A method, kit, sensor and device for analyzing intermolecular interactions

Abstract

The present invention relates to the technical field of molecular interaction analysis, and specifically provides a method for analyzing intermolecular interactions. The method comprises the following steps: using a rare earth material with a ligand molecule modified on its surface as an upconversion material, contacting the upconversion material with a target molecule in a solution system, irradiating the solution system with excitation light that matches the rare earth material, and continuously changing the intensity of the excitation light actually received by the upconversion material as the target molecule continuously binds and dissociates with the ligand molecule of the upconversion material in a dynamic process, thereby changing the intensity of the emitted light. By recording the curve of the emission light intensity changing with time, the molecular interaction kinetic constants, such as the binding rate constant (K on ), dissociation rate constant (K off ) and affinity (K D ), etc., which solves the technical problems of low accuracy of existing BLI technology and high cost of SPR technology. Based on the above principles, the present invention has also developed a series of experimental consumables, kits, sensors and analytical devices for molecular interaction analysis.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular interaction analysis, and in particular to a method, a kit, a sensor and a device for analyzing intermolecular interactions. Background Art

[0002] Molecular interaction analysis is of great significance in many fields such as biology, chemistry, medicine and pharmacy. By studying the interactions between molecules, scientists can gain a deep understanding of the internal operating mechanisms of biological systems and provide key information for new drug development, disease treatment and materials science. Molecular interaction analysis technologies are diverse and involve a wide range of instruments. According to the principles, they mainly include surface plasmon resonance technology (SPR), biomembrane interferometry technology (BLI), grating coupling interferometry technology (GCI), microthermophoresis technology (MST) and isothermal titration calorimetry technology (ITC). From the statistical analysis of successful bids in the past two years, it was found that SPR technology and BLI technology occupy a mainstream position.

[0003] Surface Plasmon Resonance (SPR) technology is a label-free real-time detection technology. Its principle is: surface plasmons is an electromagnetic wave that exists on the surface of metals. It is generated by the collective oscillation of free electrons on the metal surface. When the biological molecules in the sample solution are fixed on the metal surface (usually a gold film) and bind to another molecule, it will cause a change in the local refractive index. This change will cause the propagation conditions of the surface plasmon to change, thereby affecting the intensity of the reflected light. By measuring the change in the intensity of the reflected light, the binding of biological molecules can be indirectly known. SPR technology can monitor the binding and dissociation process between molecules in real time. By recording the curve of the change in the intensity of the reflected light over time (i.e., the SPR curve), the binding kinetic parameters such as the binding rate constant (K) can be obtained. on ), dissociation rate constant (K off ) and affinity (K D )wait.

[0004] BioLayer Interferometry (BLI) technology is a technology used to monitor biomolecular interactions in real time. Its principle is: when a beam of white light is irradiated vertically onto a transparent fiber optic probe, the light will be reflected back by the two interfaces at the end of the fiber optic probe to form two beams of coherent light. The two beams of light will interfere with each other during the return process, producing an interference phase shift pattern. When the analyte in the solution binds to the capture molecules fixed at the end of the probe, the optical thickness of the probe end will change. This change in optical thickness will cause a change in the interference pattern that can be detected. By analyzing the changes in the interference signal, the kinetic parameters of intermolecular binding can be obtained, including the binding rate constant (K on ), dissociation rate constant (K off ) and affinity (K D )wait.

[0005] The current situation in the industry is that users need convenient, low-cost, and higher-precision molecular interaction detection. BLI technology is low-cost and easy to use, but it still has the following problems: ① Insufficient precision; ② BLI technology requires the ligand to be fixed to the tip of a quartz needle, which means that each probe can only perform one detection, and different coatings need to be applied to the tip of the quartz needle to form probes for different molecules; ③ The probe needs to remain in the solution to be tested throughout the entire detection process, resulting in the solution to be tested not being in a closed environment, which is prone to solution volatilization and contamination by the experimental environment, all of which will directly affect the analysis results; ④ The BLI method uses white light, and the received light signal is the white light reflected from the molecular layer. The two lights are prone to interference, and the reflected light has energy loss, resulting in low detection signal intensity, low sensitivity and signal-to-noise ratio.

[0006] SPR technology offers the advantage of high precision, but it is also very expensive, typically tens to over a hundred times more expensive than BLI. To balance cost, convenience, and high precision, the industry often uses BLI for coarse sample screening, followed by precise SPR analysis of the screened samples. However, this approach does not fundamentally address the high cost of molecular interaction analysis.

[0007] Based on the working principles of BLI and SPR technologies, it is clear that neither SPR nor BLI is a true liquid-phase reaction, nor can it perform mixed detection and analysis of multiple target molecules. In view of the limitations of existing technologies, the inventors sought to develop a molecular interaction analysis technology that combines high precision, low cost, and practical flexibility and convenience. Summary of the Invention

[0008] (1) Technical issues to be resolved

[0009] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a method for analyzing intermolecular interactions, which uses a rare earth material with a ligand molecule modified on its surface as an upconversion material, contacts the upconversion material with a target molecule in a solution system, and irradiates the solution system with excitation light that matches the rare earth material. As the target molecule continuously binds and dissociates with the ligand molecule of the upconversion material, the excitation light intensity actually received by the upconversion material is continuously changed, thereby causing the emission light intensity to change. By recording the curve of the emission light intensity changing with time, the molecular interaction kinetic constants, such as the binding rate constant (K on ), dissociation rate constant (K off ) and affinity (K D ) and other parameters. This invention solves the technical problems of existing BLI technology, such as low precision and expensive SPR technology. Based on the above principles, the present invention has also developed a series of experimental consumables, kits, sensors and analytical devices for molecular interaction analysis.

[0010] (2) Technical solution

[0011] In a first aspect, the present invention provides a method for analyzing intermolecular interactions, comprising:

[0012] S1. Providing a surface-modified rare earth upconversion nanomaterial, wherein the surface modification includes anti-quenching modification and ligand coating; the anti-quenching modification is to encapsulate the rare earth upconversion nanomaterial with a polymer or an inert inorganic material; the ligand coating is to attach a biological ligand molecule capable of specifically (directly or indirectly) binding to a target molecule to the anti-quenching modified rare earth upconversion nanomaterial;

[0013] S2. Immersing the surface-modified rare earth up-conversion nanomaterial in a solution containing a target molecule so that the surface-modified rare earth up-conversion nanomaterial can contact the target molecule in the solution; irradiating the solution with excitation light that matches the rare earth up-conversion nanomaterial so that the rare earth up-conversion nanomaterial generates emitted light; as the target molecule continuously binds to the biological ligand molecule on the surface of the rare earth up-conversion nanomaterial, the intensity of the emitted light generated by the up-conversion nanomaterial in response to the excitation light continuously decreases until the binding of the biological ligand molecule and the target molecule reaches equilibrium or saturation, and recording a curve of the change in the emission light intensity over time during this period;

[0014] S3. Calculate the curve of the emission light intensity versus time obtained in S2 to obtain the kinetic constant of the interaction between the ligand molecule and the target molecule.

[0015] According to a preferred embodiment of the present invention, in S1, the rare earth upconversion nanomaterial is prepared by doping rare earth ions into an inorganic matrix; the rare earth ions are at least one of Yb, Er, Tm, Ho, and Nd; and the inorganic matrix is one of a fluoride, an oxide, a phosphate, and a vanadate. Preferably, the fluoride is NaYF4, NaLaF4, NaGdF4, or KLuF4; the oxide is Y2O3, Gd2O3, or La2O3; the phosphate is YPO4; and the vanadate is YVO4.

[0016] According to a preferred embodiment of the present invention, in S1, the rare earth upconversion nanomaterial is at least one of NaLnF4(Yb, Er), NaLnF4(Yb, Tm), and NaLnF4(Yb, Ho), wherein Ln is at least one of Y, Gd, La, and Lu. More preferably, it is NaYF4(Yb, Er), wherein the Yb doping ratio is 18% (by weight) and the Er doping ratio is 2% (by weight).

[0017] According to a preferred embodiment of the present invention, in S1, the rare earth upconversion nanomaterial is a nanoparticle having a particle size of 15-100 nm, preferably 20-70 nm, more preferably 20-40 nm, preferably a nanosphere. After the anti-quenching modification, the particle size of the rare earth upconversion nanomaterial is increased.

[0018] In a preferred embodiment of the present invention, in S1, the anti-quenching modification process is:

[0019] S11, dispersing the rare earth upconversion nanomaterial in an organic solvent to obtain an upconversion nanomaterial dispersion; dissolving methoxy polyethylene glycol phospholipid and an active group modifier in the organic solvent to obtain an inclusion solution; the active group modifier is at least one of methoxy polyethylene glycol phospholipid carboxyl mPEG-PLC, methoxy polyethylene glycol amino mPEG-NH2, methoxy polyethylene glycol thiol mPEG-SH, and methoxy polyethylene glycol-succinimide ester MPEG2000-NHS;

[0020] S12. After mixing the upconversion nanomaterial dispersion and the inclusion solution, the organic solvent is evaporated to obtain the anti-quenching modified rare earth upconversion nanomaterial. Pure water can be added for further dispersion, and the dispersion can be stored.

[0021] Anti-quenching modification can enhance the anti-quenching ability of rare earth upconversion nanomaterials, preventing the weakening and quenching of their luminescence intensity under the influence of water molecules. In addition to using hydrophobic phospholipids such as methoxy polyethylene glycol phospholipids for encapsulation, other hydrophobic organic polymers, non-organic polymer hydrophobic materials, or silica gel can also be used to protect the core of rare earth upconversion nanomaterials, thereby reducing the impact of water molecules on them.

[0022] In a preferred embodiment of the present invention, in S11, a rare earth upconversion nanomaterial is co-encapsulated with a methoxypolyethylene glycol phospholipid and an active group modifier. This significantly improves the rare earth upconversion nanomaterial's anti-quenching properties in water and allows for the attachment of active groups such as carboxyl, amino, sulfhydryl, polylysine, and succinimidyl groups to the surface of the rare earth upconversion nanomaterial. These groups prepare for further attachment of biological ligand molecules. The phospholipid coating exhibits good affinity for proteins (biological ligand molecules or target molecules), further facilitating subsequent modification processes.

[0023] Specifically, carboxyl groups can react with molecules containing primary amine groups to form amide bonds, making them easily coupled to biomolecules (such as proteins and antibodies). Amino groups can react with various compounds containing active ester groups (such as succinimidyl esters) to form stable amide bonds. Furthermore, amino groups can react with aldehydes or ketones via Schiff base reactions. The presence of amino groups allows for further modification of the molecule, allowing the introduction of other functional groups or the connection of other molecules. Sulfhydryl groups can react with compounds containing maleimide groups to form thioether bonds. Succinimidyl groups can react with primary amine groups to form stable amide bonds, making them easily coupled to amine-containing biomolecules (such as proteins and peptides). Succinimidyl groups can undergo coupling reactions under relatively mild conditions. Modification with these groups allows for coupling to specific biological ligand molecules (such as antibodies), generating binding-dissociation interactions with target molecules in solution.

[0024] It should be noted that the introduction of biomolecular ligands using reactive groups such as hydroxyl, carboxyl, amino, aldehyde, succinimidyl ester, and sulfonyl groups as base groups is conventional and will not be elaborated upon here. Biomolecular ligands can be proteins, antibodies, antigens, haptens, polysaccharides, nucleic acid probes, or cells; examples include Streptavidin, Avidin, BSA, Protein A, Protein G, Biotin, peptides, antibodies, various other proteins, and nucleic acid probes (DNA and RNA probes).

[0025] As a preferred embodiment of the present invention, in S1, the anti-quenching modification process is:

[0026] S11, dispersing the rare earth upconversion nanomaterial in chloroform at a concentration of 0.1-0.5M to obtain an upconversion nanomaterial dispersion; dissolving the methoxy polyethylene glycol phospholipid and the PEGylated active group modifier in chloroform at a mass ratio of 1-10:1-10 (preferably 7:3) to obtain an inclusion solution with a concentration of 20-50 mg / ml;

[0027] S12. Mix the upconversion nanomaterial dispersion and the inclusion solution, evaporate the solvent, add pure water at a concentration of 0.01-0.04 M, and disperse by ultrasonication to obtain a dispersion of the rare earth upconversion nanomaterial modified with anti-quenching.

[0028] In a second aspect, the present invention provides a kit for analyzing intermolecular interactions, comprising a surface-modified rare earth upconversion nanomaterial, wherein the surface modification comprises anti-quenching modification and ligand coating; the anti-quenching modification comprises encapsulating the rare earth upconversion nanomaterial with a polymer or an inert inorganic material; and the ligand coating comprises attaching a biological ligand molecule capable of specifically binding to a target molecule to the anti-quenching modified rare earth upconversion nanomaterial.

[0029] Alternatively, the kit comprises: a rare earth upconversion nanomaterial that has been modified with anti-quenching properties and a biological ligand molecule, wherein the anti-quenching modification comprises encapsulating the rare earth upconversion nanomaterial with a polymer or an inert inorganic material and grafting active groups onto the surface of the encapsulation layer; the active groups are at least one of carboxyl, amino, thiol and succinimide groups, and the active groups provide active connection sites for coupling the biological ligand molecule; and the biological ligand molecule is capable of specifically binding to the target molecule.

[0030] For the latter approach, it is necessary to temporarily coat the biological ligand molecule on the surface of the rare earth upconversion nanomaterial before measuring the interaction between the biological ligand molecule and the target molecule, and then perform analysis and detection.

[0031] According to a preferred embodiment of the present invention, the anti-quenching modification method is: dispersing the rare earth up-conversion nanomaterial in an organic solvent to obtain an up-conversion nanomaterial dispersion; dissolving methoxy polyethylene glycol phospholipid and an active group modifier together in an organic solvent to obtain an inclusion solution; the active group modifier is at least one of methoxy polyethylene glycol phospholipid carboxyl mPEG-PLC, methoxy polyethylene glycol amino mPEG-NH2, methoxy polyethylene glycol thiol mPEG-SH and methoxy polyethylene glycol-succinimide ester MPEG2000-NHS; after mixing the up-conversion nanomaterial dispersion and the inclusion solution, the organic solvent is evaporated to obtain the rare earth up-conversion nanomaterial modified with anti-quenching.

[0032] According to a preferred embodiment of the present invention, the rare earth upconversion nanomaterial is prepared by doping rare earth ions into an inorganic matrix; the rare earth ions are at least one of Yb, Er, Tm, Ho, and Nd; and the inorganic matrix is one of a fluoride, an oxide, a phosphate, and a vanadate. Preferably, the fluoride is NaYF4, NaLaF4, NaGdF4, or KLuF4; the oxide is Y2O3, Gd2O3, or La2O3; the phosphate is YPO4; and the vanadate is YVO4.

[0033] Preferably, the rare earth upconversion nanomaterial is at least one of NaLnF4(Yb, Er), NaLnF4(Yb, Tm), and NaLnF4(Yb, Ho), wherein Ln is at least one of Y, Gd, La, and Lu. More preferably, it is NaLnF4(Yb, Er), wherein the Yb doping ratio is 18% (by weight) and the Er doping ratio is 2% (by weight).

[0034] In a third aspect, the present invention provides a sensor for analyzing intermolecular interactions, comprising: a light-guiding material and a rare earth upconversion nanomaterial with anti-quenching modification, wherein the anti-quenching modified rare earth upconversion nanomaterial is adhered to one end face of the light-guiding material; the other end of the light-guiding material serves as an incident end for excitation light and an emitting end for emitted light; the light-guiding material serves as an excitation light conductor and an emission light conductor; the surface modification comprises anti-quenching modification and ligand coating; the anti-quenching modification comprises encapsulating the rare earth upconversion nanomaterial with a polymer or an inert inorganic material; and the ligand coating comprises attaching a biological ligand molecule capable of specifically binding to a target molecule to the anti-quenching modified rare earth upconversion nanomaterial;

[0035] Alternatively, the sensor comprises a light-guiding material and a rare earth upconversion nanomaterial with an anti-quenching modification, wherein the anti-quenching modified rare earth upconversion nanomaterial is adhered to one end face of the light-guiding material; the other end of the light-guiding material serves as the incident end for excitation light and the emission end for emitted light; the anti-quenching modification comprises encapsulating the rare earth upconversion nanomaterial with a polymer or inert inorganic material and grafting active groups onto the surface of the encapsulation layer; the active groups are at least one of carboxyl, amino, thiol, and succinimide groups; the active groups provide active attachment sites for coupling to biological ligand molecules. For the latter approach, the end face of the light-guiding material must be temporarily coated with the biological ligand molecule before the interaction between the biological ligand molecule and the target molecule is measured, and then analyzed and detected.

[0036] The sensor can be connected to the laser emitter and the emission light receiver via a coupling optical fiber. The coupling optical fiber has a main end and two branch ends, the main end is connected to the sensor, the two branch ends are connected to the main end, and the two branch ends are respectively connected to the laser emitter and the emission light receiver.

[0037] In the process of preparing the sensor, when modifying the rare earth upconversion nanomaterial, it can be first subjected to anti-quenching modification and then coated with a ligand, and then adhered to the end face of a quartz needle. In this case, some ligand molecules will face the end face of the quartz needle and cannot contact the target molecules, but this mode is easier to mass produce. Alternatively, the rare earth upconversion nanomaterial can be first subjected to anti-quenching modification, then adhered to the end face of the quartz needle, and then coated with biological ligand molecules. For example, the rare earth upconversion nanomaterial can be first subjected to anti-quenching modification, and carboxyl groups (active groups) are introduced during the anti-quenching modification process, and then the carboxyl groups are activated using an EDC / NHS mixed solution; the surface of the light-guiding material (preferably a quartz needle) is cleaned with ethanol and then dried, an appropriate amount of polylysine L-type solution is added dropwise to the end face of the quartz needle, dried, and the end face of the quartz needle is connected to the polylysine; then a dispersion of the rare earth upconversion nanomaterial treated with carboxyl group activation is added dropwise to the end face of the quartz needle, and after drying, the rare earth upconversion nanomaterial treated with carboxyl group activation is adhered to the end face of the quartz needle. Next, the carboxyl-activated rare earth upconversion nanomaterial is linked to a biological ligand molecule. Specifically, a quartz needle loaded with the rare earth upconversion nanomaterial is placed in a solution containing a biological ligand molecule (such as an antibody) and incubated at a predetermined temperature for a predetermined time. The antibody couples to the activated carboxyl groups on the surface of the rare earth upconversion nanomaterial, coating the end of the quartz needle with the antibody for subsequent analysis and detection.

[0038] In a fourth aspect, the present invention provides a device for analyzing intermolecular interactions, which includes the sensor, an excitation light emitter and an emission light receiver, wherein the excitation light emitter and the emission light receiver are correspondingly arranged at the end of the light-guiding material of the sensor opposite to the end modified with the rare earth upconversion nanomaterial.

[0039] In addition, the device also includes a data recording and analysis module for processing the recorded emission light intensity data and arranging the emission light intensity change curve over time, and calculating the kinetic constants of the interaction between the ligand molecule and the target molecule based on the change curve, including but not limited to binding kinetic parameters, such as the binding rate constant (K on ), dissociation rate constant (K off ) and affinity (K D )wait.

[0040] Furthermore, as the concentration of target molecules in the solution increases, the target molecules and the ligand molecules on the sensor surface reach binding-dissociation equilibrium more quickly. At this point, the emission light intensity no longer decreases over time, and the curve reaches a plateau. At this point, the target molecules are completely bound to the ligand molecules on the surface of the rare earth upconversion nanomaterial, and their influence on the emission light intensity ceases. Therefore, based on the time it takes to reach binding-dissociation equilibrium, the present invention can also be used to detect target molecule concentrations.

[0041] (3) Beneficial effects

[0042] The method for analyzing molecular interactions of the present invention is developed based on different technologies from existing BLI and SPR. Compared with existing technologies, its technical effects include the following aspects:

[0043] (1) Compared with BLI analysis technology, it has higher accuracy. The present invention can detect molecular interactions at the small molecule level, while BLI technology cannot detect small molecules (the signal-to-noise ratio is low, and the signal is also weaker when the molecule is too small). Therefore, BLI technology is mainly used for applications such as antigen and hybridoma screening, while the method of the present invention can detect small molecules such as antibodies.

[0044] (2) Compared with BLI analysis technology, it has a better signal-to-noise ratio, high signal intensity, and higher sensitivity. The white light used in the BLI method is also the white light received that is reflected back from the molecular layer. The non-visible excitation light used in the present invention (such as 980nm, 1550nm and other lasers) is visible light (usually visible light of about 400-700nm), which has no overlap with the wavelength of the excitation light and does not cause interference, and has a better signal-to-noise ratio. In addition, BLI relies on the difference between the white light of the received reflected light and the emitted light, and has a poor signal-to-noise ratio, especially when the target molecule is too small and its absorbance is low, which will cause the signal intensity to be further weakened. The emitted light generated by the rare earth up-conversion nanomaterial received by the present invention has a high signal intensity, higher sensitivity, and better accuracy.

[0045] (3) Since the present invention receives the emission light generated by the rare earth upconversion nanomaterial, the signal intensity is higher. Therefore, in addition to using a micro-spectrometer, the receiver of the emission light can also use other signal acquisition terminals, such as PMT, CCD, etc., which can be adjusted according to actual needs, and cost control is more flexible. Due to the high intensity of the emission light signal generated by the rare earth upconversion nanomaterial, a lower excitation light power can be used, the thermal effect is reduced, and the stability of the entire system is improved.

[0046] (4) Based on the principles of BLI and SPR, it can be seen that these two technologies currently rely on embedding on the surface of the support for testing, and cannot achieve full liquid phase detection (especially BLI cannot achieve closed detection). However, when measuring biological target molecules in some solutions, if they are not closed, the solvent in the sample is prone to volatilization and the sample is easily contaminated, and full liquid phase detection can avoid this problem. The method of the present invention can mix the modified rare earth upconversion nanomaterial with the target molecule solution and place it in a cuvette / microfluidic channel to seal it to prevent solvent volatilization or contamination, use excitation light to irradiate the cuvette and receive the emission light intensity change curve over time, and then accurately analyze and detect the molecular interaction kinetic constants.

[0047] (5) The present invention can perform simultaneous detection of multiple target molecules, while BLI and SPR cannot achieve the detection of multiple target molecules. The present invention can use different rare earth upconversion nanomaterials and embed different biological ligand molecules, and use them to detect samples containing multiple target molecules. In addition, the present invention can be used in conjunction with the use of multiple different wavelengths of excitation light (different rare earth upconversion nanomaterials match different excitation light wavelengths, and different upconversion nanomaterials must ensure that the emission spectra have separation) for irradiation, which can prompt the corresponding rare earth upconversion nanomaterials to generate emission light of different wavelengths, thereby achieving multi-channel detection and having a higher detection throughput.

[0048] (6) The present invention is more flexible in use. It can realize full liquid phase detection, or it can use a BLI-like method to embed rare earth upconversion nanomaterials and biological ligand molecules into the end face of a highly light-conducting material such as a quartz needle to prepare a sensor. It can also embed and fix rare earth upconversion nanomaterials and biological ligand molecules in a cuvette or micro-tube (transparent capillary) for detection. It is applicable to a wider range of scenarios, and according to different forms of use, a series of downstream detection consumables or instrument devices can be developed.

[0049] (7) BLI requires different end-face treatments for different target molecules to measure target molecules of different sizes. The present invention does not require consideration of target molecule size and can detect small molecules as small as less than 1 nanometer and viruses as large as hundreds of nanometers, with lower costs.

[0050] (8) Compared with SPR technology, it has high precision comparable to SPR, and has the characteristics of low cost, rapid detection, and the ability to realize multi-channel detection and full liquid phase detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Schematic diagram of the method for analyzing molecular interactions according to the present invention.

[0052] Figure 2 This is the emission light intensity-time variation curve measured in Example 1.

[0053] Figure 3 This is the emission light intensity-time variation curve obtained by testing the test samples with different concentrations in Example 2.

[0054] Figure 4 Schematic diagram of a sensor based on the principle of analyzing intermolecular interactions of the present invention.

[0055] Figure 5 This is the SEM image of the rare earth upconversion nanomaterial adhered to the bottom of the sensor. DETAILED DESCRIPTION

[0056] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation methods in conjunction with the accompanying drawings.

[0057] Example 1

[0058] This embodiment provides a method for analyzing intermolecular interactions, the steps are as follows:

[0059] (1) Preparation of NaYF4 (18% Yb, 2% Er)

[0060] NaF, YCl₃, YbCl₃, and ErCl₃ were weighed and dissolved in a stoichiometric mixture of oleic acid and 1-octadecanol. Under an argon atmosphere, the mixture was heated to 300°C and maintained at this temperature for 1.5 hours to promote the reaction. This yielded a highly dispersed and uniform rare earth upconversion nanomaterial, NaYF₄ (18% Yb, 2% Er), in the form of nanospheres with a particle size of 20-30 nm.

[0061] The preparation method of rare earth upconversion nanomaterials is an existing technology and can be prepared in the laboratory or purchased as needed. In addition to the high temperature solvent decomposition method of this embodiment, a coprecipitation method can also be used for preparation.

[0062] (2) Anti-quenching modification

[0063] 0.1 mmol of the rare earth upconversion nanomaterial NaYF4 (18% Yb, 2% Er) was dispersed in 1 ml of chloroform. Methoxypolyethylene glycol phospholipid mPEG-PL and methoxypolyethylene glycol phospholipid carboxyl mPEG-PLC were mixed in a mass ratio of 7:3 and dissolved in chloroform at 25 mg / ml. Due to their similar structures, mPEG-PL and mPEG-PLC are well soluble and mix well, resulting in a more uniform distribution of carboxyl groups on the rare earth upconversion nanomaterial. The dispersed rare earth upconversion nanomaterial, mPEG-PL, and mPEG-PLC solutions were mixed in an eggplant-shaped flask and rotary evaporated to dryness to obtain a rare earth upconversion nanomaterial co-encapsulated with mPEG-PL and mPEG-PLC. Finally, ultrapure water was added to ultrasonically disperse the mixture at a concentration of 0.1 mmol / 5 ml and stored in a refrigerator. After being encapsulated by mPEG-PL and mPEG-PLC, the rare earth upconversion nanomaterials have good anti-quenching properties, and their fluorescence properties will not be significantly weakened even when in direct contact with water. At the same time, mPEG-PLC also introduces carboxyl groups on the surface of the rare earth upconversion nanomaterials, preparing for the subsequent further encapsulation of biological ligand molecules.

[0064] (3) Ligand coating

[0065] Take 100uL of the dispersion of the anti-quenching modified rare earth upconversion nanomaterial, disperse it in pH=8 Tris buffer after centrifugation, add 5mg EDC and 5mg NHS solution, put it in a 2ml centrifuge tube and activate the carboxyl group on a shaker at room temperature for 4h, and centrifuge and wash it three times with Tris buffer to obtain the rare earth upconversion nanomaterial with activated carboxyl group.

[0066] The rare earth upconversion nanomaterial with activated carboxyl groups was placed in a 2 mL centrifuge tube, 100 uL of pH = 8 Tris buffer was added, 2 uL of 1 mg / mL interleukin-6 antibody (IL-6 antibody) was added to the centrifuge tube, and the shaker temperature was 37 ° C for 4 hours. After centrifugation, the rare earth upconversion nanomaterial coated with IL-6 antibody was collected.

[0067] (4) Analysis of molecular interaction constants

[0068] In a 2mL centrifuge tube, add 100uL of pH=8 Tris buffer and 3uL of IL-6 protein with a concentration of 1mg / mL. Add the rare earth up-conversion nanomaterial coated with IL-6 antibody prepared above to the centrifuge tube, take 5uL and transfer it to a cuvette with an optical path length of 0.1cm, seal the cuvette, excite the rare earth up-conversion nanomaterial with a 980nm laser, and use a portable spectrometer from Fuxiang Optics to receive the emitted light. The emitted light has two main peaks with good luminosity, namely 460-500nm and 520-570nm, which are clearly separated from the excitation light of 980nm wavelength. During the continuous detection process, the total detection time is 80 seconds, and the emission light intensity is continuously detected and recorded, and the received signals are tabulated every 20 seconds, so that the signal changes can be directly observed. The emission light intensity continues to decrease with the extension of the binding time. The principle is as follows. Figure 1 As shown in the figure: As the binding time increases, IL-6 in the solution continues to aggregate and connect to the IL-6 antibody on the surface of the rare earth upconversion nanomaterial, which causes the excitation light power actually received by the rare earth upconversion nanomaterial to decrease. The emission light intensity of the rare earth upconversion nanomaterial continues to decrease with the binding process until the binding of IL-6 and IL-6 antibody reaches a stable state, that is, the binding-dissociation equilibrium.

[0069] According to the original data of the recorded results, the emission light intensity (Intensity)-time (Time) change curve is made, the curve is as follows Figure 2 As shown in the figure, the molecular interaction binding rate constant (K) of IL-6 antibody and IL-6 protein was calculated based on the emission light intensity-time change curve. on ), dissociation rate constant (K off ) and affinity (K D) etc. As shown in the figure, the curve has a good linear shape, and after fitting, a smoother curve can be obtained. The calculation process can refer to the existing BLI calculation process, and other mathematical methods can also be used.

[0070] Mathematical processing methods include (1) selecting an appropriate model: for example, for a simple binding-dissociation process, a first-order reaction model can be used. (2) fitting the data: using nonlinear least squares or other appropriate fitting algorithms to find the optimal binding rate constant K on value and dissociation rate constant K off Use software tools (such as Origin, GraphPad Prism, etc.) to fit the experimental data. (3) Once K is obtained on Value and K off The value can be calculated to get K D =K off / K on .

[0071] IL-6 is a cytokine with a molecular weight of approximately 26 kilodaltons (kDa), composed of 184 amino acids, and a particle size of only 2-4 nm. The emission intensity-time curves plotted by the present method are well-defined, and the particle size of the target molecule can be as small as a few nanometers. This demonstrates the high precision of the present method, making it easy to detect small molecules with greater accuracy than existing BLI analysis techniques.

[0072] In this embodiment, the rare earth upconversion nanomaterial particles modified with anti-quenching and ligand coating can be used as detection consumables for molecular interaction analysis and are part of the test kit. Molecular interactions can be analyzed and detected in conjunction with existing laboratory equipment, optical instruments and computing devices.

[0073] Example 2

[0074] This example tests antigens at different concentrations. In this example, four gradient concentrations are set, namely:

[0075] Concentration 1: In a 2 mL centrifuge tube, add 100 uL of pH 8 Tris buffer and 1 uL of 1 mg / mL IL-6 protein.

[0076] Concentration 2: In a 2 mL centrifuge tube, add 100 uL of pH 8 Tris buffer and 2 uL of 1 mg / mL IL-6 protein.

[0077] Concentration 3: In a 2 mL centrifuge tube, add 100 uL of pH=8 Tris buffer and 3 uL of 1 mg / mL IL-6 protein.

[0078] Concentration 4: In a 2 mL centrifuge tube, add 100 uL of pH 8 Tris buffer and 4 uL of 1 mg / mL IL-6 protein.

[0079] The preparation process of the rare earth upconversion nanomaterial coated with IL-6 antibody used in this example is shown in Example 1.

[0080] Take 5uL and transfer it into a cuvette with an optical path length of 0.1cm. The cuvette is sealed and a 980nm laser is used to excite the rare earth upconversion nanomaterial. The emitted light is received using a portable spectrometer from Fuxiang Optics. During the continuous detection process, the total detection time is 80 seconds. The emission light intensity is continuously detected and recorded, and the received signal is tabulated every 20 seconds. The emission light intensity (Intensity)-time (Time) change curve is prepared according to the recorded results. After fitting, a curve with good smoothness is obtained, as shown below. Figure 3 shown.

[0081] like Figure 3 As shown, for the same primary antibody (all IL-6 antibodies), the emission light intensity-time curves for molecular interactions differ at different antigen concentrations. From bottom to top, the curve shows a decreasing gradient of sample antigen concentration. That is, the higher the target molecule concentration in the sample, the faster the emission light intensity-time curve reaches plateau. This indicates that higher concentrations of the target molecule (antigen) lead to a shorter time for equilibrium between the target molecule and the rare earth upconversion nanomaterial coated with the bioligand (IL-6 antibody). At the highest concentration (concentration 4), binding is essentially complete after 20 seconds. Based on this principle, the time it takes for the emission light intensity-time curve to reach equilibrium can be used to calculate the target molecule concentration in the solution.

[0082] Example 3

[0083] This embodiment provides a sensor based on the concept of the present invention. Figure 4 As shown, a layer of rare earth upconversion nanomaterial that has been modified with anti-quenching and coated with ligands is attached to the front end of the light-guiding material column (preferably a quartz needle in this embodiment). The preparation process of the sensor is as follows:

[0084] (1) According to the method of Example 1, nanospheres of rare earth upconversion nanomaterial NaYF4 (18% Yb, 2% Er) with a particle size of 20-30 nm were modified for anti-quenching and stored in ultrapure water at a concentration of 0.1 mmol / 5 ml.

[0085] (2) Take 100uL of the dispersion of the anti-quenching modified rare earth upconversion nanomaterial, disperse it in pH=8 Tris buffer after centrifugation, add 5mg EDC and 5mg NHS solution into it, put it in a 2ml centrifuge tube and shake it at room temperature to activate the carboxyl group for 4h, and wash it three times with Tris buffer by centrifugation to obtain the rare earth upconversion nanomaterial with activated carboxyl group, and then store it in ultrapure water at a concentration of 0.1mmol / 5ml and refrigerate it for use.

[0086] (3) Take a quartz needle with a diameter of 600 μm and a length of 15 cm (quartz needles can be selected as needed), clean its surface with alcohol, place it in a dry and ventilated place, and arrange the dried quartz needles neatly. Prepare a 10× polylysine L-type solution (original concentration 0.01% (w / v)). Immerse the end face of the quartz needle in polylysine for 3 minutes, then remove it and wash it in pH=8 Tris buffer to ensure that there is only one layer of polylysine on the surface of the quartz needle. Polylysine has strong adhesion and can easily adhere to quartz or glass products. Quartz needles have good light transmittance (light conductivity) and chemical inertness.

[0087] (4) 2 μl of rare earth upconversion nanomaterial with a concentration of 0.1 mmol / 5 ml was added dropwise to the end of the quartz needle to allow it to bind to polylysine. The rare earth upconversion nanomaterial with activated carboxyl groups was adhered to the end of the quartz needle.

[0088] (5) The end face of the quartz needle with the rare earth upconversion nanomaterial attached was placed in a 2 ml centrifuge tube, 200 ul of pH = 8 Tris buffer was added, 2 uL of interleukin-6 antibody (IL-6 antibody) with a concentration of 1 mg / ml was added to the centrifuge tube, and the shaking temperature was 37 ° C for 4 h. The IL-6 antibody formed an amide bond with the activated carboxyl group on the surface of the rare earth upconversion nanomaterial and then coated on the end face of the quartz needle. The quartz needle was then washed once with pH = 8 Tris buffer to prevent the rare earth upconversion nanomaterial from self-aggregation, and the obtained product was prepared. Figure 4 The sensor shown. The morphology of the rare earth upconversion nanomaterials adhered to the bottom of the sensor is as follows Figure 5 shown.

[0089] In other embodiments, a sensor product can be manufactured and sold simply by attaching a rare earth upconversion nanomaterial containing active groups (such as carboxyl, amino, thiol, or succinimide) to the end of a quartz needle. Before conducting molecular interaction detection and analysis, the experimenter can temporarily select a corresponding biological ligand molecule based on the target molecule to be detected and then coat the selected biological ligand molecule onto the rare earth upconversion nanomaterial on the end of the quartz needle using an appropriate method.

[0090] How to graft active groups onto the surface of materials and coat biological ligand molecules is a common method in the field of biotechnology. Those skilled in the art can choose any method that can achieve the goal according to their needs, or can refer to the methods listed in the specification of the present invention to achieve it.

[0091] Example 4

[0092] This embodiment provides a device for analyzing molecular interactions based on the concepts of the present invention, which includes integrating the sensor of Example 3 with an optical analysis device. Specifically, the quartz needle of the sensor of Example 3 is connected to an optical instrument via an optical fiber. The optical instrument includes a laser emitter and an emission light receiver. The laser emitter can adjust the laser wavelength according to the type of rare earth upconversion nanomaterial in the sensor. The emission light receiver receives the emitted light from the end of the quartz needle opposite the detection end and measures the emitted light intensity. The device should also include a data recording and processing calculation unit. Based on the recording and calculation, the emission light intensity-time curve is exported in a visual form.

[0093] It should be noted that the specific anti-quenching modification method is not limited to the specific modification process cited in the embodiments of the present invention. Among them, the hydrophobic polymer used for anti-quenching modification is mainly some lipids or ester polymers, and PVP, PE, PP, PS, PTFE, silicone rubber, etc. can also be used, but PVA (easy to dissolve in water) is not recommended; inert inorganic materials are silicon oxides, such as SiO2, titanium dioxide, carbon materials, etc.; as long as the rare earth upconversion nanomaterials can be separated from water molecules, the effect of preventing or delaying the quenching of the fluorescence of the rare earth upconversion nanomaterial by water molecules is prevented or delayed, and it is chemically inert and not easy to react chemically with the solution where the target molecule is located. Polymers or inert inorganic materials can be used for the anti-quenching modification material, and a combination of hydrophobic polymers and inert inorganic materials can also be used.

[0094] For the anti-quenching modification method, reference can be made to the anti-quenching modification method of rare earth up-conversion nanomaterials or quantum dots disclosed in the prior art. For example, polyvinyl pyrrolidone (PVP) and silicon dioxide (SiO2) can be used as encapsulating materials to encapsulate rare earth up-conversion nanomaterial particles; or oleic acid-encapsulated rare earth up-conversion nanoparticles can be first synthesized by a high-temperature solvent thermal method, and then the carboxyl arms of oleic acid are used to encapsulate them with polypropylene.

[0095] In the ligand coating modification process, one can refer to the coating process of biological ligand molecules such as magnetic beads in biotechnology. Usually, at least one basic active group is first grafted, such as one or more of hydroxyl, carboxyl, amino, aldehyde, succinimide ester and sulfonyl groups, and then the biological ligand molecules are connected and coated by forming covalent bonds.

[0096] Furthermore, anti-quenching cannot completely eliminate the quenching phenomenon of rare earth upconversion nanomaterials in water; it can only weaken the quenching effect or prolong the quenching time, making the detection results more accurate. In some environments where molecular interaction detection and analysis require long periods of time, it is often necessary to use multiple layers of wrapping materials to wrap the rare earth upconversion nanomaterials multiple times to improve their anti-quenching properties, ensuring that they are slightly quenched during the detection period, and generally controlling the quenching degree to less than 3%. However, it should be noted that anti-quenching modification can also affect the rare earth upconversion nanomaterial's reception and luminescence intensity of lasers, weakening the signal and, in some cases, causing the particle size of the rare earth upconversion nanomaterials to increase. Therefore, when performing anti-quenching modification, technicians can choose the wrapping material or the number of wrapping layers based on the type of molecules being measured by the rare earth upconversion nanomaterials and the application environment.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for analyzing intermolecular interactions, characterized in that: include: S1. Providing a surface-modified rare earth upconversion nanomaterial, wherein the surface modification includes anti-quenching modification and ligand coating; the anti-quenching modification is to coat the rare earth upconversion nanomaterial with a polymer or an inert inorganic material; The ligand coating is to connect the biological ligand molecules that can specifically bind to the target molecules to the rare earth upconversion nanomaterial modified by anti-quenching; S2. Immersing the surface-modified rare earth up-conversion nanomaterial in a solution containing a target molecule so that the surface-modified rare earth up-conversion nanomaterial can contact the target molecule in the solution; irradiating the solution with excitation light that matches the rare earth up-conversion nanomaterial so that the rare earth up-conversion nanomaterial generates emitted light; as the target molecule continuously binds to the biological ligand molecule on the surface of the rare earth up-conversion nanomaterial, the intensity of the emitted light generated by the up-conversion nanomaterial in response to the excitation light continuously decreases until the binding of the biological ligand molecule and the target molecule reaches equilibrium or saturation, and recording a curve of the change in the emission light intensity over time during this period; S3. Calculate the curve of the emission light intensity versus time obtained in S2 to obtain the kinetic constant of the interaction between the ligand molecule and the target molecule.

2. The method according to claim 1, characterized in that In S1, the rare earth upconversion nanomaterial is prepared by doping rare earth ions into an inorganic matrix; the rare earth ions are at least one of Yb, Er, Tm, Ho and Nd; and the inorganic matrix is one of fluoride, oxide, phosphate and vanadate.

3. The method according to claim 1, characterized in that In S1, the rare earth upconversion nanomaterial is at least one of NaLnF4(Yb, Er), NaLnF4(Yb, Tm) and NaLnF4(Yb, Ho), wherein Ln is at least one of Y, Gd, La and Lu.

4. The method according to claim 1, wherein In S1, the rare earth upconversion nanomaterial is a nanoparticle with a particle size of 15-100 nm.

5. The method according to claim 1, characterized in that In S1, the anti-quenching modification process is as follows: S11, dispersing the rare earth upconversion nanomaterial in an organic solvent to obtain an upconversion nanomaterial dispersion; dissolving methoxy polyethylene glycol phospholipid and an active group modifier in the organic solvent to obtain an inclusion solution; the active group modifier is at least one of methoxy polyethylene glycol phospholipid carboxyl mPEG-PLC, methoxy polyethylene glycol amino mPEG-NH2, methoxy polyethylene glycol thiol mPEG-SH, and methoxy polyethylene glycol-succinimide ester MPEG2000-NHS; S12. After mixing the upconversion nanomaterial dispersion and the inclusion solution, the organic solvent is evaporated to obtain the rare earth upconversion nanomaterial modified with anti-quenching.

6. The method according to claim 5, characterized in that In S1, the anti-quenching modification process is: S11, dispersing the rare earth upconversion nanomaterial in chloroform at a concentration of 0.1-0.5M to obtain an upconversion nanomaterial dispersion; dissolving the methoxy polyethylene glycol phospholipid and the PEGylated active group modifier in chloroform at a mass ratio of 1-10:1-10 to obtain an inclusion solution with a concentration of 20-50 mg / ml; S12. Mix the upconversion nanomaterial dispersion and the inclusion solution, evaporate the solvent, add pure water at a concentration of 0.01-0.04 M, and disperse by ultrasonication to obtain a dispersion of the rare earth upconversion nanomaterial modified with anti-quenching.

7. A kit for analyzing intermolecular interactions, characterized in that: The invention comprises a surface-modified rare earth up-conversion nanomaterial, wherein the surface modification comprises anti-quenching modification and ligand coating; the anti-quenching modification is to coat the rare earth up-conversion nanomaterial with a polymer or an inert inorganic material; The ligand coating is to connect the biological ligand molecules that can specifically bind to the target molecules to the rare earth upconversion nanomaterial modified by anti-quenching; Alternatively, the kit comprises: a rare earth upconversion nanomaterial that has been modified with anti-quenching properties and a biological ligand molecule, wherein the anti-quenching modification comprises encapsulating the rare earth upconversion nanomaterial with a polymer or an inert inorganic material and grafting active groups onto the surface of the encapsulation layer; the active groups are at least one of carboxyl, amino, thiol and succinimide groups, and the active groups provide active connection sites for coupling the biological ligand molecule; and the biological ligand molecule is capable of specifically binding to the target molecule.

8. The kit according to claim 7, characterized in that The biological ligand molecule is a protein, antibody, antigen, hapten, polysaccharide, nucleic acid probe or cell.

9. The kit according to claim 7, characterized in that The anti-quenching modification method comprises the following steps: dispersing a rare earth up-conversion nanomaterial in an organic solvent to obtain an up-conversion nanomaterial dispersion; dissolving a methoxy polyethylene glycol phospholipid and an active group modifier in the organic solvent to obtain an inclusion solution; the active group modifier is at least one of methoxy polyethylene glycol phospholipid carboxyl mPEG-PLC, methoxy polyethylene glycol amino mPEG-NH2, methoxy polyethylene glycol thiol mPEG-SH, and methoxy polyethylene glycol-succinimide ester MPEG2000-NHS; mixing the up-conversion nanomaterial dispersion and the inclusion solution, and then volatilizing the organic solvent to obtain the anti-quenching modified rare earth up-conversion nanomaterial.

10. A sensor for analyzing intermolecular interactions, characterized in that: include: A light-guiding material and a surface-modified rare earth up-conversion nanomaterial, wherein the surface-modified rare earth up-conversion nanomaterial is adhered to one end face of the light-guiding material; the other end of the light-guiding material is an incident end for excitation light and an emitting end for emission light; the light-guiding material is an excitation light conductor and an emission light conductor; the surface modification includes anti-quenching modification and ligand coating; the anti-quenching modification is to coat the rare earth up-conversion nanomaterial with a polymer or an inert inorganic material; The ligand coating is to connect the biological ligand molecules that can specifically bind to the target molecules to the rare earth upconversion nanomaterial modified by anti-quenching; Alternatively, the sensor includes: a light-guiding material and a rare earth upconversion nanomaterial with anti-quenching modification, wherein the anti-quenching modified rare earth upconversion nanomaterial is adhered to one end face of the light-guiding material; the other end of the light-guiding material is the incident end for excitation light and the emission end for emitted light; the anti-quenching modification includes wrapping the rare earth upconversion nanomaterial with a polymer or an inert inorganic material and grafting active groups on the surface of the wrapping layer; the active groups are at least one of carboxyl, amino, thiol and succinimide groups; and the active groups provide active connection sites for coupling biological ligand molecules.

11. A device for analyzing intermolecular interactions, characterized in that: It includes a surface-modified rare earth upconversion nanomaterial, an excitation light emitter and an emission light receiver; the surface modification includes anti-quenching modification and ligand coating; the anti-quenching modification is to wrap the rare earth upconversion nanomaterial with a polymer or an inert inorganic material; the ligand coating is to connect a biological ligand molecule that can specifically bind to a target molecule to the anti-quenching modified rare earth upconversion nanomaterial; or the surface modification only includes anti-quenching modification, the anti-quenching modification includes wrapping the rare earth upconversion nanomaterial with a polymer or an inert inorganic material and grafting active groups on the surface of the wrapping layer; the active group is at least one of a carboxyl group, an amino group, a thiol group and a succinimide group, and the active group provides an active connection site for coupling the biological ligand molecule.

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