Method, kit, sensor and device for analyzing intermolecular interaction

Abstract

The invention relates to the technical field of molecular interaction analysis, in particular to a method for analyzing intermolecular interaction, which comprises the following steps of: adopting a rare earth material with ligand molecules modified on the surface as an up-conversion material, contacting the up-conversion material with target molecules in a solution system, and irradiating the solution system by using exciting light matched with the rare earth material to obtain the intermolecular interaction. The intensity of the emitted light is changed by continuously changing the intensity of the exciting light actually received by the up-conversion material in the dynamic process of continuously combining and dissociating target molecules with ligand molecules of the up-conversion material, and a molecular interaction dynamic constant can be calculated by recording a change curve of the intensity of the emitted light along with time. According to the present invention, the BLI technology has the binding rate constant (Kon), the dissociation rate constant (Koff), the affinity (KD) and the like, and the technical problems of low precision of the existing BLI technology, expensive SPR technology and the like are solved. Based on the principle, the invention also develops a series of experimental consumables, kits, sensors and analysis 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 molecular 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 material science. Molecular interaction analysis technologies are diverse, involving a wide range of instruments. According to the principles, they mainly include surface plasmon resonance technology (SPR), biomembrane interferometry technology (BLI), grating coupled interferometry technology (GCI), microthermophoresis technology (MST) and isothermal titration calorimetry technology (ITC). From the statistical analysis of the winning bids in the past two years, it is 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 metal surface. 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, which in turn affects the intensity of the reflected light. By measuring the change in the intensity of the reflected light, the binding of the 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) 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 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, it changes the optical thickness of the probe end. This change in optical thickness will cause changes 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 there are still the following problems: ① Insufficient accuracy; ② BLI technology requires the ligand to be fixed to the tip of the quartz needle, indicating that each probe can only perform one detection, and different coatings need to be coated on the tip of the quartz needle to form probes for different molecules; ③ The probe needs to be continuously in the solution to be tested during the entire detection process, resulting in the solution to be tested being unable to be in a closed environment, which is prone to solution volatilization and pollution by the experimental environment, 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] The advantage of SPR technology is its high accuracy, but its cost is very high, which is basically dozens to more than 100 times that of BLI. In order to balance low cost, convenience and high accuracy, the industry often uses BLI for rough screening of samples, and then uses SPR for precise detection of the samples after rough screening, but this method cannot fundamentally solve the problem of high cost of molecular interaction analysis.

[0007] Based on the working principles of BLI and SPR technologies, it can be seen 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 problems of the prior art, the inventors hope to propose a molecular interaction analysis technology that combines high precision, low cost, and high flexibility and convenience in practical use. 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 molecular interactions, wherein a rare earth material with a ligand molecule modified on the surface is used as an upconversion material, the upconversion material is contacted with a target molecule in a solution system, and an excitation light matching the rare earth material is used to irradiate the solution system. As the target molecule continuously binds to and dissociates from the ligand molecule of the upconversion material, the excitation light intensity and excitation light power actually received by the upconversion material are 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, the present invention solves the technical problems of low accuracy of existing BLI technology and expensive SPR technology. Based on the above principles, the present invention also develops a series of experimental consumables, kits, sensors and analytical devices for molecular interaction analysis.

[0010] (II) Technical solution

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

[0012] S1. Providing a surface-modified rare earth up-conversion nanomaterial, wherein the surface modification includes anti-quenching modification and ligand coating; the anti-quenching modification is to encapsulate the rare earth up-conversion nanomaterial with a polymer or an inert inorganic material; the ligand coating is to connect a biological ligand molecule that can specifically (directly or indirectly) bind to a target molecule to the anti-quenching modified rare earth up-conversion nanomaterial;

[0013] S2, immersing the surface-modified rare earth up-conversion nanomaterial in a solution containing target molecules, so that the surface-modified rare earth up-conversion nanomaterial can contact the target molecules in the solution; irradiating the solution with excitation light matching the rare earth up-conversion nanomaterial, so that the rare earth up-conversion nanomaterial generates emission light; as the target molecules continuously combine with the biological ligand molecules on the surface of the rare earth up-conversion nanomaterial, the emission light intensity generated by the up-conversion nanomaterial under the excitation light continuously decreases until the combination of the biological ligand molecules and the target molecules reaches equilibrium or saturation, and recording the change curve of 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; the inorganic matrix is ​​one of fluoride, oxide, phosphate and 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 up-conversion 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% (mass ratio) and the Er doping ratio is 2% (mass ratio).

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

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

[0019] S11, 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 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;

[0020] S12, after mixing the upconversion nanomaterial dispersion and the inclusion solution, evaporate the organic solvent to obtain the rare earth upconversion nanomaterial modified with anti-quenching. Pure water can be added for dispersion and stored in the form of dispersion.

[0021] After anti-quenching modification treatment, the anti-quenching ability of rare earth upconversion nanomaterials can be improved to prevent the luminescence intensity of rare earth upconversion nanomaterials from being weakened and quenched under the action of water molecules. In addition to using phospholipid hydrophobic materials such as methoxy polyethylene glycol phospholipids for packaging, other hydrophobic organic polymer materials, non-organic polymer hydrophobic materials or silica gel can also be used to protect the core of rare earth upconversion nanomaterials, thereby reducing the influence of water molecules on them.

[0022] As a preferred embodiment of the present invention, in S11, methoxy polyethylene glycol phospholipids and active group modifiers are used to co-encapsulate the rare earth up-conversion nanomaterials, which greatly improves the anti-quenching performance of the rare earth up-conversion nanomaterials in water, and realizes the connection of active groups such as carboxyl, amino, thiol, polylysine, succinimide, etc. on the surface of the rare earth up-conversion nanomaterials, which are prepared for further connection of biological ligand molecules. The phospholipid encapsulation layer has good affinity with proteins (biological ligand molecules or target molecules), which is more conducive to the subsequent modification process.

[0023] Specifically, carboxyl groups can react with molecules containing primary amine groups by forming amide bonds, making it easy for carboxyl groups to couple with biomolecules (such as proteins and antibodies); amino groups can react with a variety of compounds containing active ester groups (such as succinimidyl esters) to form stable amide bonds. In addition, amino groups can also react with aldehydes or ketones through Schiff base reactions. The presence of amino groups makes it possible to further modify the molecule, and other functional groups can be introduced or connected to 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, which are easy to couple with amine-containing biomolecules (such as proteins and peptides). Succinimidyl groups can undergo coupling reactions under milder conditions. Through the modification of these groups, they can be coupled with specific biological ligand molecules (such as antibodies) and produce binding-dissociation interactions with target molecules in the solution.

[0024] It should be noted that the technology of introducing biomolecular ligands with active groups such as hydroxyl, carboxyl, amino, aldehyde, succinimidyl ester and sulfonyl as basic groups is a conventional technology and will not be described in detail here. The biomolecular ligands can be proteins, antibodies, antigens, haptens, polysaccharides, nucleic acid probes or cells; such as Streptavidin, Avidin, BSA, Protein A, Protein G, Biotin, polypeptides, antibodies, various other proteins and nucleic acid probes (DNA, RNA probes), etc.

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

[0026] S11, the rare earth upconversion nanomaterial is dispersed in chloroform at a concentration of 0.1-0.5M to obtain an upconversion nanomaterial dispersion; the methoxy polyethylene glycol phospholipid and the PEGylated active group modifier are dissolved 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, mixing the upconversion nanomaterial dispersion and the inclusion solution, rotary evaporating the solvent, adding pure water at a concentration of 0.01-0.04M, and ultrasonically dispersing 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, which comprises a surface-modified rare earth upconversion nanomaterial, wherein the surface modification comprises 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 connect a biological ligand molecule that can specifically bind to a target molecule to the anti-quenching modified rare earth upconversion nanomaterial;

[0029] Alternatively, the kit comprises: a rare earth up-conversion nanomaterial modified with anti-quenching and a biological ligand molecule, wherein the anti-quenching modification comprises encapsulating the rare earth up-conversion nanomaterial with a polymer or an inert inorganic material and grafting active groups on 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 can specifically bind to the target molecule.

[0030] For the latter scheme, it is necessary to temporarily coat the biological ligand molecule on the surface of the rare earth conversion nanomaterial before determining the interaction between the biological ligand molecule and the target molecule, and then perform analytical detection.

[0031] According to a preferred embodiment of the present invention, the method for anti-quenching modification 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 by 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; the inorganic matrix is ​​one of fluoride, oxide, phosphate and 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% (mass ratio) and the Er doping ratio is 2% (mass ratio).

[0034] In a third aspect, the present invention provides a sensor for analyzing intermolecular interactions, which comprises: a light-guiding material and a rare earth up-conversion nanomaterial modified with anti-quenching, wherein the rare earth up-conversion nanomaterial modified with anti-quenching 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 comprises anti-quenching modification and ligand coating; the anti-quenching modification is to wrap the rare earth up-conversion 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 rare earth up-conversion nanomaterial modified with anti-quenching;

[0035] Alternatively, the sensor includes: a light-guiding material and a rare earth up-conversion nanomaterial with anti-quenching modification, wherein the rare earth up-conversion nanomaterial with anti-quenching modification is adhered to one end face of the light-guiding material; the other end of the light-guiding material is the incident end of the excitation light and the emission end of the emission light; the anti-quenching modification includes encapsulating the rare earth up-conversion nanomaterial with a polymer or an inert inorganic material and grafting active groups on 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 connection sites for coupling biological ligand molecules. For the latter scheme, it is necessary to temporarily coat the end face of the light-guiding material with biological ligand molecules before determining the interaction between the biological ligand molecules and the target molecules, and then perform analysis and detection.

[0036] The sensor can be connected to the laser transmitter and the emission light receiver through 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 transmitter and the emission light receiver.

[0037] In the process of preparing the sensor, when modifying the rare earth upconversion nanomaterial, the anti-quenching modification can be performed first, and then the ligand can be coated, and then the ligand can be adhered to the end face of the quartz needle. In this case, some ligand molecules will face the end face of the quartz needle and cannot contact the target molecule, but this mode is easier to mass produce; the rare earth upconversion nanomaterial can also be first modified for anti-quenching, and then adhered to the end face of the quartz needle, and then the biological ligand molecule can be coated. For example, the rare earth upconversion nanomaterial can be first modified for anti-quenching, and a carboxyl group (active group) can be introduced during the anti-quenching modification process, and then the carboxyl group can be activated using an EDC / NHS mixed solution; the surface of the light-guiding material (preferably a quartz needle) is washed with ethanol and then dried, and an appropriate amount of polylysine L-type solution is added dropwise to the end face of the quartz needle, and dried to connect the polylysine to the end face of the quartz needle; then the dispersion of the rare earth upconversion nanomaterial treated with carboxyl activation is added dropwise to the end face of the quartz needle, and after drying, the rare earth upconversion nanomaterial treated with carboxyl activation is adhered to the end face of the quartz needle. Next, the rare earth upconversion nanomaterials treated with carboxyl activation are connected to biological ligand molecules. Specifically, the quartz needle with the rare earth upconversion nanomaterial can be placed in a solution containing biological ligand molecules (such as antibodies), incubated at a certain temperature for a preset time, and the antibodies are coupled with the activated carboxyl groups on the surface of the rare earth upconversion nanomaterials. At this point, the antibodies are coated on the end surface of the quartz needle 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 an end of the light-guiding material of the sensor opposite to the end modified with rare earth up-conversion nanomaterials.

[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 the binding kinetic parameters, such as the binding rate constant (K on ), dissociation rate constant (K off ) and affinity (K D )wait.

[0040] In addition, as the concentration of the target molecule in the solution increases, the target molecule and the ligand molecule on the sensor end surface can reach the binding-dissociation equilibrium faster, at which time the emission light intensity no longer decreases with time, and the curve reaches a plateau; at this time, the target molecule and the ligand molecule on the surface of the rare earth upconversion nanomaterial are completely bound, and the influence on the emission light intensity stops. Therefore, according to the time to reach the binding-dissociation equilibrium, the present invention can also be used to detect the concentration of the target molecule.

[0041] (III) Beneficial effects

[0042] The method for analyzing molecular interactions of the present invention is developed based on BLI and SPR technologies that are different from the existing ones. Compared with the 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 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 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, does not cause interference, and has a better signal-to-noise ratio. In addition, BLI relies on the difference between the received reflected light and the white light of 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 nanomaterials 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 up-conversion nanomaterial, the signal strength 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 the cost control is more flexible. Since the emission light signal intensity generated by the rare earth up-conversion nanomaterial is high, a lower excitation light power can be used, and the thermal effect is lower, so that the stability of the entire system is better.

[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, so as to 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 detect multiple target molecules. The present invention can use different rare earth up-conversion nanomaterials and embed different biological ligand molecules, and use them to detect samples containing multiple target molecules, and use multiple different wavelengths of excitation light (different rare earth up-conversion nanomaterials match different excitation light wavelengths, and different up-conversion nanomaterials must ensure that the emission spectra have separation) for irradiation, which can prompt the corresponding rare earth up-conversion 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) For different target molecules, BLI needs to process its end surface differently to achieve the measurement of target molecules of different sizes. The present invention does not need to consider the size of the target molecule, 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 this Example 1.

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

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

[0055] Figure 5 This is a 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 modes 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] According to the stoichiometric ratio, NaF, YCl3, YbCl3 and ErCl3 were weighed and dissolved in a mixed solution of oleic acid and 1-octadecanol. Under an argon atmosphere, the mixed solution was heated to 300°C and kept at this temperature for 1.5 hours to promote the reaction, thereby obtaining a rare earth upconversion nanomaterial NaYF4 (18% Yb, 2% Er) with good dispersion and uniformity, which was a nanosphere with a particle size of 20-30 nm.

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

[0062] (2) Anti-quenching modification

[0063] Take 0.1mmol of rare earth up-conversion nanomaterial NaYF4 (18% Yb, 2% Er) and disperse it in 1ml of chloroform. Mix methoxy polyethylene glycol phospholipid mPEG-PL and methoxy polyethylene glycol phospholipid carboxyl mPEG-PLC in a mass ratio of 7:3 and dissolve them in chloroform at 25mg / ml. Since mPEG-PL and mPEG-PLC have similar structures, the two substances can be well soluble and mixed, making the distribution of carboxyl groups on the rare earth up-conversion nanomaterial more uniform. Mix the dispersed rare earth up-conversion nanomaterial and the solution of mPEG-PL and mPEG-PLC in an eggplant-shaped bottle, and after rotary evaporation and drying, obtain a rare earth up-conversion nanomaterial co-encapsulated by mPEG-PL and mPEG-PLC. Finally, add ultrapure water at a concentration of 0.1mmol / 5ml for ultrasonic dispersion and store 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 rare earth upconversion nanomaterial modified with anti-quenching, disperse it in pH=8 Tris buffer after centrifugation, add 5mg EDC and 5mg NHS solution, 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 to obtain the rare earth upconversion nanomaterial with activated carboxyl group.

[0066] The rare earth upconversion nanomaterial with activated carboxyl group was placed in a 2mL centrifuge tube, 100uL of pH=8 Tris buffer was added, 2uL of 1mg / mL interleukin-6 antibody (IL-6 antibody) was added to the centrifuge tube, incubated at 37°C for 4h, and 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 nanomaterials coated with IL-6 antibodies 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 nanomaterials 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; there is a clear separation between the 980nm wavelength light of the excitation light. 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 decreases continuously with the extension of the binding time. The principle is as follows Figure 1 As shown: As the binding time increases, the 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 during 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, a curve of emission light intensity (Intensity)-time (Time) is prepared. The curve is as follows Figure 2 As shown in Figure 2, 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 line shape, and a smoother curve can be obtained after fitting. The calculation process can refer to the existing BLI calculation process, and other mathematical methods can also be used for processing.

[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 of K can be calculated D =K off / K on .

[0071] IL-6 is a cytokine with a molecular weight of about 26 kilodaltons (kDa), composed of 184 amino acids, and a particle size of only 2-4nm. The emission light intensity-time variation curve drawn by the method of the present invention has a regular linear shape and the particle size of the target molecule to be detected can be as small as a few nanometers. It can be seen that the present invention has the advantage of high precision and can easily detect small molecules with higher precision than the existing BLI analysis technology.

[0072] In this embodiment, the rare earth upconversion nanomaterial particles modified with anti-quenching and coated with ligands can be used as detection consumables for molecular interaction analysis and are a component 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 IL-6 protein with a concentration of 1 mg / mL.

[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, seal the cuvette, use a 980nm laser to excite the rare earth upconversion nanomaterial, and use a portable spectrometer from Fuxiang Optics to receive the emitted light. During the continuous detection process, the total detection time is 80 seconds, and the emission light intensity is continuously detected and recorded. The received signal is tabulated every 20 seconds, and the emission light intensity (Intensity)-time (Time) change curve is prepared according to the recorded results. After fitting, a curve graph with good smoothness is obtained, as shown below: Figure 3 shown.

[0081] like Figure 3 As shown in the figure, under the same primary antibody (all IL-6 antibodies), different antigen concentrations have different emission light intensity-time variation curves of molecular interactions. The curve has a decreasing sample antigen concentration gradient from bottom to top, that is, the higher the concentration of the target molecule in the sample, the faster the emission light intensity-time variation curve reaches a plateau, which means that the higher the concentration of the target molecule (antigen) to be tested, the shorter the time it takes to reach equilibrium with the rare earth upconversion nanomaterial coated with the biological ligand molecule (IL-6 antibody). Among them, at the highest concentration (concentration 4), the binding is basically completed after 20 seconds. Based on this principle, the concentration of the target molecule in the solution can be calculated according to the time it takes for the emission light intensity-time variation curve to reach equilibrium.

[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 nanomaterials that have 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 rare earth upconversion nanomaterial modified with anti-quenching, disperse it in pH=8 Tris buffer after centrifugation, add 5mg EDC and 5mg NHS solution, put it in a 2ml centrifuge tube and shake it at room temperature to activate the carboxyl group for 4h, wash it three times by centrifugation with Tris buffer 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 later 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, and place it in a dry and ventilated place. Arrange the dried quartz needles neatly. Prepare 10× poly-lysine L-type solution (original concentration 0.01% (w / v)). Immerse the end face of the quartz needle in poly-lysine, soak for 3 minutes, then take it out and wash it in pH=8 Tris buffer to ensure that there is only one layer of poly-lysine on the surface of the quartz needle. Poly-lysine 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) Take 2ul of rare earth upconversion nanomaterial with a concentration of 0.1mmol / 5ml and drop it onto the end of the quartz needle to make it combine with poly-lysine. The rare earth upconversion nanomaterial with activated carboxyl groups is adhered to the end of the quartz needle.

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

[0089] In addition, in other embodiments, the sensor product can be made and sold only by adhering the rare earth upconversion nanomaterial containing active groups (such as carboxyl, amino, thiol or succinimide) to the end face of the quartz needle. Before the experimenter conducts molecular interaction detection and analysis, the corresponding biological ligand molecule is temporarily selected according to the target molecule to be detected, and the selected biological ligand molecule is coated on the rare earth upconversion nanomaterial on the end face of the quartz needle in an appropriate manner.

[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 as needed, 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 concept 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 through an optical fiber, and the optical instrument includes a laser transmitter and an emission light receiver. The laser transmitter can adjust the laser wavelength according to the type of rare earth upconversion nanomaterial in the sensor, and the emission light receiver receives the emission light from the end of the quartz needle opposite to the detection end and measures the emission light intensity. The device should also include a data recording and processing calculation unit, and 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 polymers used for anti-quenching modification are 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, it can prevent or delay the effect of water molecules quenching the fluorescence of the rare earth upconversion nanomaterials and have chemical inertness, and it is not easy to react chemically with the solution where the target molecule is located. Polymers or inert inorganic materials can be used on the anti-quenching modification materials, 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 solvothermal 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, succinimidyl ester and sulfonyl groups, and then the biological ligand molecules are connected and coated by forming covalent bonds.

[0096] In addition, 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 to make the detection results more accurate. In some environments where molecular interaction detection and analysis require a long time, it is often necessary to use multiple layers of wrapping materials to wrap the rare earth upconversion nanomaterials multiple times to improve the anti-quenching property, so as to ensure that the quenching is slight during the detection time. Generally, the quenching degree is controlled within 3%. However, it should be noted that anti-quenching modification will also affect the reception and luminescence intensity of the rare earth upconversion nanomaterials for lasers, weaken the signal, and in some cases cause 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 according to the type of molecules 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by 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 up-conversion nanomaterial, wherein 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 molecule that can specifically bind to the target molecule to the rare earth up-conversion nanomaterial modified by anti-quenching; S2, immersing the surface-modified rare earth up-conversion nanomaterial in a solution containing target molecules, so that the surface-modified rare earth up-conversion nanomaterial can contact the target molecules in the solution; irradiating the solution with excitation light matching the rare earth up-conversion nanomaterial, so that the rare earth up-conversion nanomaterial generates emission light; as the target molecules continuously combine with the biological ligand molecules on the surface of the rare earth up-conversion nanomaterial, the emission light intensity generated by the up-conversion nanomaterial under the excitation light continuously decreases until the combination of the biological ligand molecules and the target molecules reaches equilibrium or saturation, and recording the change curve of 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 up-conversion 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, characterized in that: In S1, the rare earth up-conversion 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 up-conversion nanomaterial in an organic solvent to obtain an up-conversion nanomaterial dispersion; dissolving methoxy polyethylene glycol phospholipid and an active group modifier 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; S12, after mixing the upconversion nanomaterial dispersion and the inclusion solution, evaporate the organic solvent 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, the rare earth upconversion nanomaterial is dispersed in chloroform at a concentration of 0.1-0.5M to obtain an upconversion nanomaterial dispersion; methoxy polyethylene glycol phospholipid and PEGylated active group modifier are dissolved 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, mixing the upconversion nanomaterial dispersion and the inclusion solution, rotary evaporating the solvent, adding pure water at a concentration of 0.01-0.04M, and ultrasonically dispersing to obtain a dispersion of the rare earth upconversion nanomaterial modified with anti-quenching.

7. A kit for analyzing molecular 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 and ligand coating are to encapsulate the rare earth up-conversion nanomaterial with a polymer or an inert inorganic material; the ligand coating is to connect a biological ligand molecule capable of specifically binding to a target molecule to the anti-quenching modified rare earth up-conversion nanomaterial; Alternatively, the kit comprises: a rare earth up-conversion nanomaterial modified with anti-quenching and a biological ligand molecule, wherein the anti-quenching modification comprises encapsulating the rare earth up-conversion nanomaterial with a polymer or an inert inorganic material and grafting active groups on 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 can specifically bind to the target molecule.

8. The kit according to claim 7, characterized in that The biomolecule ligand is a protein, an antibody, an antigen, a hapten, a polysaccharide, a nucleic acid probe or a 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 nano material in an organic solvent to obtain an up-conversion nano material dispersion; dissolving a methoxy polyethylene glycol phospholipid and an active group modifier 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; mixing the up-conversion nano material dispersion and the inclusion solution, and then evaporating the organic solvent to obtain the rare earth up-conversion nano material after anti-quenching modification.

10. A sensor for analyzing intermolecular interactions, characterized in that: include: A light-guiding material and a rare earth up-conversion nanomaterial with anti-quenching modification, wherein the rare earth up-conversion nanomaterial with anti-quenching modification is adhered to one end face of the light-guiding material; the other end of the light-guiding material is an incident end of the excitation light and an emitting end of the 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 use a polymer or an inert inorganic material to encapsulate the rare earth up-conversion nanomaterial; The ligand coating is to connect the biological ligand molecule that can specifically bind to the target molecule to the rare earth up-conversion nanomaterial modified by anti-quenching; Alternatively, the sensor comprises: a light-guiding material and a rare earth up-conversion nanomaterial with anti-quenching modification, wherein the rare earth up-conversion nanomaterial with anti-quenching modification is adhered to one end face of the light-guiding material; the other end of the light-guiding material is the incident end of the excitation light and the emission end of the emission light; the anti-quenching modification comprises wrapping the rare earth up-conversion 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; the active groups provide active connection sites for coupling biological ligand molecules.

11. A device for analyzing molecular interactions, characterized in that: It includes a surface-modified rare earth up-conversion nanomaterial, an excitation light emitter and an emission light receiver; the surface modification includes anti-quenching modification and ligand coating; the anti-quenching modification and ligand coating are to wrap the rare earth up-conversion 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 up-conversion nanomaterial; or the surface modification only includes anti-quenching modification, the anti-quenching modification includes wrapping the rare earth up-conversion 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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