Dissolve the functionalized polymer in a weakly acidic buffer solution, add a rare earth ion solution, and react at a suitable temperature to form a water-soluble polymer. Characterize the product using FT-IR or NMR, and adjust the synthesis parameters based on the results.

[0011] Antibody coupling and purification: The polymer with maleimide groups was dissolved in a pH 7.0 buffer solution, and the antibody was added in proportion and stirred at room temperature for reaction. After the reaction, the polymer was separated and purified using an ultrafiltration tube. The coupling ratio was determined using various instruments and the reaction conditions were optimized to obtain a rare earth element labeled probe.

[0012] Furthermore, in the step of grafting the ligand to the dendrimer skeleton, the weighed PAMAM dendrimer is dissolved in an appropriate amount of buffer solution to prepare a solution of a certain concentration, DOTA or DTPA ligand is added according to a molar ratio of the polymer terminal amino group to the ligand of 1:20-1:50, and a condensation agent is added according to a molar ratio of 1:2-1:5. The reaction is carried out at 25-37°C under stirring conditions to allow the ligand to undergo a condensation reaction with the terminal amino group of the dendrimer. After the reaction is completed, the product is transferred to a dialysis bag and dialyzed in deionized water for 1-3 days, during which the deionized water is replaced several times, and the dialyzed product is freeze-dried to obtain a ligand-grafted dendrimer.

[0013] Furthermore, in the polymer core reduction and coupling agent connection step, the ligand-grafted dendrimer is dissolved in a solvent, DTT or TCEP is added at a molar ratio of 1:5-1:20, and the reaction is carried out at room temperature for 1-4 hours under nitrogen protection to reduce the cystamine core of the dendrimer to thiol.

[0014] Furthermore, in the polymer core reduction and coupling agent connection step, after the reduction is completed, a bifunctional coupling agent is added at a molar ratio of 1:5-1:20, and the reaction is continued for 1-3 hours to allow the thiol and the bifunctional coupling agent to react to generate a functionalized polymer with a maleimide group at the end.

[0015] Furthermore, in the rare earth ion chelation step, the functionalized polymer is dissolved in a weakly acidic buffer solution, and the rare earth ion solution is added at a molar ratio of 1:5-1:20. The reaction is stirred at 30-50°C to allow the rare earth ions to undergo a chelation reaction with the ligands on the functionalized polymer to form a water-soluble polymer carrying multiple metal chelate groups. The product is characterized by FT-IR or NMR, and the parameters are adjusted to optimize the synthesis based on the results.

[0016] Furthermore, in the antibody coupling and purification step, a polymer with a maleimide group is dissolved in a buffer solution with a pH of 7.0, and the antibody is added at a molar ratio of 10:1-50:1. The reaction is stirred at 37°C to allow the maleimide group to react with the cysteine-SH group of the Fc part of the antibody. After the reaction is completed, separation and purification are performed using an ultrafiltration tube to remove unreacted antibodies and impurities, and the reaction conditions are optimized based on the measurement results to obtain a rare earth element labeled probe.

[0017] Compared with the existing technology, this rare earth tree probe and its preparation method have the following beneficial effects:

[0018] 1. Based on the unique highly branched structure and clear three-dimensional framework of the dendrimer backbone, the present invention significantly increases the metal ion loading sites and effectively improves the metal loading capacity. At the same time, the rigid structure greatly reduces nonspecific adsorption through the steric effect, enhancing the detection specificity. It successfully solves the key problems of existing probes such as the contradiction between metal loading capacity and steric hindrance, and nonspecific adsorption, and provides a highly sensitive and precise detection tool for single-cell mass spectrometry flow cytometry analysis.

[0019] 2. Through detailed product characterization and reaction condition optimization, the present invention ensures the stability and uniformity of probe quality to meet the needs of large-scale production. In addition, the complete application technology system designed for different cell detection needs can flexibly adjust probe preparation conditions and detection parameters, significantly improving the sensitivity and accuracy of single-cell mass spectrometry flow cytometry analysis, providing reliable technical support for scientific research and clinical diagnosis.

[0020] Other advantages, objects and features of the present invention will be described in part in the following description and, in part, will be apparent to those skilled in the art based on an examination of the following or may be learned from the practice of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0022] Figure 1 This is a schematic diagram of the construction of a rare earth tree probe;

[0023] Figure 2 Schematic diagram of mass spectrometry flow cytometry detection;

[0024] Figure 3 The present invention is a flow chart of a method for preparing a rare earth tree probe. DETAILED DESCRIPTION

[0025] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, the specific implementation methods, structures, features and effects of the present invention are described in detail below in conjunction with the accompanying drawings and preferred embodiments.

[0026] Example 1

[0027] Accurately weigh 0.1 g of cystamine core G3 generation PAMAM dendrimer, select pH 7.4 PBS buffer solution, and dissolve it in 10 mL of the buffer solution to ensure that the polymer can be dispersed in a suitable environment. Then, add 1.3 g of DOTA ligand, 0.3 g of DCC and 0.1 g of NHS as condensation agents, and continue stirring at 25 ° C for 12 hours to allow the DOTA ligand to fully condense with the terminal amino groups of the PAMAM dendrimer. Figure 1 As shown, after the reaction is completed, the product is transferred to a dialysis bag with a molecular weight cutoff of 10,000 Da and placed in deionized water for dialyzation. The dialysis process lasts for 2 days, during which the deionized water is replaced 3 times a day to fully remove impurities such as unreacted DOTA ligand, DCC and NHS. Finally, the dialyzed product is freeze-dried to obtain a ligand-grafted dendrimer.

[0028] The freeze-dried product obtained in the previous step was dissolved in 5 mL of DMSO to form a uniform solution. 0.19 g of DTT was then added and the reaction was carried out at room temperature under nitrogen for 2 hours to reduce the cystamine core of the dendrimer to a thiol. After the reaction was completed, 0.35 g of 1,2-bis(maleimidoethoxy)ethane was immediately added and the reaction was continued for 4 hours to allow the thiol to fully react with the bifunctional coupling agent to form a functionalized polymer with a maleimide group at the end. After the reaction was completed, the product was ultrafiltered using an ultrafiltration tube with a molecular weight cutoff of 10,000 Da. During the ultrafiltration process, PBS buffer solution was added and washed three times to remove excess impurities. Finally, the ultrafiltered product was freeze-dried and stored at -20°C to prevent deterioration.

[0029] The functionalized polymer was dissolved in 10 mL of acetic acid-sodium acetate buffer solution at pH 5.0 to create a weakly acidic environment suitable for rare earth ion chelation. Then, 0.26 g of EuCl3 was added and the reaction was stirred continuously at 40°C for 8 hours to allow the rare earth ions to fully chelate with the DOTA ligands on the functionalized polymer to form a water-soluble polymer carrying multiple metal chelate groups. After the reaction, the product was structurally characterized using Fourier transform infrared absorption spectrometry (FT-IR) and nuclear magnetic resonance spectrometry (NMR). By analyzing the characterization results, it was determined whether the rare earth ions were successfully chelated and the degree of chelation, and the reaction conditions, such as temperature and time, were adjusted to optimize the synthesis process.

[0030] A polymer with a maleimide group was dissolved in 5 mL of PBS buffer solution (pH 7.0). An antibody targeting a specific tumor cell surface marker was added at a molar ratio of 1:20. The reaction was stirred continuously at room temperature for 2 hours to allow the maleimide group to fully react with the cysteine-SH group of the antibody Fc portion, achieving coupling between the antibody and the polymer. After the reaction, the polymer was separated and purified using an ultrafiltration tube with a molecular weight cutoff of 50,000 Da. During the ultrafiltration process, PBS buffer solution was added and washed three times to remove unreacted antibody and other impurities. The coupling ratio of the antibody to the polymer was measured using various instruments such as a UV-Vis spectrophotometer, a fluorophotometer, and an inductively coupled plasma mass spectrometer. Based on the measurement results, the reaction conditions, such as reactant concentration and reaction time, were further optimized to improve coupling efficiency and quality.

[0031] Tumor cell lines were selected and their cell surface marker expressions were measured in detail. Tumor cells were labeled with prepared rare earth element labeled probes, such as Figure 2 As shown, during the labeling process, PBS containing 1% BSA was selected as the buffer solution, the ionic strength was controlled at 150 mM, and the cells were incubated at 4°C for 1 hour. The amount of antibody was reasonably adjusted according to the number of cells. After labeling, the labeled cells were detected using a mass spectrometry flow cytometer, and the relevant test data were recorded. The test results were compared with those of existing commercial kits to evaluate the performance of the probe in terms of sensitivity, specificity, accuracy, etc., providing a basis for subsequent optimization.

[0032] Example 2

[0033] 0.15 g of cystamine core G4 generation PAMAM dendrimer was accurately weighed and dissolved in 15 mL of pH 7.4 PBS buffer solution. 0.68 g of DTPA ligand was added, and 0.03 g of DCC and 0.02 g of NHS were added as condensing agents. The reaction was stirred continuously at 30° C. for 18 hours to allow the DTPA ligand to fully condense with the terminal amino groups of the PAMAM dendrimer. After the reaction, the product was transferred to a suitable dialysis bag and dialyzed in deionized water for 2 days. The deionized water was regularly replaced during the dialysis period to ensure that impurities were fully removed. Finally, the dialyzed product was freeze-dried to obtain a ligand-grafted dendrimer.

[0034] The resulting freeze-dried product was dissolved in 8 mL of DMSO to form a homogeneous solution, to which 0.45 g of TCEP was added. The solution was reacted at room temperature under nitrogen for 3 hours to reduce the cystamine core of the dendrimer to a thiol. After the reaction was complete, 0.38 g of 1,2-bis(maleimidoethoxy)ethane was added, and the reaction was continued for 5 hours to allow the thiol to react with the bifunctional coupling agent to form a functionalized polymer with a maleimide group at the end. After the reaction was complete, the product was ultrafiltered using an ultrafiltration tube with an appropriate molecular weight cutoff. PBS buffer solution was added during the ultrafiltration process to wash the product multiple times to remove impurities. The ultrafiltered product was finally freeze-dried and stored at low temperature.

[0035] The functionalized polymer was dissolved in 15 mL of acetic acid-sodium acetate buffer solution with a pH of 5.5 to provide a suitable weakly acidic environment for rare earth ion chelation. 0.32 g of DyCl3 was added and the reaction was stirred continuously at 45°C for 10 hours to allow the rare earth ions to fully chelate with the DTPA ligand on the functionalized polymer. After the reaction, the product was structurally characterized using instruments such as FT-IR and NMR. Based on the characterization results, the chelation of rare earth ions was analyzed, and reaction parameters such as reaction temperature and time were adjusted to optimize the synthesis process and improve the chelation efficiency.

[0036] Take a polymer with a maleimide group and dissolve it in 8 mL of PBS buffer solution at pH 7.0. Add antibodies targeting immune cell surface markers at a molar ratio of 1:30. Continue stirring at room temperature for 3 hours to allow the antibody and polymer to fully couple. After the reaction is completed, use an ultrafiltration tube with an appropriate molecular weight cutoff for separation and purification. During the ultrafiltration process, add PBS buffer solution for washing to remove unreacted antibodies and impurities. Use multiple instruments to measure the coupling ratio of antibody to polymer. Based on the measurement results, optimize the reaction conditions, such as reactant concentration and reaction time, to improve the coupling quality.

[0037] Immune cell samples were selected and the expression of their cell surface markers was analyzed. The immune cells were labeled with the prepared rare earth element-labeled probes. During the labeling process, PBS was selected as the buffer solution, the ionic strength was controlled at 200 mM, and the cells were incubated at room temperature for 1.5 hours. The antibody dosage was reasonably adjusted according to the number of cells. After labeling, the labeled cells were detected using a mass spectrometry flow cytometer, the test data was recorded, and the test results were compared with the test results of existing commercial kits. The performance of the probe in immune cell analysis was evaluated from multiple aspects, providing a reference for further optimization of the probe.

[0038] In summary, the rare earth element labeled probes prepared by different generations of PAMAM dendrimers have different effects in various aspects. In the ligand grafting link, the G3 and G4 generation polymers have different molecular structures and active site numbers due to their different generations, which results in different amounts of ligands and condensing agents required, and different reaction times. The G4 generation has larger molecules and more active sites, so more ligands and condensing agents are used, and the reaction time is longer. In the core reduction and coupling agent connection steps, different generations of polymers have different reactivity to reducing agents and coupling agents. The G4 generation has a complex structure, a long reaction time, and a large amount of reagents. When rare earth ions are chelated, the spatial structure of different generations of polymers affects the chelation efficiency and stability of rare earth ions. The G3 generation polymer provides a more suitable spatial environment, which makes the rare earth ions chelated more fully and has better stability. Under the same reaction conditions, the chelation of the G3 generation is better. The signal of the composite product is more stable in subsequent detection. In terms of antibody coupling, the generations of different polymers affect the coupling ratio and antibody activity retention. The coupling ratio of G3 generation to antibody is more stable after optimization, and has less effect on antibody activity, which is beneficial to improve detection sensitivity. Although G4 generation has a strong antibody-carrying capacity, steric hindrance may affect antibody activity, resulting in changes in detection sensitivity. In single-cell mass spectrometry flow cytometry applications, different generations of polymer probes have different detection effects for different cell types. G3 generation probes have a higher sensitivity for recognizing specific markers in tumor cell detection and can effectively distinguish tumor cell subpopulations. G4 generation can detect more types of markers in immune cell analysis, but there may be non-specific adsorption problems. Rare earth element labeled probes prepared from different generations of PAMAM dendrimers have their own characteristics and are suitable for different detection scenarios and needs.

[0039] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the scope of the technical solution of the present invention, make some changes or modifications to equivalent embodiments using the technical contents disclosed above. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A rare earth tree probe, characterized in that: The probe uses a polyamide-amine dendrimer as a skeleton, with 1,4,7,10-tetraazadodecane-1,4,7,10-tetraacetic acid or diethyltriaminepentaacetic acid multidentate ligands grafted onto its surface. The ligands chelate with rare earth ions, and the ends of the dendrimer skeleton are connected to specific antibodies through a bifunctional coupling agent.

2. A rare earth tree probe according to claim 1, characterized in that: The rare earth ions include one or more of Y, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu, and the bifunctional coupling agent is 1,2-bis(maleimidoethoxy)ethane.

3. A method for preparing a rare earth tree probe, the method being used to prepare a rare earth tree probe according to any one of claims 1 to 2, characterized in that: The method comprises the following specific steps: Ligand grafting onto the dendrimer backbone: Dissolve the cystamine core G1-G5 generation PAMAM dendrimer in a buffer solution, add DOTA or DTPA ligands at a certain molar ratio, and add a condensing agent. Stir the reaction at a suitable temperature. After the reaction is completed, dialyze to remove impurities and freeze-dry to obtain the ligand-grafted dendrimer. Polymer core reduction and coupling agent connection: The ligand-grafted dendrimer is dissolved in a solvent, and DTT or TCEP is added to reduce the cystamine core to a thiol under specific conditions. After the reduction is completed, a bifunctional coupling agent is added in proportion to react. After the reaction is completed, impurities are removed by ultrafiltration, freeze-dried, and stored at low temperature to obtain a functionalized polymer with a maleimide group at the end; Rare earth ion chelation: Dissolve the functionalized polymer in a weakly acidic buffer solution, add a rare earth ion solution, and react at a suitable temperature to form a water-soluble polymer. Characterize the product using FT-IR or NMR, and adjust the synthesis parameters based on the results. Antibody coupling and purification: The polymer with maleimide groups was dissolved in a pH 7.0 buffer solution, and the antibody was added in proportion and stirred at room temperature for reaction. After the reaction, the polymer was separated and purified using an ultrafiltration tube. The coupling ratio was determined using various instruments and the reaction conditions were optimized to obtain a rare earth element labeled probe.

4. The method for preparing a rare earth tree probe according to claim 3, characterized in that: In the step of grafting the ligand onto the dendrimer backbone, the weighed PAMAM dendrimer is dissolved in an appropriate amount of buffer solution to prepare a solution of a certain concentration, DOTA or DTPA ligand is added at a molar ratio of polymer terminal amino group to ligand of 1:20-1:50, and a condensing agent is added at a molar ratio of 1:2-1:

5. The reaction is carried out at 25-37° C. under stirring to cause a condensation reaction between the ligand and the terminal amino group of the dendrimer. After the reaction is completed, the product is transferred to a dialysis bag and dialyzed in deionized water for 1-3 days, during which the deionized water is replaced multiple times. The dialyzed product is freeze-dried to obtain a ligand-grafted dendrimer.

5. The method for preparing a rare earth tree probe according to claim 3, characterized in that: In the polymer core reduction and coupling agent connection step, the ligand-grafted dendrimer is dissolved in a solvent, DTT or TCEP is added at a molar ratio of 1:5-1:20, and the reaction is carried out at room temperature for 1-4 hours under nitrogen protection to reduce the cystamine core of the dendrimer to thiol.

6. The method for preparing a rare earth tree probe according to claim 3, characterized in that: In the polymer core reduction and coupling agent connection step, after the reduction is completed, a bifunctional coupling agent is added at a molar ratio of 1:5-1:20, and the reaction is continued for 1-3 hours to allow the thiol and the bifunctional coupling agent to react to generate a functionalized polymer with a maleimide group at the end.

7. The method for preparing a rare earth tree probe according to claim 3, characterized in that: In the rare earth ion chelation step, the functionalized polymer is dissolved in a weakly acidic buffer solution, and the rare earth ion solution is added at a molar ratio of 1:5-1:

20. The reaction is stirred at 30-50° C. to allow the rare earth ions to undergo a chelation reaction with the ligands on the functionalized polymer to form a water-soluble polymer carrying multiple metal chelate groups. The product is characterized by FT-IR or NMR, and the parameters are adjusted according to the results to optimize the synthesis.

8. The method for preparing a rare earth tree probe according to claim 3, characterized in that: In the antibody coupling and purification step, a polymer with a maleimide group is dissolved in a buffer solution with a pH of 7.0, and an antibody is added at a molar ratio of 10:1 to 50:

1. The reaction is stirred at 37°C to allow the maleimide group to react with the cysteine-SH group of the Fc portion of the antibody. After the reaction is completed, separation and purification are performed using an ultrafiltration tube to remove unreacted antibody and impurities, and the reaction conditions are optimized based on the measurement results to obtain a rare earth element labeled probe.

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