Method, kit, sensor and device for analyzing intermolecular interaction

Abstract

A method, kit, sensor and device for analyzing intermolecular interaction. In the method for analyzing intermolecular interaction, a rare-earth material of which the surface is modified with ligand molecules is used as an up-conversion material; the up-conversion material comes into contact with target molecules in a solution system; excitation light matching the rare-earth material is used to irradiate the solution system; in a dynamic process of continuous association / dissociation between the target molecules and the ligand molecules on the up-conversion material, the intensity of excitation light actually received by the up-conversion material continuously changes, such that the intensity of emitted light changes; and by recoding a curve of the intensity of the emitted light changing with time, kinetic constants of molecular interaction, such as an association rate constant (Kon), a dissociation rate constant (Koff), and affinity (KD), are calculated. In this way, existing technical problems of low accuracy of BLI technology and high costs of SPR technology.

Description

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

[0001] The present application relates to the technical field of molecular interaction analysis, and particularly relates to a method, kit, sensor and device for analyzing intermolecular interactions. BACKGROUND

[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 operation mechanism of biological systems, and provide key information for new drug development, disease treatment and material science. Molecular interaction analysis techniques are diverse, involving a wide range of instruments. According to the principle, it mainly includes surface plasmon resonance technology (SPR), biological membrane interference technology (BLI), grating coupling interference technology (GCI), microscale thermophoresis technology (MST) and isothermal titration calorimetry technology (ITC) and the like. From the statistical analysis of the winning bid in the past two years, it is found that SPR technology and BLI technology occupy the mainstream position.

[0003] Surface plasmon resonance (SPR) technology is a label-free real-time detection technology. Its principle is that surface plasmons are electromagnetic waves existing on the surface of metal, which are generated by the collective oscillation of free electrons on the metal surface. When the biomolecules in the sample solution are fixed on the metal surface (usually gold film) and combined with another molecule, the local refractive index will change. This change will cause the propagation conditions of surface plasmons to change, and then affect the intensity of reflected light. By measuring the change of reflected light intensity, the binding situation of biomolecules can be indirectly obtained. SPR technology can monitor the binding and dissociation process of molecules in real time, and by recording the change curve of reflected light intensity with time (i.e. SPR curve), the binding kinetic parameters such as binding rate constant (K on ), dissociation rate constant (K off ) and affinity (K D ) can be obtained.

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

[0005] At present, the user needs a convenient, low-cost and higher-precision molecular interaction detection. BLI technology is low in cost and convenient to use, but still has the following problems: ① insufficient precision, ② BLI technology needs to fix ligands to the end of the quartz needle, each probe can only detect one kind of detection, and different plating layers need to be formed on the end 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 whole detection process, which causes the solution to be tested to be unable to be in a closed environment, and is easy to produce solution volatilization and experimental environment pollution, which will directly affect the analysis results; ④ BLI method uses white light, and the received light signal is the white light reflected by the molecular layer, the two kinds of light are easy to interfere with each other, and the reflected light exists energy loss, resulting in low detection signal strength, low sensitivity and low signal-to-noise ratio.

[0006] The advantage of SPR technology is high precision, but the cost is very high, which is basically several tens to one hundred times of BLI. In order to balance the low cost and convenience and high precision, BLI is often used for sample screening, and SPR is used for accurate detection of samples screened by BLI, but this method cannot fundamentally solve the problem of high cost of molecular interaction analysis.

[0007] Based on the working principles of BLI technology and SPR technology, neither SPR nor BLI is a real liquid-phase reaction, and it is also impossible to perform mixed detection analysis of multiple target molecules. In view of the problems of the prior art, the inventors expect to propose a molecular interaction analysis technology which balances high precision, low cost and high flexibility and convenience in actual use. SUMMARY

[0008] (I) Technical problems to be solved

[0009] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present application provides a method for analyzing intermolecular interaction, which uses a rare earth material modified with ligand molecules as an upconversion material, contacts the upconversion material with target molecules in a solution system, uses excitation light matched with the rare earth material to irradiate the solution system, changes the excitation light intensity and power actually received by the upconversion material in the dynamic process of continuous binding and dissociation of the target molecules with the ligand molecules on the surface of the upconversion material, and further changes the emission light intensity, so that the emission light intensity-time curve can be recorded, and the molecular interaction kinetic constants, such as the binding rate constant (K on ), the dissociation rate constant (K off ) and the affinity (K D ) and other parameters can be calculated. The present application solves the technical problems of low precision of the existing BLI technology and high cost of the SPR technology. Based on the foregoing principle, a series of experimental consumables, kits, sensors and analysis devices for molecular interaction analysis are also developed.

[0010] (II) Technical solutions

[0011] In a first aspect, the present application provides a method for analyzing intermolecular interaction, which comprises:

[0012] S1, providing a rare earth upconversion nanomaterial modified on the surface, wherein the surface modification comprises 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 capable of specifically (directly or indirectly) binding with a target molecule on the rare earth upconversion nanomaterial modified by the anti-quenching modification;

[0013] S2, immersing the rare earth upconversion nanomaterial modified on the surface in a solution containing target molecules, so that the rare earth upconversion nanomaterial modified on the surface can contact the target molecules in the solution; using excitation light matched with the rare earth upconversion nanomaterial to irradiate the solution, so that the rare earth upconversion nanomaterial generates emission light; as the target molecules continuously bind with the biological ligand molecules on the surface of the rare earth upconversion nanomaterial, the emission light intensity generated by the upconversion nanomaterial under the excitation light continuously decreases until the binding of the biological ligand molecules and the target molecules reaches equilibrium or saturation, and the emission light intensity-time curve during this period is recorded;

[0014] S3, calculating the emission light intensity-time curve obtained in S2 to obtain the kinetic constants of the interaction between the ligand molecules and the target molecules.

[0015] According to a preferred embodiment of the present application, 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. 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 application, 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, the rare earth upconversion nanomaterial is NaYF4(Yb,Er), wherein the doping ratio of Yb is 18% (mass ratio) and the doping ratio of Er is 2% (mass ratio).

[0017] According to a preferred embodiment of the present application, in S1, the rare earth upconversion nanomaterial is a nanoparticle with a particle size of 15-100 nm, preferably 20-70 nm, and more preferably 20-40 nm, and is preferably a nanosphere. The particle size of the rare earth upconversion nanomaterial after the anti-quenching modification process increases.

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

[0019] S11, dispersing the rare earth upconversion nanomaterial in an organic solvent to obtain an upconversion nanomaterial dispersion; dissolving the methoxy polyethylene glycol phospholipid and an active group modifier in the organic solvent to obtain a coating 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 succinimidyl ester MPEG2000-NHS;

[0020] S12, mixing the upconversion nanomaterial dispersion and the coating solution, and evaporating the organic solvent to obtain the rare earth upconversion nanomaterial after the anti-quenching modification process. Pure water can be added for dispersion, and the dispersion is stored.

[0021] After the anti-quenching modification process, the anti-quenching ability of the rare earth upconversion nanomaterial is improved, and the light intensity of the rare earth upconversion nanomaterial is prevented from being weakened and quenched under the action of water molecules. In addition to being coated with methoxy polyethylene glycol phospholipid and other phospholipid hydrophobic materials, other hydrophobic organic polymer materials, non-organic polymer hydrophobic materials or silica gel can also be used to protect the core of the rare earth upconversion nanomaterial, thereby reducing the influence of water molecules on the rare earth upconversion nanomaterial.

[0022] As a preferred embodiment of the present application, in S11, the rare earth upconversion nanomaterial is co-wrapped with methoxyl polyethylene glycol phospholipid and active group modifier. On one hand, the anti-quenching performance of the rare earth upconversion nanomaterial in water is greatly improved. On the other hand, carboxyl, amino, mercapto, polylysine, succinimidyl and other active groups are connected to the surface of the rare earth upconversion nanomaterial, which are prepared for further connection of biological ligand molecules. The phospholipid wrapping layer has better affinity with proteins (biological ligand molecules or target molecules), which is more conducive to the subsequent modification process.

[0023] Specifically, the carboxyl group can react with molecules containing primary amine groups by forming amide bonds, so that the carboxyl group is easy to couple with biological molecules (such as proteins, antibodies); the amino group can react with various compounds containing active ester groups (such as succinimidyl ester) to form stable amide bonds. In addition, the amino group can also react with aldehydes or ketones through Schiff base reaction. The presence of amino group makes the molecule have the possibility of further modification, and other functional groups or other molecules can be introduced. The mercapto group can react with compounds containing maleimide groups to form thioether bonds. The succinimidyl group can react with primary amine groups to form stable amide bonds, which is easy to couple with biological molecules (such as proteins, polypeptides) containing amine groups. The succinimidyl group can be coupled under relatively mild conditions. Through the modification of these groups, specific biological ligand molecules (such as antibodies) can be coupled, and binding-dissociation and other interactions between the target molecules in the solution can be generated.

[0024] It should be noted that the technology of introducing biological molecule ligands based on active groups such as hydroxyl, carboxyl, amino, aldehyde, succinimidyl ester and sulfonyl is a conventional technology, which will not be described here. The biological molecule ligand can be protein, antibody, antigen, hapten, polysaccharide, nucleic acid probe or cell; such as Streptavidin, Avidin, BSA, Protein A, Protein G, Biotin, polypeptide, antibody, other various proteins and nucleic acid probes (DNA, RNA probes) and the like.

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

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

[0027] S12, mixing the up-conversion nanomaterial dispersion liquid and the coating solution, rotary evaporation to evaporate the solvent, adding pure water with a concentration of 0.01-0.04M, ultrasonic dispersion, and obtaining the dispersion liquid of the anti-quenching modified rare earth up-conversion nanomaterial.

[0028] In a second aspect, the present application provides a kit for analyzing the interaction between molecules, which comprises a surface modified rare earth up-conversion nanomaterial, the surface modification comprising an anti-quenching modification and a ligand coating; the anti-quenching modification is that the rare earth up-conversion nanomaterial is coated with a polymer or an inert inorganic material; the ligand coating is that a biological ligand molecule capable of specifically binding to a target molecule is connected to the anti-quenching modified rare earth up-conversion nanomaterial.

[0029] Alternatively, the kit comprises an anti-quenching modified rare earth up-conversion nanomaterial and a biological ligand molecule, the anti-quenching modification comprising that the rare earth up-conversion nanomaterial is coated with a polymer or an inert inorganic material and active groups are grafted on the surface of the coating layer; the active group is at least one of carboxyl, amino, thiol and succinimidyl, the active group provides an active connection site for coupling the biological ligand molecule; the biological ligand molecule is capable of specifically binding to a target molecule.

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

[0031] According to a preferred embodiment of the present application, the method for the anti-quenching modification is that the rare earth up-conversion nanomaterial is dispersed in an organic solvent to obtain an up-conversion nanomaterial dispersion liquid; methoxyl polyethylene glycol phospholipid and an active group modifier are dissolved in the organic solvent to obtain a coating solution; the active group modifier is at least one of methoxyl polyethylene glycol phospholipid carboxyl mPEG-PLC, methoxyl polyethylene glycol amino mPEG-NH2, methoxyl polyethylene glycol thiol mPEG-SH and methoxyl polyethylene glycol succinimidyl ester MPEG2000-NHS; after mixing the up-conversion nanomaterial dispersion liquid and the coating solution, the organic solvent is evaporated to obtain the anti-quenching modified rare earth up-conversion nanomaterial.

[0032] According to a preferred embodiment of the present application, the rare earth up-conversion nanomaterial is prepared by doping rare earth ions into an inorganic matrix; the rare earth ion is 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; 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, the rare earth upconversion nanomaterial is NaLnF4(Yb,Er), wherein the doping ratio of Yb is 18% (mass ratio) and the doping ratio of Er is 2% (mass ratio).

[0034] In a third aspect, the present application provides a sensor for analyzing intermolecular interaction, comprising: a light guide material and a surface-modified rare earth upconversion nanomaterial adhered to one end face of the light guide material; the other end of the light guide material is an incident end of excitation light and an emission end of emission light; the light guide material is an excitation light conductor and an emission light conductor; the surface modification comprises an anti-quenching modification and a ligand coating; the anti-quenching modification is a wrapping of the rare earth upconversion nanomaterial with a polymer or an inert inorganic material; the ligand coating is a connection of a biological ligand molecule capable of specifically binding to a target molecule on the anti-quenching modified rare earth upconversion nanomaterial.

[0035] Alternatively, the sensor comprises: a light guide material and an anti-quenching modified rare earth upconversion nanomaterial adhered to one end face of the light guide material; the other end of the light guide material is an incident end of excitation light and an emission end of emission light; the anti-quenching modification comprises a wrapping of the rare earth upconversion nanomaterial with a polymer or an inert inorganic material and a grafting of active groups on the surface of the wrapping layer; the active groups are at least one of carboxyl, amino, thiol and succinimidyl; the active groups are active connection sites for coupling biological ligand molecules. For the latter scheme, it is necessary to coat the biological ligand molecules on the end face of the light guide material temporarily before determining the interaction between the biological ligand molecules and the target molecules, and then performing analysis and detection.

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

[0037] In the preparation of the sensor, the rare earth upconversion nanomaterials are modified by anti-quenching modification, then coated with ligands, and then adhered to the end face of the quartz needle. In this case, part of the ligand molecules are directed to 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 nanomaterials are modified by 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 nanomaterials are modified by anti-quenching modification, and a carboxyl group (an active group) is introduced during the anti-quenching modification process. Then, the carboxyl group is activated by using an EDC / NHS mixed solution. The surface of the light guide material (preferably a quartz needle) is cleaned with ethanol and dried. An appropriate amount of polylysine L 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 liquid of the carboxyl group-activated rare earth upconversion nanomaterials is added dropwise to the end face of the quartz needle, and dried to adhere the carboxyl group-activated rare earth upconversion nanomaterials to the end face of the quartz needle. Then, the biological ligand molecules are connected to the carboxyl group-activated rare earth upconversion nanomaterials. Specifically, the quartz needle with the rare earth upconversion nanomaterials is placed in a solution containing biological ligand molecules (such as antibodies) and incubated at a certain temperature for a preset time. The antibodies are coupled to the activated carboxyl groups on the surface of the rare earth upconversion nanomaterials. Thus, the antibodies are coated to the end face of the quartz needle, so as to facilitate subsequent analysis and detection.

[0038] In a fourth aspect, the present application provides a device for analyzing intermolecular interactions, which comprises the sensor, an excitation light emitter and an emission light receiver. The excitation light emitter and the emission light receiver are arranged on opposite ends of the light guide material of the sensor, which is modified with the rare earth upconversion nanomaterials.

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

[0040] In addition, the higher the concentration of the target molecules in the solution, the faster the target molecules and the ligand molecules on the end face of the sensor reach the binding-dissociation equilibrium. At this time, the emission light intensity no longer decreases with time, and the curve reaches a plateau. At this time, the target molecules are completely combined with the ligand molecules on the surface of the rare earth upconversion nanomaterials, and the influence on the emission light intensity stops. Therefore, according to the time to reach the binding-dissociation equilibrium, the present application can also be used for detecting the concentration of the target molecules. (Three) beneficial effects

[0041] The method for analyzing intermolecular interaction of the present application is developed based on a principle different from the existing BLI and SPR technologies, and compared with the existing technologies, the technical effects include the following aspects:

[0042] (1) Compared with the BLI analysis technology, the precision is higher. The present application can detect the molecular interaction of small molecules, while the 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), so the BLI technology is mainly used for antigen, hybridoma screening and other applications, while the method of the present application can detect small molecules such as antibodies.

[0043] (2) Compared with the BLI analysis technology, the signal-to-noise ratio is better, the signal intensity is high, and the sensitivity is higher. The BLI method uses white light, and the received is also the white light reflected by the molecular layer. While the non-visible excitation light (such as 980nm, 1550nm laser) used in the present application, the received emission light is visible light (usually 400-700nm visible light), which does not overlap with the excitation light wavelength and does not interfere, and the signal-to-noise ratio is better. In addition, the BLI relies on the difference between the received reflected light and the emitted light, and the signal-to-noise ratio is poor, especially when the target molecule is too small, the absorbance is low, which will further weaken the signal intensity. While the emission light generated by the rare earth upconversion nanomaterials received by the present application has high signal intensity, high sensitivity and high precision.

[0044] (3) Since the emission light generated by the rare earth upconversion nanomaterials received by the present application has higher signal intensity, the receiver of the emission light can use not only a miniature spectrometer, but also other signal collection terminals such as PMT, CCD, etc., which can be adjusted according to actual needs, and the cost control is more flexible. Because the emission light generated by the rare earth upconversion nanomaterials has high signal intensity, lower excitation light power can be used, and the thermal effect is lower, so the stability of the whole system is better.

[0045] (4) Based on the principle of BLI and SPR, both technologies currently rely on embedding in the surface of the support for testing, and cannot realize full liquid phase detection (especially BLI cannot realize closed detection). However, when measuring biological target molecules in some solutions, if not closed, the solvent in the sample is easy to volatilize, and the sample is easy to be contaminated, and full liquid phase detection can avoid this problem. The method of the present application can mix the modified rare earth upconversion nanomaterials with the target molecule solution and place it in a sealed cuvette / microfluidic channel to prevent solvent volatilization or contamination, use excitation light to irradiate the cuvette and receive the emission light intensity curve with time, and then accurately analyze and detect the molecular action kinetics constant.

[0046] (5) The present application can perform synchronous detection of multiple target molecules, while BLI and SPR cannot achieve detection of multiple target molecules. The present application can use different rare earth upconversion nanomaterials and embed different biological ligand molecules, and use the same to detect samples containing multiple target molecules, and cooperate with irradiation of multiple different wavelength excitation lights (different rare earth upconversion nanomaterials are matched with different excitation light wavelengths, and different upconversion nanomaterials need to ensure that the emission spectrum has a separation degree), which can promote the corresponding rare earth upconversion nanomaterials to produce emission lights of different wavelengths, so that multi-channel detection can be achieved, and the detection throughput is higher.

[0047] (6) The present application is more flexible to use, and can achieve full liquid phase detection, and can also embed the rare earth upconversion nanomaterials and the biological ligand molecules into the end face of a high light guide material such as a quartz needle to prepare a sensor in a manner similar to BLI, or can embed and fix the rare earth upconversion nanomaterials and the biological ligand molecules in a cuvette, a micro pipeline (transparent tube) for detection, which is more suitable for a wider scene, and according to different use forms, a series of downstream detection consumables or instrument devices can be developed.

[0048] (7) For different target molecules, BLI needs to process the end face for different target molecules to achieve measurement of target molecules of different sizes. The present application does not need to consider the size of the target molecules, and can detect small molecules smaller than 1 nanometer and viruses up to several hundred nanometers, and has a lower cost.

[0049] (8) Compared with the SPR technology, the present application has a high precision comparable to the SPR, and has the characteristics of low cost, rapid detection, multi-channel detection and full liquid phase detection. BRIEF DESCRIPTION OF DRAWINGS

[0050] Fig. 1 is a schematic diagram of the method for analyzing the interaction between molecules according to the present application.

[0051] Fig. 2 is a curve of the change of the emission light intensity-time detected in Example 1.

[0052] Fig. 3 is a curve of the change of the emission light intensity-time obtained by detecting samples with different concentrations in Example 2.

[0053] Fig. 4 is a schematic diagram of a sensor based on the principle of analyzing the interaction between molecules according to the present application.

[0054] Fig. 5 is a SEM diagram of the rare earth upconversion nanomaterials adhered to the bottom of the sensor. DETAILED DESCRIPTION

[0055] In order to better explain the present application and facilitate understanding, the present application is described in detail in combination with the drawings and through specific embodiments.

[0056] Example 1

[0057] The present embodiment provides a method for analyzing intermolecular interaction, and the steps are as follows:

[0058] (1) Preparation of NaYF4(18% Yb, 2% Er)

[0059] According to the stoichiometric ratio, NaF, YCl3, YbCl3 and ErCl3 are weighed and dissolved in the mixed solution of oleic acid and 1-octadecanol, respectively. Under the argon atmosphere, the above mixed solution is heated to 300°C, and kept at this temperature for 1.5 h to promote the reaction to occur, thereby preparing the rare earth upconversion nanomaterial NaYF4(18% Yb, 2% Er) with good dispersity and uniformity, which is a nanosphere with a particle size of 20-30 nm.

[0060] The preparation method of the rare earth upconversion nanomaterial is prior art, which can be prepared by the laboratory itself or purchased according to the needs. In addition to the high-temperature solvothermal method of the present embodiment, a coprecipitation method can also be used for preparation.

[0061] (2) Anti-quenching modification

[0062] Take 0.1 mmol of rare earth upconversion nanomaterial NaYF4(18% Yb, 2% Er) and disperse it in 1 ml of chloroform. Mix methoxypolyethylene glycol phospholipid mPEG-PL and methoxypolyethylene glycol phospholipid carboxyl mPEG-PLC according to the mass ratio of 7:3, and dissolve them in chloroform at 25 mg / ml. Since mPEG-PL and mPEG-PLC have similar structures, the two substances can be well miscible and mixed, so that the distribution of carboxyl on the rare earth upconversion nanomaterial is more uniform. Mix the dispersed rare earth upconversion nanomaterial and the solution of mPEG-PL and mPEG-PLC in an Erlenmeyer flask, and after rotary evaporation and drying, the rare earth upconversion nanomaterial coated with mPEG-PL and mPEG-PLC is obtained. Finally, add ultrapure water to a concentration of 0.1 mmol / 5 ml and ultrasonic dispersion, and store in the refrigerator. After being coated with mPEG-PL and mPEG-PLC, the rare earth upconversion nanomaterial has good anti-quenching property, and even if it is directly contacted with water, its fluorescence performance will not be significantly weakened. At the same time, mPEG-PLC also introduces carboxyl groups on the surface of the rare earth upconversion nanomaterial, which prepares for further coating of biological ligand molecules.

[0063] (3) Ligand coating

[0064] Take 100 uL of anti-quenching modified rare earth upconversion nanomaterial dispersion, centrifuge and disperse in pH = 8 Tris buffer, add 5 mg EDC and 5 mg NHS solution to the solution, and shake in a 2 ml centrifuge tube at room temperature for 4 h. Wash with Tris buffer for 3 times to obtain the activated carboxyl group of the rare earth upconversion nanomaterial.

[0065] Put the activated carboxyl group of the rare earth upconversion nanomaterial into a 2 mL centrifuge tube, add 100 uL of pH = 8 Tris buffer, take 2 uL of 1 mg / mL interleukin-6 antibody (IL-6 antibody) and incubate at 37°C for 4 h. After centrifugation, collect the IL-6 antibody coated rare earth upconversion nanomaterial.

[0066] (4) Analysis of molecular interaction constant

[0067] In a 2 mL centrifuge tube, add 100 uL of pH = 8 Tris buffer and 3 uL of 1 mg / mL IL-6 protein. Add the prepared IL-6 antibody coated rare earth upconversion nanomaterial to the centrifuge tube, take 5 uL and transfer to a cuvette with an optical path length of 0.1 cm, seal the cuvette, use a 980 nm laser to excite the rare earth upconversion nanomaterial, and use a portable spectrometer of complex optical to receive the emitted light. The emitted light has two main peaks with good luminosity, which are 460-500 nm and 520-570 nm, and is obviously separated from the excitation light 980 nm wavelength light. In the continuous detection process, the total detection time is 80 seconds, the emission intensity is continuously detected and recorded, and the received signal is tabulated every 20 seconds, which can be directly observed to observe the signal change. The emission intensity decreases with the increase of the binding time, and the principle is shown in Figure 1: with the increase of the binding time, IL-6 in the solution continuously aggregates and connects to the IL-6 antibody on the surface of the rare earth upconversion nanomaterial, which leads to the decrease of the actual excitation light power received by the rare earth upconversion nanomaterial, and the emission intensity of the rare earth upconversion nanomaterial continuously decreases with the binding process, until the binding of IL-6 and IL-6 antibody reaches a stable state, i.e. the binding-dissociation equilibrium.

[0068] According to the recorded raw data of the results, the emission intensity-time (Intensity-Time) change curve is made, as shown in Figure 2, and the molecular interaction binding rate constant (K on ), dissociation rate constant (K off ) and affinity (K D ) of IL-6 antibody and IL-6 protein are calculated based on the emission intensity-time change curve. As shown in the figure, the curve is good, and a smoother curve can be obtained after fitting. The calculation process can refer to the calculation process of existing BLI, or other mathematical methods can be used for processing.

[0069] The mathematical processing method includes (1) selecting a suitable model: for example, for a simple binding-dissociation process, a first-order reaction model can be used. (2) Fitting data: using a non-linear least squares method or other appropriate fitting algorithm to find the best association rate constant K on and dissociation rate constant K off ; using a software tool (such as Origin, GraphPad Prism, etc.) to fit the experimental data. (3) Once the K on and K off values are obtained, the K D = K off / K on .

[0070] IL-6 is a cytokine with a molecular weight of about 26 kilodaltons (kDa), consisting of 184 amino acids, and a particle size of only 2-4 nm. The emission intensity-time variation curve drawn by the method of the present application is linear and the particle size of the target molecule to be measured can be as small as a few nanometers. Therefore, the present application has the advantage of high precision and can easily perform small molecule detection with higher precision than existing BLI analysis techniques.

[0071] The rare earth upconversion nanomaterial particles modified by anti-quenching and coated with ligands in the embodiment can be used as detection consumables for molecular interaction analysis and are components of a kit. In combination with existing laboratory equipment, optical instruments and computing devices, the molecular interaction can be analyzed and detected.

[0072] Embodiment 2

[0073] In this embodiment, different concentrations of antigens are detected. In this embodiment, four gradient concentrations are set, which are:

[0074] Concentration 1: In a 2 mL centrifuge tube, 100 uL of pH = 8 Tris buffer and 1 uL of IL-6 protein with a concentration of 1 mg / mL are added.

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

[0076] Concentration 3: In a 2 mL centrifuge tube, 100 uL of pH = 8 Tris buffer and 3 uL of IL-6 protein with a concentration of 1 mg / mL are added.

[0077] Concentration 4: In a 2 mL centrifuge tube, 100 uL of pH = 8 Tris buffer and 4 uL of IL-6 protein with a concentration of 1 mg / mL are added.

[0078] The preparation process of the rare earth upconversion nanomaterial coated with IL-6 antibody used in this embodiment is described in Embodiment 1.

[0079] 5 uL of the rare earth upconversion nanomaterial was taken into a cuvette with an optical path length of 0.1 cm, and the cuvette was sealed. The rare earth upconversion nanomaterial was excited by a 980 nm laser, and the emitted light was received by a portable spectrometer of Complex Optics. During continuous detection, the total detection time was 80 seconds. The detection and recording of the intensity of the emitted light were continuously performed, and the received signals were tabulated every 20 seconds. The intensity-time curve of the emitted light was plotted according to the recorded results, and a smooth curve was obtained after fitting, as shown in FIG. 3.

[0080] As shown in FIG. 3, under the same primary antibody (all IL-6 antibodies), the intensity-time curves of the molecular interaction of different antigen concentrations are different. The sample antigen concentration gradient decreases from bottom to top, that is, the higher the concentration of the target molecule in the sample, the faster the intensity-time curve of the emitted light reaches the plateau. This indicates that the higher the concentration of the target molecule (antigen) to be detected, the shorter the time required for the combination of the rare earth upconversion nanomaterial coated with the biological ligand molecule (IL-6 antibody) to reach equilibrium. In the case of the highest concentration (concentration 4), the combination 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 at which the intensity-time curve of the emitted light reaches equilibrium.

[0081] Embodiment 3

[0082] This embodiment provides a sensor based on the inventive concept. As shown in FIG. 4, a layer of rare earth upconversion nanomaterial modified against quenching and coated with a ligand is adhered to the front end surface of a light guide material column (preferably quartz needle in this embodiment). The preparation process of the sensor is as follows:

[0083] (1) The rare earth upconversion nanomaterial NaYF4(18% Yb, 2% Er) nanosphere with a particle size of 20-30 nm was modified against quenching according to the method of Embodiment 1, and was stored in ultrapure water at a concentration of 0.1 mmol / 5 ml.

[0084] (2) 100 uL of the dispersion of the rare earth upconversion nanomaterial modified against quenching was centrifuged and dispersed in a Tris buffer solution with pH = 8. 5 mg of EDC and 5 mg of NHS solution were added, and the mixture was placed in a 2 ml centrifuge tube and shaken at room temperature for 4 h to activate the carboxyl group. The activated rare earth upconversion nanomaterial was washed with Tris buffer solution for 3 times to obtain the activated carboxyl group, and was stored in ultrapure water at a concentration of 0.1 mmol / 5 ml for cold storage.

[0085] (3) Take the quartz needle with diameter of 600 μm and length of 15 cm (the quartz needle can be selected according to the need), clean the surface with alcohol, place in a dry and ventilated place, and arrange the dried quartz needles in order. Prepare 10x polylysine L solution (original concentration 0.01% (w / v)). Dip the end face of the quartz needle into the polylysine, take it out after 3 min and clean it in pH = 8 Tris buffer to ensure that the surface of the quartz needle has only a layer of polylysine. Polylysine has strong adhesion and can easily adhere to quartz or glass products. The quartz needle has good light transmission (light transmission) and chemical inertness.

[0086] (4) Take 2ul of rare earth upconversion nanomaterial with a concentration of 0.1 mmol / 5ml and drop it on the end face of the quartz needle to combine it with the polylysine. The rare earth upconversion nanomaterial with activated carboxyl groups is adhered to the end face of the quartz needle.

[0087] (5) Put the quartz needle end face with rare earth upconversion nanomaterial adhered into a 2ml centrifuge tube, add 200ul of pH = 8 Tris buffer, take 2uL of interleukin-6 antibody (IL-6 antibody) with a concentration of 1mg / ml and add it into the centrifuge tube, incubate at 37℃ on a shaking bed for 4h, and the IL-6 antibody is coated on the end face of the quartz needle by forming an amide bond with the activated carboxyl groups on the surface of the rare earth upconversion nanomaterial. Then wash it once with pH = 8 Tris buffer to avoid self-aggregation of the rare earth upconversion nanomaterial, and prepare the sensor as shown in Figure 4. The morphology of the rare earth upconversion nanomaterial adhered to the bottom of the sensor is shown in Figure 5.

[0088] In addition, in other embodiments, the sensor product can also be prepared and sold only after the rare earth upconversion nanomaterial containing active groups (such as carboxyl, amino, thiol or succinimide) is adhered to the end face of the quartz needle. Before the experimental personnel perform the molecular interaction detection analysis, the corresponding biological ligand molecules are selected according to the target molecules to be detected, and the selected biological ligand molecules are coated on the rare earth upconversion nanomaterial on the end face of the quartz needle by using a suitable method.

[0089] As for how to graft active groups on the surface of the material and coat biological ligand molecules, it is a common means in the field of biotechnology, and those skilled in the art can select any method that can achieve the goal according to the need, or refer to the method listed in the specification of the present application to achieve it.

[0090] Example 4

[0091] The present embodiment provides a device for analyzing molecular interaction based on the inventive concept, which comprises integrating the sensor of embodiment 3 with an optical analysis device. Specifically, the quartz needle of the sensor of embodiment 3 is connected to an optical instrument through an optical fiber, which comprises a laser emitter and an emission light receiver. The laser emitter can adjust the laser wavelength according to the type of the 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 contain a data recording and processing calculation unit, and according to the recording and calculation, the emission light intensity-time curve is derived in a visual form.

[0092] It should be noted that the specific anti-quenching modification method is not limited to the specific modification process presented in the embodiments of the present application. The hydrophobic polymer used in the anti-quenching modification is mainly some lipid or ester polymer, and PVP, PE, PP, PS, PTFE, silicone rubber, etc. can also be used, but PVA (easy to dissolve in water) is not recommended; the inert inorganic material is a silicon oxide compound, such as SiO2, titanium dioxide, carbon material, etc.; as long as it can separate the rare earth upconversion nanomaterial from water molecules, prevent or delay the quenching of the fluorescence of the rare earth upconversion nanomaterial by water molecules, has chemical inertness, and is not easy to chemically react with the solution in which the target molecules are located. In the anti-quenching modification material, polymers or inert inorganic materials can be used, or a combination of hydrophobic polymers and inert inorganic materials can be used.

[0093] For the anti-quenching modification method, reference can be made to the anti-quenching modification methods of rare earth upconversion nanomaterials or quantum dots disclosed in the prior art, for example, polyvinylpyrrolidone (PVP) and silicon dioxide (SiO2) can be used as wrapping materials to wrap rare earth upconversion nanomaterial particles; or first use high-temperature solvothermal method to synthesize oleic acid-wrapped rare earth upconversion nanoparticles, and then use polypropylene to wrap them using the carboxyl arm of oleic acid.

[0094] In the ligand coating modification process, reference can be made to the coating process of biological ligand molecules such as magnetic beads in biotechnology. Typically, at least one basic active group such as hydroxyl, carboxyl, amino, aldehyde, succinimidyl ester and sulfonyl is first grafted, and then the biological ligand molecules are connected and coated by forming a covalent bond.

[0095] In addition, anti-quenching cannot completely eliminate the quenching of rare earth upconversion nanomaterials in water, but can only weaken or prolong the quenching time, so as to make the detection result more accurate. In some environments that require long-time detection and analysis of molecular interaction, the rare earth upconversion nanomaterials are often wrapped with multiple layers of materials for multiple times to improve the anti-quenching property, so as to ensure that the quenching is slight during the detection time, and generally the quenching degree is controlled within 3%. However, it should be noted that the anti-quenching modification also affects the receiving of laser and the luminescence intensity of the rare earth upconversion nanomaterials, so that the signal becomes weak, and in some cases it also causes the particle size of the rare earth upconversion nanomaterials to become large. Therefore, when the anti-quenching modification is performed, the technical personnel can select the wrapping material or the number of wrapping layers according to the type of molecule to be detected by the rare earth upconversion nanomaterials and the application environment.

[0096] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method of analyzing intermolecular interactions, characterized by, The method comprises the following steps: S1, providing surface-modified rare earth upconversion nanomaterials, wherein the surface modification comprises anti-quenching modification and ligand coating; the anti-quenching modification is that the rare earth upconversion nanomaterials are wrapped with polymers or inert inorganic materials; The ligand coating is that biological ligand molecules capable of specifically binding to target molecules are connected to the rare earth upconversion nanomaterials after the anti-quenching modification; S2, immersing the surface-modified rare earth upconversion nanomaterials in a solution containing target molecules, so that the surface-modified rare earth upconversion nanomaterials can contact the target molecules in the solution; irradiating the solution with excitation light matched with the rare earth upconversion nanomaterials, so that the rare earth upconversion nanomaterials generate emission light; as the target molecules continuously combine with the biological ligand molecules on the surface of the rare earth upconversion nanomaterials, the emission light intensity generated by the upconversion nanomaterials under the excitation light decreases continuously until the combination of the biological ligand molecules and the target molecules reaches equilibrium or saturation, and a curve of the emission light intensity changing with time during this period is recorded; S3, calculating the curve of the emission light intensity changing with time obtained in S2 to obtain the kinetic constant of the interaction between the ligand molecules and the target molecules.

2. The method of claim 1, wherein, In S1, the rare earth upconversion nanomaterials are 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 of claim 1, wherein, In S1, the rare earth upconversion nanomaterials are 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 of claim 1, wherein, In S1, the rare earth upconversion nanomaterials are nanoparticles with a particle size of 15-100 nm.

5. The method of claim 1, wherein, In S1, the anti-quenching modification process is as follows: S11, dispersing the rare earth upconversion nanomaterials in an organic solvent to obtain an upconversion nanomaterial dispersion; dissolving methoxyl polyethylene glycol phospholipid and an active group modifier in the organic solvent to obtain a wrapping solution; the active group modifier is at least one of methoxyl polyethylene glycol phospholipid carboxyl mPEG-PLC, methoxyl polyethylene glycol amino mPEG-NH2, methoxyl polyethylene glycol thiol mPEG-SH and methoxyl polyethylene glycol succinimidyl ester MPEG2000-NHS; S12, mixing the upconversion nanomaterial dispersion and the wrapping solution, and then evaporating the organic solvent to obtain the rare earth upconversion nanomaterials after the anti-quenching modification.

6. The method of claim 5, wherein, In S1, the anti-quenching modification process is as follows: S11, dispersing the rare earth upconversion nanomaterials in chloroform at a concentration of 0.1-0.5 M to obtain an upconversion nanomaterial dispersion; dissolving methoxyl polyethylene glycol phospholipid and a PEGylated active group modifier in chloroform at a mass ratio of 1-10:1-10 to obtain a wrapping solution with a concentration of 20-50 mg / ml; S12, mixing the up-conversion nanomaterial dispersion liquid and the coating solution, rotary evaporation to evaporate the solvent, adding pure water with a concentration of 0.01-0.04M, ultrasonic dispersion, and obtaining the dispersion liquid of the anti-quenching modified rare earth up-conversion nanomaterial.

7. A kit for analyzing intermolecular interactions, characterized in that, The surface modification includes anti-quenching modification and ligand coating; the anti-quenching modification is that the rare earth up-conversion nanomaterial is coated with a polymer or an inert inorganic material; The ligand coating is that a biological ligand molecule capable of specifically binding to a target molecule is connected to the anti-quenching modified rare earth up-conversion nanomaterial; Alternatively, the kit comprises: anti-quenching modified rare earth up-conversion nanomaterial and biological ligand molecules, the anti-quenching modification includes that the rare earth up-conversion nanomaterial is coated with a polymer or an inert inorganic material and active groups are grafted on the surface of the coating layer; the active groups are at least one of carboxyl, amino, thiol and succinimidyl, the active groups provide active connection sites for coupling biological ligand molecules; the biological ligand molecules can specifically bind to target molecules.

8. The kit of claim 7, wherein The biological molecule ligand is protein, antibody, antigen, hapten, polysaccharide, nucleic acid probe or cell.

9. The kit of claim 7, wherein The method for the anti-quenching modification is: dispersing the rare earth up-conversion nanomaterial in an organic solvent to obtain an up-conversion nanomaterial dispersion liquid; dissolving methoxyl polyethylene glycol phospholipid and an active group modifier in the organic solvent to obtain a coating solution; the active group modifier is at least one of methoxyl polyethylene glycol phospholipid carboxyl mPEG-PLC, methoxyl polyethylene glycol amino mPEG-NH2, methoxyl polyethylene glycol thiol mPEG-SH and methoxyl polyethylene glycol-succinimidyl ester MPEG2000-NHS; mixing the up-conversion nanomaterial dispersion liquid and the coating solution, and evaporating the organic solvent to obtain the anti-quenching modified rare earth up-conversion nanomaterial.

10. A sensor for analyzing intermolecular interactions, characterized in that It comprises: The light guide material and the surface modified rare earth up-conversion nanomaterial are adhered to one end face of the light guide material; the other end of the light guide material is the incident end of the excitation light and the emission end of the emission light; the light guide 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 that the rare earth up-conversion nanomaterial is coated with a polymer or an inert inorganic material; The ligand coating is that a biological ligand molecule capable of specifically binding to a target molecule is connected to the anti-quenching modified rare earth up-conversion nanomaterial; Alternatively, the sensor comprises a light guide material and an anti-quenching modified rare earth upconversion nanomaterial adhered to one end face of the light guide material; the other end of the light guide material is an incident end of excitation light and an emission end of emission light; the anti-quenching modification comprises 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 succinimidyl groups; the active groups provide active connection sites for coupling of biological ligand molecules.

11. An apparatus for analyzing intermolecular interactions, characterized in that, The sensor comprises a surface-modified rare earth upconversion nanomaterial, an excitation light emitter and an emission light receiver; the surface modification comprises an anti-quenching modification and a ligand coating; the anti-quenching modification comprises wrapping the rare earth upconversion nanomaterial with a polymer or an inert inorganic material; the ligand coating comprises connecting biological ligand molecules capable of specifically binding to target molecules on the anti-quenching modified rare earth upconversion nanomaterial; or the surface modification only comprises an anti-quenching modification, which comprises 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 succinimidyl groups, which provide active connection sites for coupling of biological ligand molecules.

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