Method and system for collaborative analysis of molecular interaction state based on multi-mode optical parameters of rare earth up-conversion nano material

Abstract

The invention belongs to the technical field of biomolecular interaction detection, and particularly relates to a method for synergistically analyzing a molecular interaction state based on multimodal optical parameters of a rare earth up-conversion nano material. The method comprises the following steps: S1, providing a ligand coated rare earth up-conversion nano material UCNPs probe; s2, enabling the rare earth up-conversion nano material UCNPs probe to be in contact with a solution sample to be detected; s3, exciting the rare earth up-conversion nano material UCNPs probe by using near-infrared pulse laser, and collecting the following optical parameters: luminous intensity change, molecular layer thickness / mass change delta D, luminous life change delta tau and emission peak displacement delta lambda; and S4, based on at least two of the parameters, cooperatively analyzing and judging the interaction state of the ligand on the surface of the probe and the molecules in the solution sample to be detected. The molecular interaction state is synergistically analyzed based on changes of multiple optical parameters, and the technical problem that an existing molecular interaction analysis technology cannot be applied to complex biological samples (serum and cell lysis buffer) is solved.

Description

Technical Field

[0001] This invention belongs to the field of biomolecular interaction detection technology, specifically relating to a method and system for synergistic analysis of molecular interaction states based on multimodal optical parameters of rare earth upconversion nanomaterials. Background Technology

[0002] Molecular interaction detection technology refers to a technique for detecting key information such as the interaction process, rate, affinity, and dissociation constant of molecules. Based on this technology, interactions between various biomolecules can be studied, such as the affinity detection of common antigens and antibodies. Molecular interaction analysis is of great significance in many fields, including biology, chemistry, medicine, and pharmacy. By studying intermolecular interactions, scientists can gain a profound understanding of the internal mechanisms of biological systems and provide crucial information for new drug development, disease treatment, and materials science.

[0003] Currently used techniques for detecting biomolecular interactions include surface plasmon resonance (SPR) and biomembrane interferometry (BLI). Both SPR and BLI essentially measure changes in the optical path, i.e., physical changes. SPR measures changes in the refractive index (RI), while BLI measures changes in optical thickness. Both are macroscopic physical-optical properties and cannot directly reflect the state of molecular interactions. Techniques like SPR and BLI indirectly characterize molecular interactions by monitoring changes in refractive index or optical thickness at the sensor interface caused by molecular bonding in real time. However, these techniques cannot directly obtain photophysical parameters (such as luminescence lifetime) that reflect changes in the molecular microenvironment and conformation, and their signals are susceptible to interference from non-specific adsorption.

[0004] Compared to traditional SPR and BLI technologies, luminescence based on rare-earth nano-upconversion particles (UCNPs) can simultaneously output multiple sets of optical parameters, including luminescence intensity, fluorescence lifetime, and emission wavelength, from multiple emission peaks. Its main characteristics are as follows: First, the emission peaks are narrow. Because the 4f-4f electronic transitions of rare-earth ions are protected by the 5s and 5d orbitals, the rare-earth energy levels are less disturbed by the crystal field, resulting in minimal energy level broadening. Therefore, the rare-earth upconversion emission peaks are very narrow, with no crosstalk between peaks, leading to more accurate detection. Second, the rare-earth luminescence lifetime τ is much longer than that of quantum dots and organic dyes used in other technologies, exhibiting strong resistance to external interference and high detection accuracy. After the rare-earth nano-upconversion particles bind to molecules, energy resonance transfer occurs, establishing more non-radiative relaxation channels and influencing the rare-earth luminescence lifetime. Furthermore, the more target molecules specifically bound to the surface of the rare-earth nanoparticles, the greater the change in luminescence lifetime. Therefore, the lifetime change (Δτ) can directly and quantitatively characterize the strength and tightness of molecular interactions (tightness is positively correlated with specific binding). However, traditional SPR and BLI analysis techniques cannot capture this dynamic information.

[0005] Based on the aforementioned preliminary research, the applicant has proposed a molecular interaction sensor based on upconversion nanoparticles (UCNPs) (Publication No. CN119198650B). This patented approach primarily relies on detecting the change in luminescence intensity over time and calculating the molecular interaction relationships. However, this approach may be more suitable for pure samples but has limitations for complex samples. Complex samples, in addition to containing target molecules that bind to ligand molecules on UCNPs, also contain impurities such as non-target molecules or cell debris. These impurities may be non-specifically adsorbed onto the UCNPs, causing changes in luminescence intensity and leading to false positives.

[0006] Fluorescence lifetime is one of the "fingerprint" characteristics of materials. It is mainly affected by the specific binding of target molecules and ligand molecules, which may help to exclude non-specific adsorption. However, to date, there are no reports on how to use the fluorescence lifetime or other parameters fed back by UCNP-based molecular interaction sensors for molecular interaction analysis. This also limits the compatibility of UCNP-based molecular interaction sensors with complex samples in terms of molecular affinity and kinetic detection. Summary of the Invention

[0007] (a) Technical problems to be solved

[0008] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a method and system for synergistic analysis of molecular interaction states based on multimodal optical parameters of rare earth upconversion nanomaterials. Using ligand-coated UCNPs as probes, the probes are brought into contact with the sample solution to be tested, and near-infrared pulsed laser excitation is used to collect changes in probe luminescence intensity, molecular layer thickness / mass, luminescence lifetime, and emission peak shift. Based on these changes, the molecular interaction states are synergistically analyzed, fundamentally solving the technical problems of false positives, compatibility with complex samples, small molecule interaction mechanism research, and existing SPR / BLI analysis techniques that cannot be solved.

[0009] (II) Technical Solution

[0010] In a first aspect, the present invention provides a method for synergistic analysis of molecular interaction states based on multimodal optical parameters of rare-earth upconversion nanomaterials, comprising:

[0011] S1. Provides ligand-coated rare-earth upconversion nanomaterials (UCNPs) probes;

[0012] S2. Contact the rare earth upconversion nanomaterial UCNPs probe with the sample solution to be tested;

[0013] S3. Excite the rare-earth upconversion nanomaterial UCNPs probe with near-infrared pulsed laser and simultaneously collect the following optical parameters: change in luminescence intensity, change in molecular layer thickness / mass ΔD, change in luminescence lifetime Δτ, and emission peak shift Δλ.

[0014] S4. Based on at least two of the aforementioned parameters, jointly analyze and determine the interaction state between the ligands on the probe surface and the molecules in the test solution sample.

[0015] When conducting synergistic analysis, the types and number of parameters involved in the analysis can be determined based on the defined analytical objectives. If the compositional characteristics of the test solution sample are completely unknown, it is usually necessary to combine more parameters or signals for synergistic analysis in order to obtain as much information as possible.

[0016] According to a preferred embodiment of the present invention, in step S1, the surface of the rare earth upconversion nanomaterial UCNPs probe is further coated with an anti-quenching inert layer, and ligands are coated on the surface of the anti-quenching inert layer.

[0017] According to a preferred embodiment of the present invention, in step S2, the test solution sample is a pretreated sample, a purified sample, an untreated sample, or an unpurified complex biological sample, such as serum or cell lysate.

[0018] According to a preferred embodiment of the present invention, in step S2, the rare earth upconversion nanomaterial UCNPs probe is directly immersed in the test solution sample, so that the test solution sample is in contact with the rare earth upconversion nanomaterial UCNPs probe; or the rare earth upconversion nanomaterial UCNPs probe is modified onto the end face of a light guide rod to form a sensor, and the end face of the sensor is immersed in the test solution sample; or the rare earth upconversion nanomaterial UCNPs probe is modified into a microfluidic chip, so that the test solution sample is in contact with the rare earth upconversion nanomaterial UCNPs probe.

[0019] According to a preferred embodiment of the present invention, in step S3, the near-infrared pulsed laser is a laser with a wavelength of 808nm, 915nm, 940nm, 980nm or 1143nm, and the spectrometer collects the emission spectrum of UCNPs after excitation to obtain the optical parameters. The laser pulse is realized by a data acquisition card (DAQ) or a signal generator.

[0020] According to a preferred embodiment of the present invention, in step S3, the optical parameters are qualitative values ​​of changes in luminescence intensity, molecular layer thickness / mass change ΔD, luminescence lifetime change Δτ, and emission peak shift Δλ, where the qualitative value is one of negative, zero, or positive (the qualitative value can be used for qualitative judgment and analysis of molecular interactions); or the optical parameters are quantitative values ​​of changes in luminescence intensity, molecular layer thickness / mass change ΔD, luminescence lifetime change Δτ, and emission peak shift Δλ (the quantitative value can be used for qualitative judgment of molecular interactions or quantitative calculation of affinity).

[0021] The change in luminescence intensity is defined as either absolute luminescence intensity ΔI or relative luminescence intensity ΔR after internal reference calibration. Absolute luminescence intensity ΔI is the change in light intensity before and after the probe binds to molecules in the test solution. Relative luminescence intensity ΔR is the change in R before and after the probe binds to molecules in the test solution, where R = I1 / I2, and I1 and I2 are the intensities of two selected characteristic emission peaks corresponding to UCNPs, respectively. The emission peak shift Δλ is either a red shift or a blue shift, with a positive Δλ indicating a red shift and a negative Δλ indicating a blue shift.

[0022] According to a preferred embodiment of the present invention, in step S3, I1 and I2 are the intensities of two characteristic peaks corresponding to single-doped UCNPs (i.e., internal reference ratios), or I1 and I2 are the intensities of two characteristic peaks corresponding to double-doped UCNPs (i.e., external reference ratios). The relative luminescence intensity ΔR, compared to the absolute luminescence intensity ΔI, can eliminate system interference (e.g., excitation power fluctuations, excitation instability due to temperature in the optical path, etc.). The internal reference ratio mechanism is achieved by calculating the intensity ratio of two characteristic peaks of the same UCNP (e.g., NaYF4:Yb). 3+ Er 3+ I 540 / I 660The fluorescence at 540nm and 660nm originates from Er 3+ (Ions) can eliminate systematic errors such as excitation source fluctuations and concentration differences, improving detection stability. External reference mechanisms can use two types of UCNPs (such as Er...). 3+ with Ho 3+ Independently doped UCNPs can be used to construct ratio signals with a wider bandwidth (such as I). 540 / I 1180 ), of which 540nm comes from Er 3+ The emission; the 1180nm emission comes from rare earth Ho. 3+ Ions, external references can expand dynamic range or avoid spectral overlap.

[0023] According to a preferred embodiment of the present invention, in step S3, when the four optical parameters are subjected to synergistic analysis, their priority or weight is determined based on the parameter quality. The criteria for evaluating whether the optical parameter is of good or poor quality are as follows:

[0024] The evaluation criteria for good quality of the molecular layer thickness / mass change ΔD are: continuous and unidirectional signal change, the change amplitude is related to the target molecule concentration or reaction time, and there is synergy between ΔD and Δτ or ΔR. The evaluation criteria for poor quality of the ΔD parameter are: sudden step or violent fluctuation.

[0025] The evaluation criteria for good quality of luminous intensity ΔI or ΔR parameters are: ΔI or ΔR changes in synergy with Δτ; the evaluation criteria for poor quality of ΔI or ΔR parameters are: ΔI or ΔR remains unchanged when Δτ changes.

[0026] The evaluation criteria for good quality of the parameter Δτ of the change in luminescence lifetime are: a significant and repeatable shortening of the luminescence lifetime and a high degree of fit of the decay curve; the evaluation criteria for poor quality of the parameter Δτ of the change in luminescence lifetime are: the decrease in luminescence lifetime is less than the instrument error or close to zero.

[0027] The evaluation criteria for good emission peak displacement Δλ are: the emission peak undergoes a significant and continuous unidirectional displacement, the displacement amount is >0.5nm, and it is coordinated with the change in luminescence lifetime Δτ; the evaluation criteria for poor emission peak displacement Δλ are: the emission peak displacement amount is small and fluctuates within the instrument noise range, and the curve of emission peak displacement change over time cannot be fitted and converged.

[0028] According to a preferred embodiment of the present invention, in step S4, during qualitative judgment, based on at least two optical parameters obtained in S3, and simultaneously according to a preset judgment rule library, the interaction state between the ligands on the probe surface and the molecules in the test solution sample is analyzed and judged collaboratively; the judgment rule library includes, but is not limited to, the following rules:

[0029] When ΔD is positive, the luminescence intensity becomes negative, and when Δτ is negative, it indicates that the probe has a specific binding with molecules in the sample solution.

[0030] When ΔD is positive, the luminescence intensity becomes negative, and Δτ is zero, it is determined that the probe surface produces non-specific adsorption between molecules in the sample solution and the test solution.

[0031] When ΔD is positive, the luminescence intensity is reduced to zero; when Δτ is negative, it indicates that the probe has a specific binding with molecules in the sample solution.

[0032] ΔD is positive, the luminescence intensity is positive, Δτ is negative, Δλ is negative, indicating that the probe specifically binds to molecules in the sample solution and undergoes a change in molecular conformation.

[0033] When ΔD is positive, the luminescence intensity is positive, and when Δτ is zero, it is determined that the probe surface produces non-specific adsorption between molecules in the sample solution.

[0034] When ΔD is positive, the luminescence intensity is negative; when Δτ is negative, Δλ is positive, it indicates that the probe has a specific binding with the molecules in the sample solution and that there are changes in the microenvironment.

[0035] When ΔD is positive, the luminescence intensity becomes zero, Δτ is zero, and pure physical adsorption occurs;

[0036] When ΔD is zero, the luminous intensity is zero, and Δτ is zero, it is determined that there is no binding event.

[0037] When ΔD is zero, the luminescence intensity becomes negative, and Δτ is zero, a quencher is present in the sample solution to be tested.

[0038] When ΔD is negative, the luminescence intensity becomes positive; when Δτ is positive, it indicates that the ligands on the probe surface dissociate from or undergo molecular allosteric activation with the molecules in the sample solution.

[0039] When ΔD is negative, the luminous intensity becomes negative; when Δτ is negative, it is an abnormal signal.

[0040] The main innovation of this invention lies in using the unique multiple optical parameters of UCNP probes for mutual verification and synergistic analysis. The analytical objectives can be qualitative analysis (in which qualitative or quantitative values ​​of these optical parameters can be used), such as analyzing whether the probe has specifically bound or non-specifically adsorbed molecules in the solution, or whether molecular conformational changes occur after specific binding, etc. It can also be quantitative analysis, such as calculating the absolute or relative affinity between the probe ligand and the target molecule. Quantitative calculations depend on the quantitative values ​​of these optical parameters and the fitted curves.

[0041] Secondly, the present invention provides a system for synergistic analysis of molecular interaction states based on multimodal optical parameters of rare-earth upconversion nanomaterials, comprising:

[0042] A ligand-coated rare-earth upconversion nanomaterial UCNP probe is in contact with the sample to be tested.

[0043] The reaction site provides a place for the sample to be tested and the rare earth upconversion nanomaterial UCNPs probe to react in contact;

[0044] The laser module is used to provide near-infrared pulsed laser light to rare-earth upconversion nanomaterial UCNPs probes;

[0045] The detection module is used to collect the fluorescence signals emitted by the rare earth upconversion nanomaterial UCNPs probe: changes in luminescence intensity, changes in molecular layer thickness / mass ΔD, changes in luminescence lifetime Δτ, and emission peak shift Δλ.

[0046] The analysis module provides analysis results based on the signals collected by the detection module and the preset judgment rule base.

[0047] Preferably, the reaction site is a microplate, a microfluidic chip, or a cuvette, etc., and can also be used for various in vivo imaging scenarios.

[0048] Explanation of proper nouns

[0049] 1. Molecular layer thickness / mass change ΔD

[0050] The core physical significance of molecular layer thickness / mass is the accumulation of molecular layer mass / thickness on the probe surface. A good quality of this parameter indicates continuous, unidirectional signal change and coordination with changes in Δτ or ΔR. Conversely, sudden signal jumps or violent fluctuations suggest the presence of bubbles or precipitates. When ΔD > 0, it indicates adsorption has occurred, meaning molecules have bound to the probe surface. Therefore, changes in molecular layer thickness / mass can provide macroscopic information on the total amount of binding, which is related to both the composition and concentration of the test solution (and thus the OD value of the solution). During molecular interaction detection, the state of molecular interactions is also affected by the concentration of the test solution sample, and the change in molecular layer thickness / mass ΔD can serve as an analytical parameter to help obtain accurate analytical results. However, ΔD cannot be used alone as a judgment parameter because it cannot distinguish between specific binding and non-specific adsorption. In the acquired optical signal, the increment of molecular layer thickness / mass manifests as the overall offset (ΔOD) of the acquired broadband background signal, i.e., a baseline shift.

[0051] 2. Change in luminous intensity ΔI or ΔR

[0052] Changes in luminescence intensity are essentially the result of energy transfer on the probe surface. These changes typically trend inversely to ΔD; that is, when ΔD is positive, ΔI or ΔR is negative (except in cases where fluorescent impurities adsorb or form hydrophobic pockets, reducing defects). After reference calibration, ΔI becomes ΔR, and the trends of ΔI or ΔR are consistent with the trends of luminescence lifetime Δτ. When ΔD is positive while ΔI remains unchanged or is also positive, it suggests possible optical path obstruction or non-specific luminescence interference, resulting in poorer quality of the luminescence intensity change parameter. A luminescence intensity change ΔI or ΔR greater than 0 initially indicates an energy transfer event on the probe surface. However, when luminescence intensity changes ΔI or ΔR are used alone for analysis, they are easily affected by various non-specific factors, leading to unreliable results. Luminescence intensity can be directly acquired; ΔI is obtained by subtracting the light intensity at the beginning and end of a predetermined observation time; ΔR is obtained by subtracting the light intensity ratio of two characteristic peaks at the beginning and end of a predetermined observation time.

[0053] Furthermore, compared to ΔI, ΔR can also reflect changes in the polarity of the probe's microenvironment (after calibration with an internal reference). As a signal of internal reference change, ΔR, in conjunction with Δτ, can reflect changes in microenvironment polarity. When ΔR is used as a microenvironment probe, its direction of change (e.g., negative) has chemical rationale (e.g., indicating enhanced hydrophobicity of the probe surface). Because wavelengths are relatively more stable and lower wavelengths are more sensitive, the ratio of low-wavelength light intensity to high-wavelength light intensity is usually used when calculating R. ΔR provides an interference-resistant binding signal (the calibration value of light intensity, resistant to system interference) and provides information on the binding mechanism (e.g., a negative ΔR indicates enhanced hydrophobic interaction).

[0054] 3. Change in luminescence lifetime Δτ

[0055] Changes in luminescence lifetime reflect the molecular microenvironment and relaxation dynamics. Better quality luminescence lifetime variation parameters are characterized by: a significant and repeatable shortening of the luminescence lifetime (Δτ being negative); and a high goodness of fit (R²) in the luminescence lifetime decay curve function. 2 A value closer to 1 indicates a higher goodness of fit (e.g., >0.8); or a multi-exponential fit can resolve reasonable lifetime components. Conversely, the following characteristics indicate poor quality of the luminescence lifetime variation parameter: Δτ is less than instrument error or its own drift (e.g., within 1% of the luminescence lifetime, in which case Δτ is considered approximately zero or no change in lifetime); or the decay curve function of the luminescence lifetime has poor goodness of fit (cannot fit convergence), making it impossible to extract reliable lifetime values. When the luminescence lifetime variation Δτ is greater than zero, it indicates that specific binding has occurred on the probe surface. Δτ also uniquely reflects and detects the conformational dynamics of molecules.

[0056] 4. Emission peak displacement change Δλ (red-blue shift)

[0057] The change in emission peak wavelength reflects the change in the local electromagnetic field intensity at the luminescence center, originating from the polarity of the microenvironment, stress, or lattice field disturbance. A reliable evaluation method for the emission peak shift change Δλ is as follows: a significant and continuous unidirectional shift in the emission peak wavelength (>instrument resolution, e.g., >0.5 nm); or a shift direction with physicochemical rationality: such as a blue shift (Δλ is negative), indicating a decrease in microenvironment polarity after binding (increased hydrophobicity), or compression stress; for example, a red shift (Δλ↑), indicating an increase in microenvironment polarity, or the formation of specific interactions such as hydrogen bonds. It must strongly correlate with the change in Δτ; when Δτ is negative, Δλ must not be zero, but either negative or positive. When Δλ occurs in the following situations, its priority or weight can be ignored or reduced in the judgment rules: For example: Δλ is small and fluctuates within the instrument noise range, then it is considered approximately equal to 0; the wavelength shift direction is erratic and cannot be fitted; it is not correlated with the change in Δτ, for example, a significant blue shift in wavelength but no change in Δτ (indicating that it may only be physical adsorption rather than specific adsorption), or a significant red shift in wavelength but no change in Δτ.

[0058] Δλ is a microscopic characterization, directly revealing specific physicochemical changes (such as polarity and stress) within the binding pocket. It provides the most direct mechanistic evidence for "how molecules and probes bind," surpassing the information provided by ΔR. Δλ and ΔR corroborate each other, enhancing the reliability of binding mechanism assessments. When using Δλ to determine specific binding or non-specific adsorption, it must be analyzed in conjunction with Δτ, as its use alone is easily affected by factors such as temperature and pH.

[0059] The above parameters can corroborate each other. When the quality of the collected parameters is poor, their priority or weight in collaborative analysis and judgment can be reduced; conversely, their priority or weight should be increased. It should be noted that zero change does not mean the value change is absolutely zero, but rather that the change is less than the system error, typically within 1% of the corresponding index value. For example, if the luminescence lifetime τ is 1 ms, then 1% of 1 ms is considered a very small change (or even no change or negligible change). Furthermore, a change less than 0.5% is considered zero.

[0060] (III) Beneficial Effects

[0061] Compared with the prior art, the present invention has the following significant advantages:

[0062] 1. Addressing the problems of existing technologies, this invention, for the first time, incorporates fluorescence lifetime τ into the molecular interaction analysis system of UCNPs. Since fluorescence lifetime τ is a "fingerprint" parameter, only local microenvironmental changes caused by specific binding (such as molecular packing density and polarity) significantly alter the τ value, while non-specific adsorption has minimal impact on τ. Therefore, by combining τ with other parameters, it is suitable for detecting complex samples and eliminating false positives (non-specific adsorption). Furthermore, the change in fluorescence lifetime Δτ / initial fluorescence lifetime τ0 is co-correlated with the change in ligand binding ratio, eliminating the need for model transformation.

[0063] 2. This invention collects at least two parameters for synergistic analysis and compares the collected multi-parameter optical information with a pre-stored rule base to reveal more biological significance beyond molecular interaction information (including coverage of specific and non-specific events). The method of this invention is applicable to all-liquid phase environments (immersing the probe into the sample solution); it is also applicable to solid-phase detection environments, including: modifying the probe onto the end face of a light guide column to create a sensor, using this sensor to detect and analyze the sample solution; or encapsulating the probe in the reaction cell of a microfluidic chip, using this fluidic chip to detect and analyze the sample solution, eliminating the solid-phase interface defects of traditional techniques.

[0064] 3. The analytical method and system of the present invention can be used for in situ analysis of undiluted or purified pretreated serum samples or cell lysates. Information on the changing trends of at least two parameters can be cross-validated, and the false positive rate is lower than that of traditional SPR / BLI detection technology. Attached Figure Description

[0065] Figure 1 This is a schematic diagram of the working mode of whole-liquid phase molecular interaction analysis and detection based on UCNPs.

[0066] Figure 2 This is a schematic diagram of the working mode of solid-phase molecular interaction analysis and detection based on UCNPs.

[0067] Figure 3 It is the shape of the fitted curve of four optical parameters: change in luminous intensity ΔI or ΔR, change in molecular layer thickness / mass ΔD, change in luminous lifetime Δτ, and change in wavelength Δλ. Detailed Implementation

[0068] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0069] 1. Preparation of ligand-coated rare-earth upconversion nanomaterial UCNPs probes

[0070] For the preparation of ligand-coated rare earth upconversion nanomaterials (UCNPs) probes, please refer to the prior patent CN119198650B, in which the surface of rare earth upconversion nanomaterials (UCNPs) can be coated with an anti-quenching layer, and then ligands are connected to the surface of the anti-quenching layer.

[0071] Rare earth upconversion nanomaterials are prepared by doping rare earth ions into an inorganic matrix. The rare earth ions are selected from Yb. 3+ Er 3+ Tm 3+ Ho 3+ and Nd 3+The rare earth upconversion nanomaterials contain at least one rare earth ion, and the inorganic matrix can be selected from fluorides, oxides, phosphates, and vanadates. The rare earth upconversion nanomaterials are nanoscale particles, such as nanospheres, cubes, or hexagonal phases, with a particle size of 15-100 nm, preferably 20-70 nm, and more preferably 20-40 nm. In practical applications, 22 nm rare earth upconversion nanoparticles show superior detection performance and are currently the preferred choice for commercial conversion.

[0072] When the test solution is aqueous, the vibration of water molecules easily leads to the quenching of rare earth upconversion nanomaterials. The vibration of the hydroxyl groups of two water molecules (~3400 cm⁻¹) -1 ) and Er 3+ Excited state energy level (~6500cm) -1 Rare-earth upconversion nanomaterials are highly susceptible to emission resonance, leading to non-radiative energy dissipation and a quenching effect on their luminescence. This problem does not exist when the test solution is in an oil phase. Most applications of molecular interaction detection are in aqueous environments. To reduce the quenching caused by water molecule vibrations and improve signal intensity, anti-quenching modification of the rare-earth upconversion nanomaterials can be considered. However, quenching modification may also weaken the emission intensity of the rare-earth upconversion nanomaterials and may increase the distance between the rare-earth upconversion nanomaterials and ligand and target molecules, affecting energy transfer. Therefore, thin-layer modification / coating is preferred to achieve anti-quenching modification.

[0073] In this application, anti-quenching modification means treating rare earth upconversion nanoparticles using any of the following methods: inert shell coating (NaYF4, NaLuF4, NaGdF4, SiO2, Al2O3), metal-organic framework (MOF) encapsulation, or polymer encapsulation. Whether or not to pre-treat rare earth upconversion nanomaterials with anti-quenching modification depends not only on whether the test solution is aqueous, but also on the fact that if the intensity of the emitted light signal is sufficient in actual detection, anti-quenching modification of the rare earth upconversion nanoparticles may not be necessary.

[0074] Rare-earth upconversion nanomaterials can couple ligand molecules to their surfaces. These ligand molecules interact specifically with target molecules in the test solution through various mechanisms, such as electrostatic adsorption, coordination, covalent bonding, mechanical interlocking, and chimerism. The ligand molecules can be selected from protein ligands, nucleic acid ligands, small molecule ligands, cellular ligands, carbohydrate and lipid ligands, and biomimetic synthetic ligands. When the rare-earth upconversion nanomaterials have a quench-resistant modified shell, active groups (such as hydroxyl, amino, and carboxyl groups) can be formed on the shell to connect the ligand molecules.

[0075] Among them, protein ligands include natural receptor proteins (such as cell surface receptors, enzyme active cores), engineered binding proteins (such as Affibody, DARPin, and Anticalin), polypeptide ligands (such as phage display-screened target peptides), and monoclonal antibodies; nucleic acid ligands include single-stranded DNA or RNA (such as primers and probes), and nucleic acid aptamers; small molecule ligands include natural ligands (such as hormones, vitamins, and metabolites) or synthetic compounds (such as drug molecules or fluorescently labeled probes); carbohydrate and lipid ligands include polysaccharides / oligosaccharides (such as lectins binding to glycans), glycolipids / phospholipids, etc.; and biomimetic synthetic ligands include molecularly imprinted polymers (artificially mimicking antibody binding sites) and peptide nucleic acids (PNAs), etc.

[0076] 2. Fabrication of a sensor using end-face modified rare-earth upconversion nanomaterial UCNPs probes

[0077] For the fabrication of sensors with end-face modified rare-earth upconversion nanomaterial (UCNP) probes, please refer to the prior patent CN119198650B. Since the rare-earth upconversion nanomaterials are fixed on the outer surface of the detection end of the light guide post, the upper surface of the rare-earth upconversion nanomaterials will not come into contact with water and is less affected by water molecule vibrational quenching. Therefore, it is also possible to consider only modifying / coating the exposed surface of the rare-earth upconversion nanomaterials with a thin layer, such as 2-6 nm Al₂O₃ALD or oleic acid coating (oleic acid coating thickness approximately 2 nm). The modification of the rare-earth upconversion nanomaterial (UCNP) probes on the end face of the sensor's light guide post can be achieved using electrostatic adsorption or chemical bond coupling methods. For example, it can be carried out as follows.

[0078] (1) Electrostatic adsorption method

[0079] The surface of a quartz light guide column is typically negatively charged (isoelectric point approximately pH 2-3), and rare earth upconversion nanoparticles (NaYF4: 20% Yb) are used. 3+ 2% Er 3+ In solution, quartz can become negatively charged through surface modification (such as adding carboxylic acid groups). To achieve electrostatic adsorption, the quartz surface can be pre-treated to be positively charged. The specific steps are as follows:

[0080] Step 1: Surface treatment of quartz light guide column (positive electrochemical modification)

[0081] ① Substrate cleaning: Ultrasonic cleaning: Use acetone, ethanol and deionized water in sequence for ultrasonic cleaning to remove organic matter and particulate contamination.

[0082] ② Plasma treatment: Activate the surface hydroxyl groups (-OH) with oxygen plasma.

[0083] ③ Aminosilanization (introduction of positive charge): Immerse the quartz light guide post 6 substrate in an ethanol solution containing APTES and react at room temperature. After the reaction, remove it, rinse with ethanol, dry with ammonia, anneal, and cure. Measure the Zeta potential of the modified quartz surface using a Zeta potentiometer; it must reach the expected value.

[0084] Step 2: Dispersion and charge regulation of rare earth upconversion nanoparticles

[0085] Rare earth upconversion nanoparticles (NaYF4:20%Yb) 3+ 1% Ho 3+ @NaYF4: 20% Yb 3+ 2% Er 3+ Er 3+ Used to emit 540nm fluorescence, Ho 3+ Used to emit 1180nm fluorescence. Er 3+ with Ho 3+ The ion-layer design can reduce energy quenching between ions on the one hand, and on the other hand, Ho 3+ In nano-core layers, it is less affected by the environment and can be used to generate doping ratio signals; Er 3+ Ions in the outer layer are influenced by the environment and connect to ligand molecules (interacting with target molecules), which can serve as detection signals. Nanoparticles are dispersed in phosphate-buffered saline (PBS). Sodium citrate is added as a surface ligand, and the mixture is sonicated to form a stable dispersion. By adjusting the pH of the solution, the carboxylic acid groups on the particle surface are ionized (-COO-), becoming negatively charged. The Zeta potential of the modified quartz surface is measured using a Zeta potential meter to confirm that the particle surface potential reaches the expected value. The method for synthesizing core-shell upconversion nanocrystals is an existing technique; for example, refer to the paper titled "An Emerging Toolkit of Ho" published by Xusheng Wang, Wenxiao Wu et al. in the Journal of the American Chemical Society in 2025. 3+ Sensitized Lanthanide Nanocrystals with NIR-II Excitation and Emission for in Vivo Bioimaging" article.

[0086] Step 3: Electrostatic adsorption coating

[0087] The amino-aluminate light guide column is vertically immersed in the nanoparticle dispersion, and allowed to stand for adsorption and electrostatic bonding.

[0088] Step 4: Post-processing and fixation

[0089] Rinse the detection end of the light guide column gently with deionized water to remove physically adsorbed particles, and then dry the surface with ammonia gas.

[0090] (2) Chemical bond coupling method

[0091] First, the detection end of the quartz light guide and the surface of rare-earth upconversion nanoparticles (UCNPs) are chemically modified separately to introduce specific, reactive functional groups. On the detection end surface of the quartz light guide, amino (-NH2, such as APTES), carboxyl (-COOH, such as carboxyl-containing silanes), or epoxy (such as GPTMS) groups are introduced through silanization. On the UCNPs surface, groups complementary to the functional groups on the quartz end face are introduced. Common combinations include:

[0092] Quartz -NH2+UCNPs-COOH (forms amide bonds by activating carboxyl groups via EDC / NHS)

[0093] Quartz -COOH + UCNPs -NH2 (quartz carboxyl groups are also activated via EDC / NHS)

[0094] Quartz-epoxy group + UCNPs-NH2 (epoxy group ring-opening reaction with amino group)

[0095] Coupling reaction: Under specific reaction conditions (common catalysts such as EDC / NHS), the functional groups on the end face of quartz undergo a covalent chemical reaction with the functional groups on the surface of UCNPs (such as forming amide bonds), thereby firmly bonding UCNPs to the quartz surface.

[0096] The modification method of ligand-coated rare-earth upconversion nanomaterials (UCNPs) probes on microfluidic chips can be found in the sensor fabrication process.

[0097] 3. Detection Mode

[0098] Liquid phase detection mode: Ligand-coated rare earth upconversion nanomaterials (UCNPs) probes are inserted into the solution sample to be tested.

[0099] Solid-phase detection mode: A sensor is obtained by modifying a ligand-containing rare-earth upconversion nanomaterial (UCNP) probe onto the end face of a quartz light guide column. The sensor's detection is then applied to the sample solution to be tested. Alternatively, a ligand-containing rare-earth upconversion nanomaterial (UCNP) probe is modified into the flow channel or reaction cell of a microfluidic chip, and the sample solution to be tested is loaded into the flow channel or reaction cell of the microfluidic chip.

[0100] See Figure 1 The full liquid phase detection mode shown and Figure 2 The sensor is shown in solid-phase detection mode. Near-infrared pulsed lasers of 808nm, 915nm, 940nm, 980nm, or 1143nm are used as excitation light.

[0101] like Figure 1 As shown, a ligand-coated rare-earth upconversion nanomaterial (UCNP) probe is inserted into the test solution sample. A lens is placed on one side of the transparent container holding the test solution sample. A PWM generator produces a pulse width modulation signal (square wave signal) as the system's "synchronization reference." The emitter, driven by the PWM signal, emits modulated laser light (red arrow). The lens is used to focus the visible light emitted by the UCNP probe (to improve the emission light collection efficiency). The receiver receives optical parameters, and the MCP (microchannel plate) is used for synchronous acquisition time.

[0102] like Figure 2 As shown, a UCNPs probe, a rare-earth upconversion nanomaterial with ligands, is modified onto the end face of a quartz light guide to obtain the sensor's detection end, which is then immersed in a container holding the sample solution. The non-detection end of the sensor is connected to the optical fiber at the red position (transmitting laser light), the optical fiber at the blue position (transmitting emitted light signal), and the optical fiber at the green position, and is equipped with corresponding optical devices (such as a bandpass filter of 520-600nm, which only receives the emitted light signal intensity of the 540nm peak, used as a lifetime light source for MCP measurement).

[0103] The signal flow of the two detection modes mentioned above:

[0104] (1) Dual function of PWM signal. Driving the emitter: The PWM signal directly controls the emitter (such as a laser) to emit pulsed light (red "excitation light") synchronized with the PWM, which illuminates the sample solution. Synchronizing the receiver: The PWM signal is simultaneously transmitted to the receiver as a "reference clock" to identify the "useful signal". (2) Reception and differentiation of optical signals. Useful signal: The laser emitted by the emitter is reflected by the target and received by the receiver. Since the optical signal is synchronized with the PWM (same frequency and phase), the receiver can identify it through the PWM reference. Interference light: Unmodulated light (blue "visible light") such as natural light and other light sources in the environment will also be received by the receiver, but they are not synchronized with the PWM signal and will therefore be regarded as "noise". The key function of the PWM signal is to suppress self-emission interference: Only light synchronized with the PWM signal (pulsed light emitted by the emitter itself) will be recognized as a valid signal by the receiver. Asynchronous interference light (including the weak "self-emission" noise that the emitter may generate) will be filtered out because it is not synchronized with the PWM, thereby improving the detection signal-to-noise ratio.

[0105] 4. Signal Acquisition and Processing

[0106] Near-infrared light with a frequency of 500 Hz was used for excitation. The following parameters were collected during the detection process:

[0107] Fluorescence lifetime Δτ: obtained using a microchannel plate detector (MCP);

[0108] Change in luminous intensity ΔI: collected by photomultiplier tube or spectrometer 540 with I 1180 (or I) 540 / I 1180 Peak intensity, I 1180 It is believed that the fundamentals will remain unchanged;

[0109] Emission intensity change ΔR: The change in emission peak ratio recorded by the spectrometer, such as I 540 / I 1180 .

[0110] To address signal drift and interference from internal system factors (such as excitation power fluctuations and temperature-induced excitation instability in the optical path), a correction can be achieved using the ratio of the 540nm sensitive peak I1 to the 1180nm inert peak I2. To eliminate systematic errors caused by probe concentration fluctuations, optical path drift, and matrix absorption, the intensity I of the 540nm sensitive peak is used as the reference value. 540 With the 1180nm inert peak intensity I 1180 The ratio ΔR / R0 is used as the intrinsic strength parameter, and the formula for calculating the intrinsic strength parameter is as follows:

[0111] Intensity intrinsic parameter ΔR=(I 540 / I 1180 )-(I 0540 / I 01180 )

[0112] Among them: I 540 and I 1180 These are the light intensities of the two characteristic peaks measured in real time; I 0540 and I 01180 This is the baseline value (initial value) at the start of a certain observation period.

[0113] All the above changes are relative to the initial signal and can be negative, positive, or unchanged (approximately equal to zero).

[0114] 5. The data dimensions, core significance, and evaluation criteria for the quality of each of the above parameters.

[0115] Table 1:

[0116]

[0117] Note: (1) In the table above, “↓” indicates a negative change and “↑” indicates a positive change; it only represents the trend of change, not the rate of change.

[0118] (2) "≈0" means that the change is less than the system error value or less than 1% of the index value, which can be ignored.

[0119] In the parameter quality evaluation of the above four parameters (ΔI and ΔR are both luminescence intensity parameters), the higher the percentage of parameters meeting the evaluation criteria for "good" quality, the better the parameter quality; conversely, the higher the percentage meeting the evaluation criteria for "poor" quality, the worse the parameter quality. In molecular interaction detection, higher quality parameters have higher reliability. When using the same probe and the same sample, parameters with higher quality are preferred. In the four-parameter synergistic analysis, besides Δλ being an important reference option, the higher the quality of the other three parameters, the greater their weight in subsequent analysis processes.

[0120] 6. Judgment Rules

[0121] Table 2: Judgment Rules

[0122]

[0123] Note: (1) In the table above, “↓” indicates a negative change and “↑” indicates a positive change.

[0124] (2) When performing co-analysis, Δλ is a reference option, and "-" indicates that its displacement can be ignored;

[0125] (3) "≈0" means that the change is less than the system error value or less than 1% of the index value, which can be ignored.

[0126] The judgment rules and combinations listed in the table above are merely examples and not an exhaustive list of judgment or analysis rules. For example, in addition to the rule base shown in Table 2, when ΔD is zero and ΔI is positive, the luminescence intensity increases while the molecular layer thickness remains unchanged. This means that there are no molecules bound (specifically bound or non-specifically adsorbed) on the UCNPs probe surface in the test solution, but the UCNPs probe surface may have undergone defect repair, reducing non-radiative relaxation channels and showing a trend of increasing luminescence intensity. Therefore, the rule base listed in Table 2 is only a partial list of judgment rules in this invention and is not a limitation on the present invention. Any method for synergistic analysis to determine the molecular interaction state based on at least two parameters of the multimodal signals (ΔD, ΔI / ΔR, Δτ, Δλ) disclosed in this application should fall within the protection scope of this invention.

[0127] Signal acquisition revealed that the fitting curves of the four optical parameters over time exhibited different functional characteristics. (See [link to relevant documentation]). Figure 3 The image shows an example curve obtained during actual sample testing. Figure 3As shown in (a), the curve of change in molecular layer thickness / mass ΔD over time follows an exponential curve; as shown in (b), the curve of change in luminescence intensity ΔI over time follows an exponential curve; as shown in (d), the curve of change in wavelength ratio ΔR over time follows an exponential curve; and as shown in (c), the curve of change in fluorescence lifetime Δτ over time follows an exponential curve (overall S-shaped in the full variation scenario). As shown in Figure (e), the curve of change in fluorescence lifetime Δτ over time cannot be fitted and converged, indicating that the fluorescence lifetime Δτ has not changed substantially. When the R² of the fitted data is closer to 1, it indicates that the parameter is of good quality, and it is considered that the parameter has changed substantially during analysis, and can be used as a relatively reliable analytical parameter. Of course, whether to use a certain parameter depends on the specific analytical objective. For example, if the analytical objective is to screen antibodies, when comparing antibody affinity, non-specific adsorption issues do not need to be considered. In this case, when the changes in luminescence intensity or molecular layer thickness can be used to draw conclusions, the change in luminescence lifetime may not need to be included in the analysis.

[0128] The following description is based on preferred embodiments of the present invention. The UCNPs probes in the following embodiments utilize upconversion nanoparticles NaYF4:Yb. 3+ Er 3+ Through a multi-epitaxy synthesis method, the particle size of upconversion nanocrystals NaYF4:Yb,Er can be controlled within 52nm ± 2nm, which can be used to improve the brightness and detection sensitivity of nanoparticles. Antibodies are then chemically coated onto the upconversion nanoparticles.

[0129] Example 1

[0130] 1.1 Preparation of Experimental Materials

[0131] Test sample: Peripheral blood sample taken from suspected colorectal cancer patients. Serum was obtained by centrifugation and detected by protein quantification kit. The concentration of carcinoembryonic antigen (CEA) in the serum was 5.2 ng / mL.

[0132] Preparation of UCNPs probes: NaYF4:Yb was synthesized by hydrothermal method. 3+ Er 3+ Upconversion nanoparticles (average particle size 20 nm) were modified by introducing amino groups on the particle surface through silanization. Then, an anti-CEA monoclonal antibody (anti-CEAmAb, which specifically recognizes the C-terminal domain of CEA) was coated onto the particle surface using a glutaraldehyde cross-linking method to prepare an anti-CEAmAb-UCNPs probe. The probe was characterized by dynamic light scattering (DLS) and the hydrated particle size was 55 nm with a zeta potential of -18 mV.

[0133] Detection Instruments: A spectrometer equipped with a 980nm pulsed laser (using dual-mode processing, output power 50mW, single acquisition cycle 500ms, 500Hz excitation frequency in the 0ms-249ms range for MCP lifetime acquisition, and continuous excitation in the 250ms-499ms range for spectral data reception by the spectrometer), an MCP (MCP microchannel plate) system, and a detection timescale of greater than 10μs and less than 1ms (determined by the material's designed lifetime range and the pulse excitation frequency of 500Hz) for the fluorescence lifetime of the 540 emission peak. A delay time of 100μs and a time gate of 800μs are set to suppress short-lived autofluorescence (<10ns, such as the fluorescence of bilirubin and hemoglobin derivatives) and background noise in serum.

[0134] 1.2 Experimental Procedure

[0135] Mix 100 μL of serum sample with 100 μL of anti-CEAmAb-UCNPs probe solution (concentration 0.1 mg / mL) and incubate at 37℃ for 30 min, gently shaking every 5 min to ensure sufficient contact between the probe and the sample. Transfer the mixture to a quartz cuvette and place it in the detection optical path of the spectrometer. Excite with a 980 nm pulsed laser and simultaneously acquire the following optical parameters:

[0136] Ellipsometry was used to monitor the change in molecular layer thickness (ΔD) on the probe surface in real time; 540 nm (Er) was recorded. 3+ of 4 S3 / 2→ 4 I 15 The change in luminescence intensity (ΔI) at the / 2 transition is collected; the change in luminescence lifetime (Δτ) at 540nm is collected using MCP technology.

[0137] 1.3 Experimental Results

[0138] Changes in molecular layer thickness: After incubation, the molecular layer thickness on the probe surface increased compared to the initial state, ΔD=+8.5nm, indicating the presence of substances in the sample that bind to anti-CEAmAb, forming a “UCNP-antibody-antigen” complex, which leads to the thickening of the molecular layer on the probe surface.

[0139] Changes in luminescence intensity: The luminescence intensity at 540 nm decreased significantly, with ΔI = -32% at 540 nm and ΔI = -28% at 660 nm. This is because after CEA binds to the antibody, the large protein molecules affect the local optical environment of UCNPs, enhancing fluorescence resonance energy transfer (FRET) or non-radiative transition processes, leading to fluorescence quenching.

[0140] Changes in fluorescence lifetime: The fluorescence lifetime at 540 nm decreased from the initial 480 μs to 390 μs, Δτ = -90 μs, indicating that the specific binding of the antibody to CEA altered the electronic transition environment on the UCNP surface, shortening the luminescence lifetime. 3+ The excited state lifetime.

[0141] 1.4 Conclusion

[0142] The ligand of the anti-CEAmAb-UCNPs probe (anti-CEA monoclonal antibody) specifically bound to carcinoembryonic antigen (CEA) in the blood sample, and the result was consistent with expectations.

[0143] Example 2

[0144] The only difference between this embodiment and Example 1 is that the UCNPs probe was prepared by coating its surface with an anti-human serum albumin monoclonal antibody (anti-HSAmAb, which specifically recognizes serum albumin but does not bind to CEA), resulting in an anti-HSAmAb-UCNPs probe. DLS characterization showed a hydrated particle size of 52 nm and a zeta potential of -16 mV. The detection instruments and parameters were the same as in Example 1. The detection steps, experimental results, and conclusions are as follows.

[0145] 1. Experimental steps:

[0146] Take 100 μL of serum sample and mix it with 100 μL of anti-HSAmAb-UCNP probe solution (concentration 0.1 mg / mL). Incubate at 37℃ for 30 min, following the same procedure as in Example 1. Transfer the mixture to a quartz cuvette and excite it with a 980 nm pulsed laser. Simultaneously acquire the following optical parameters: ΔD, ΔI (540 nm), and Δτ (540 nm, MCP technique).

[0147] 2. The experimental results are as follows:

[0148] Changes in molecular layer thickness: The molecular layer thickness on the probe surface increased slightly, ΔD=+2.1nm. It is speculated that a small amount of non-specific proteins (such as globulins) in the serum were adsorbed onto the probe surface through van der Waals forces or electrostatic interactions, resulting in a slight increase in molecular layer thickness, but the increase was much lower than that in Example 1.

[0149] Changes in luminescence intensity: ΔI = -8% at 540nm, the fluorescence quenching is significantly weaker than in Example 1. This is because the non-specifically adsorbed protein does not specifically bind to the antibody on the probe surface, and has little impact on the optical environment of UCNPs, resulting in only weak non-radiative quenching.

[0150] Changes in luminescence lifetime: The initial luminescence lifetime at 540 nm was 543 μs. After incubation, an attempt was made to fit the result using a double exponential decay function. Δτ approached zero (calculated value: +3 μs, error range: ±5 μs), indicating that nonspecific adsorption did not change Er.3+ The excited-state lifetime is long, and there is no specific interaction between the probe and the sample.

[0151] 3. Conclusion

[0152] The ligand of the anti-HSAmAb-UCNPs probe could not form a specific binding with carcinoembryonic antigen (CEA) in the blood sample; only a weak non-specific protein adsorption was observed, which was consistent with the expected result.

[0153] Example 3

[0154] The steps in this embodiment are as follows:

[0155] (1) Sample to be tested: Prepare “protein-free serum” (i.e., serum matrix sample without any protein). Specifically, peripheral blood serum from healthy individuals is taken and all protein components are removed by ultracentrifugation (120000×g, 4℃ for 24h) combined with molecular sieve chromatography (Sephadex G-25 column). The protein concentration is confirmed to be <0.01μg / mL by BCA protein quantification kit, and only small molecules (such as glucose, electrolytes, lipids, etc.) in the serum are retained.

[0156] (2) Synthesis of NaYF4:20%Yb 3+ 1% Ho 3+ @NaYF4: 20% Yb 3+ 2% Er 3+ Upconversion nanoparticles (average particle size 25 nm) were modified with carboxyl groups via silanization. Then, a polyclonal antibody targeting common serum proteins (anti-human total serum protein pAb, capable of broadly recognizing multiple antigenic epitopes of common serum proteins such as albumin and globulin) was coated onto the particle surface using an EDC / NHS activation method to prepare an anti-serum protein pAb-UCNPs probe. DLS characterization showed a hydrated particle size of 58 nm and a zeta potential of -17 mV. Coomassie brilliant blue staining confirmed an antibody coating amount of approximately 12 μg / mg UCNPs, with the characteristic peak of UCNPs at 540 nm (Er). 3+ ) and 1180nm (Ho 3+ ).

[0157] (3) The testing instruments and parameters are the same as in Example 1.

[0158] (4) Mix 100 μL of protein-free serum sample with 100 μL of antiserum protein pAb-UCNPs probe solution (concentration 0.1 mg / mL), incubate at 37℃ for 30 min, and simultaneously collect ΔD and ΔR (R = I 540 / I 1180 ) and Δτ(540nm, curve fitting).

[0159] (5) Experimental results:

[0160] The molecular layer thickness ΔD = +8.5 nm; ΔR is -0.18; the initial luminescence lifetime at 540 nm is 485 μs; and Δτ = 0 μs is calculated (error range ±2 μs).

[0161] (6) Conclusion: The ligands of the anti-serum protein pAb-UCNPs probe do not specifically bind to the components in the protein-free serum sample. The positive ΔD and negative ΔR observed in the experiment are due to the non-specific adsorption of small molecules in the serum matrix, rather than the specific interaction between "antibody and protein", which is consistent with the expected results.

[0162] Example 4

[0163] The experiment in this embodiment is as follows:

[0164] 1. Sample to be tested: Peripheral blood serum from a pancreatic cancer patient during postoperative follow-up, with a CA19-9 concentration of 38 U / mL.

[0165] 2. Preparation of UCNPs probes: Synthesis of NaYF4:20% Yb 3+ 1% Ho 3+ @NaYF4: 20% Yb 3+ 2% Er 3+ (Average particle size 25 nm), after PEGylation modification, coated with anti-CA19-9 mAb, DLS characterization showed a hydrated particle size of 62 nm, a zeta potential of -21 mV, and characteristic emission peaks of 540 nm / 1180 nm. Refer to Example 1 (with an additional wavelength locking module).

[0166] 3. Experimental Procedure: Referring to Example 1, mix 100 μL of serum with 100 μL of probe solution (0.12 mg / mL), incubate at 37°C for 40 min, and collect ΔD and ΔR (R = I). 450 / I 1180 ), Δτ.

[0167] 4. Experimental Results

[0168] ΔD = +6.8nm (±0.4nm); ΔR = -0.01 (±0.008, considered as zero change in light intensity); Δτ = -55μs (±4μs).

[0169] 5. Experimental Conclusions

[0170] The probe was determined to bind specifically to CA19-9, and the result was consistent with expectations.

[0171] Example 5

[0172] The experiment in this embodiment is as follows:

[0173] 1. Sample to be tested: Peripheral blood samples were taken from suspected colorectal cancer patients. Serum was obtained by centrifugation and detected by a protein quantification kit. The concentration of carcinoembryonic antigen in the serum was 5.2 ng / mL.

[0174] 2. UCNPs probe: prepared according to Example 1.

[0175] 3. The testing instrument is the same as in Example 1.

[0176] 4. Referring to Example 1, mix 150 μL of serum with 150 μL of probe solution (0.1 mg / mL), incubate at 37°C for 35 min, and collect ΔD, ΔI, Δτ, and Δλ.

[0177] 5. Experimental Results

[0178] ΔD=+9.2nm (±0.3nm); ΔI=+18% (±1.5%); Δτ=-70μs (±3μs); Δλ=-0.5nm (±0.03nm).

[0179] 6. Conclusion

[0180] The probe was found to specifically bind to CEA, accompanied by a change in antibody conformation, which was consistent with the expected results.

[0181] Example 6

[0182] The experiment in this embodiment is as follows:

[0183] 1. Sample to be tested: Peripheral blood samples were taken from suspected colorectal cancer patients. Serum was obtained by centrifugation and detected by a protein quantification kit. The concentration of carcinoembryonic antigen (CEA) in the serum was 5.2 ng / mL. See Example 1.

[0184] 2. UCNPs probes: UCNPs were prepared according to Example 1, but their surfaces were only hydroxylated without ligands (no specific antibodies).

[0185] 3. The testing instrument is the same as in Example 1.

[0186] 4. Referring to Example 1, mix 100 μL of serum with 100 μL of probe solution (0.08 mg / mL), incubate at 37°C for 30 min, and collect ΔD, ΔI, and Δτ.

[0187] 5. Experimental Results:

[0188] ΔD = +2.5nm (±0.2nm); ΔI = +12% (±1.0%); Δτ = +2μs (±3μs, considered zero).

[0189] 6. Conclusion: The probe non-specifically adsorbs to non-target proteins in serum, which is consistent with the expected results.

[0190] Example 7

[0191] The experiment in this embodiment is as follows:

[0192] 1. Sample to be tested: Serum from breast cancer patients (HER2 concentration 8 ng / mL).

[0193] 2. UCNPs probes: UCNPs were prepared according to Example 1, and after silanization modification, they were coated with anti-HER2 mAb. DLS characterization showed that the hydrated particle size was 60 nm, the zeta potential was -20 mV, and the characteristic emission peaks were 540 nm / 660 nm.

[0194] 3. Testing instruments: Refer to Example 1 (equipped with a low-temperature control module).

[0195] 4. Referring to Example 1, 120 μL of serum was mixed with 120 μL of probe solution (0.11 mg / mL), and incubated at 37°C for 38 min. ΔD, ΔI, Δτ, and Δλ were collected.

[0196] 5. Experimental Results:

[0197] ΔD=+7.6nm (±0.3nm); ΔI=-25% (±1.2%); Δτ=-75μs (±4μs); Δλ=+0.5nm (±0.02nm).

[0198] 6. Conclusion: The probe was found to specifically bind to HER2 and be accompanied by changes in the microenvironment, which is consistent with the expected results.

[0199] Example 8

[0200] The experiment in this embodiment is as follows:

[0201] 1. Sample to be tested: 5% sucrose PBS solution (protein-free).

[0202] 2. UCNPs probe: UCNPs probe was prepared according to Example 4.

[0203] 3. Testing instruments: Refer to Example 4.

[0204] 4. Referring to Example 4, mix 100 μL of sucrose solution with 100 μL of probe solution (0.09 mg / mL), incubate at 37°C for 25 min, and collect ΔD and ΔR (R = I). 450 / I 1180 ), Δτ.

[0205] (5) Experimental results:

[0206] ΔD = +1.9nm (±0.2nm); ΔR = -0.01 (±0.007, considered as zero light intensity change); Δτ = +1μs (±2μs, considered as zero).

[0207] (6) Conclusion: The adsorption of the probe to sucrose molecules was determined to be purely physical, which is consistent with the expected result.

[0208] Example 9

[0209] The experiment in this embodiment is as follows:

[0210] 1. Sample to be tested: Ultrapure water (18.2 MΩ·cm, free of CEA and other interfering substances).

[0211] 2. Preparation of UCNP probes: See Example 1.

[0212] 3. Testing instrument: Refer to Example 1 (ellipsometry set to high sensitivity mode).

[0213] 4. Referring to Example 1, mix 100 μL of ultrapure water with 100 μL of probe solution (0.1 mg / mL), incubate at 37°C for 30 min, and collect ΔD, ΔI, and Δτ.

[0214] 5. Experimental Results

[0215] ΔD = +0.1nm (±0.08nm, considered zero); ΔI = -2counts / s (±3counts / s, considered zero); Δτ = +1μs (±2μs, considered zero).

[0216] 6. Conclusion: The probe was found to have no binding / adsorption events with ultrapure water, which is consistent with the expected results.

[0217] Example 10

[0218] The experiment in this embodiment is as follows:

[0219] 1. Sample to be tested: serum dilution of colorectal cancer patients containing 0.1 mmol / L Congo red (fluorescence quencher) (CEA concentration 5.2 ng / mL, maintaining the presence of the target antigen but introducing the quencher).

[0220] 2. Preparation of UCNP probes: See Example 1.

[0221] 3. Testing instruments: Refer to Example 1.

[0222] 4. Referring to Example 1, mix 100 μL of Congo red serum dilution with 100 μL of probe solution (0.1 mg / mL), incubate at room temperature for 10 min, and collect ΔD, ΔI, and Δτ.

[0223] 5. Experimental Results

[0224] ΔD = +0.2nm (±0.1nm, considered zero); ΔI = -30% (±1.5%); Δτ = +2μs (±3μs, considered zero).

[0225] 6. The presence of a fluorescence quencher (Congo red) in the sample was determined. The decrease in light intensity was caused by quenching, not by a binding event, which is consistent with the expected result.

[0226] Example 11

[0227] The experiment in this embodiment is as follows:

[0228] 1. Sample to be tested: 5% SDS PBS solution (which can destroy the probe structure), with serum from a colon cancer patient added (CEA concentration 5.2 ng / mL).

[0229] 2. Preparation of UCNP probes: See Example 1.

[0230] 3. Testing instrument: Refer to Example 1 (synchronous DLS monitoring of particle size).

[0231] 4. Experimental Procedure

[0232] Referring to Example 1, 100 μL of SDS serum solution was mixed with 100 μL of probe solution (0.1 mg / mL), and incubated at room temperature for 8 min. ΔD, ΔI, and Δτ were collected.

[0233] 5. Experimental Results

[0234] ΔD = -1.8 nm (±0.3 nm); ΔI = -70% (±2%); Δτ = -300 μs (±5 μs); DLS showed that the probe particle size distribution was irregular (20-100 nm).

[0235] 6. Conclusion: The abnormal signal was detected because SDS damaged the probe structure, resulting in abnormal binding / adsorption events, which is consistent with the expected results.

[0236] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. These modifications or substitutions, or combinations of technical features in the above embodiments that do not conflict with each other, can be made in accordance with the manner described in the embodiments. These modifications, substitutions or combinations 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 synergistic analysis of molecular interaction states based on multimodal optical parameters of rare-earth upconversion nanomaterials, characterized in that, include: S1. Provides ligand-coated rare-earth upconversion nanomaterials (UCNPs) probes; S2. Contact the rare earth upconversion nanomaterial UCNPs probe with the sample solution to be tested; S3. Excite the rare-earth upconversion nanomaterial UCNPs probe with near-infrared pulsed laser and simultaneously collect the following optical parameters: change in luminescence intensity, change in molecular layer thickness / mass ΔD, change in luminescence lifetime Δτ, and emission peak shift Δλ. S4. Based on at least two of the aforementioned parameters, jointly analyze and determine the interaction state between the ligands on the probe surface and the molecules in the test solution sample.

2. The method according to claim 1, characterized in that, In step S1, the surface of the rare earth upconversion nanomaterial UCNPs probe is also coated with an anti-quenching inert layer, and ligands are coated on the surface of the anti-quenching inert layer.

3. The method according to claim 1, characterized in that, In step S2, the test solution sample is a pretreated sample, a purified sample, an untreated sample, or an unpurified complex biological sample.

4. The method according to claim 1, characterized in that, In step S2, the rare earth upconversion nanomaterial UCNPs probe is directly immersed in the test solution sample, so that the test solution sample comes into contact with the rare earth upconversion nanomaterial UCNPs probe; or the rare earth upconversion nanomaterial UCNPs probe is modified onto the end face of a light guide rod to form a sensor, and the end face of the sensor is immersed in the test solution sample; or the rare earth upconversion nanomaterial UCNPs probe is modified into a microfluidic chip, so that the test solution sample comes into contact with the rare earth upconversion nanomaterial UCNPs probe.

5. The method according to claim 1, characterized in that, In step S3, the near-infrared pulsed laser is a laser with a wavelength of 808nm, 915nm, 940nm, 980nm or 1143nm. The spectrometer collects the emission spectrum of the excited UCNPs to obtain the optical parameters. The laser pulse is generated by a data acquisition card (DAQ) or a signal generator.

6. The method according to claim 1, characterized in that, In step S3, the optical parameters are qualitative values ​​of changes in luminescence intensity, molecular layer thickness / mass change ΔD, luminescence lifetime change Δτ, and emission peak shift Δλ, such as negative, zero, or positive; or the optical parameters are quantitative values ​​of changes in luminescence intensity, molecular layer thickness / mass change ΔD, luminescence lifetime change Δτ, and emission peak shift Δλ. The change in luminescence intensity is defined as either absolute luminescence intensity ΔI or relative luminescence intensity ΔR after calibration with an internal reference. Absolute luminescence intensity ΔI is the change in light intensity before and after the probe binds to molecules in the test solution. Relative luminescence intensity ΔR is the change in R before and after the probe binds to molecules in the test solution, where R = I1 / I2, and I1 and I2 are the intensities of the two characteristic emission peaks corresponding to the UCNPs, respectively. The emission peak shift Δλ is either a redshift or a blueshift; a positive Δλ indicates a redshift, and a negative Δλ indicates a blueshift.

7. The method according to claim 6, characterized in that, In step S3, I1 and I2 are the intensities of two characteristic peaks corresponding to single-doped UCNPs, or I1 and I2 are the intensities of two characteristic peaks corresponding to double-doped UCNPs.

8. The method according to claim 1, characterized in that, In step S3, when the four optical parameters are analyzed collaboratively, their priority or weight is determined based on their quality. The criteria for evaluating the quality of an optical parameter as good or bad are as follows: The evaluation criteria for good quality of the molecular layer thickness / mass change ΔD are: continuous and unidirectional signal change, the change amplitude is related to the target molecule concentration or reaction time, and there is synergy between ΔD and Δτ or ΔR. The evaluation criteria for poor quality of the ΔD parameter are: sudden step or violent fluctuation. The evaluation criteria for good quality of luminous intensity ΔI or ΔR parameters are: ΔI or ΔR changes in synergy with Δτ; the evaluation criteria for poor quality of ΔI or ΔR parameters are: ΔI or ΔR remains unchanged when Δτ changes. The evaluation criteria for good quality of the parameter Δτ of the change in luminescence lifetime are: a significant and repeatable shortening of the luminescence lifetime and a high degree of fit of the decay curve; the evaluation criteria for poor quality of the parameter Δτ of the change in luminescence lifetime are: the decrease in luminescence lifetime is less than the instrument error or close to zero. The evaluation criteria for good emission peak displacement Δλ are: the emission peak undergoes a significant and continuous unidirectional displacement, the displacement amount is >0.5nm, and it is coordinated with the change in luminescence lifetime Δτ; the evaluation criteria for poor emission peak displacement Δλ are: the emission peak displacement amount is small and fluctuates within the instrument noise range, and the curve of emission peak displacement change over time cannot be fitted and converged.

9. The method according to claim 1 or 8, characterized in that, In step S4, during qualitative judgment, based on at least two optical parameters obtained in S3, and according to a preset judgment rule base, the interaction state between the ligands on the probe surface and the molecules in the test solution sample is analyzed and judged collaboratively; the judgment rule base includes, but is not limited to, the following rules: When ΔD is positive, the luminescence intensity becomes negative, and when Δτ is negative, it indicates that the probe has a specific binding with molecules in the sample solution. When ΔD is positive, the luminescence intensity becomes negative, and Δτ is zero, it is determined that the probe surface produces non-specific adsorption between molecules in the sample solution and the test solution. When ΔD is positive, the luminescence intensity is reduced to zero; when Δτ is negative, it indicates that the probe has a specific binding with molecules in the sample solution. ΔD is positive, the luminescence intensity is positive, Δτ is negative, Δλ is negative, indicating that the probe specifically binds to molecules in the sample solution and undergoes a change in molecular conformation. When ΔD is positive, the luminescence intensity is positive, and when Δτ is zero, it is determined that the probe surface produces non-specific adsorption between molecules in the sample solution. When ΔD is positive, the luminescence intensity is negative; when Δτ is negative, Δλ is positive, it indicates that the probe has a specific binding with the molecules in the sample solution and that there are changes in the microenvironment. When ΔD is positive, the luminescence intensity becomes zero, Δτ is zero, and pure physical adsorption occurs; When ΔD is zero, the luminous intensity is zero, and Δτ is zero, it is determined that there is no binding event. When ΔD is zero, the luminescence intensity becomes negative, and Δτ is zero, a quencher is present in the sample solution to be tested. When ΔD is negative, the luminescence intensity becomes positive; when Δτ is positive, it indicates that the ligands on the probe surface dissociate from or undergo molecular allosteric activation with the molecules in the sample solution. When ΔD is negative, the luminous intensity becomes negative; when Δτ is negative, it is an abnormal signal.

10. A system for synergistic analysis of molecular interaction states based on multimodal optical parameters of rare-earth upconversion nanomaterials, characterized in that, include: A ligand-coated rare-earth upconversion nanomaterial UCNP probe is in contact with the sample to be tested. The reaction site provides a place for the sample to be tested and the rare earth upconversion nanomaterial UCNPs probe to react in contact; The laser module is used to provide near-infrared pulsed laser light to rare-earth upconversion nanomaterial UCNPs probes; The detection module is used to collect the fluorescence signals emitted by the rare earth upconversion nanomaterial UCNPs probe: changes in luminescence intensity, changes in molecular layer thickness / mass ΔD, changes in luminescence lifetime Δτ, and emission peak shift Δλ. The analysis module provides analysis results based on the signals collected by the detection module and the preset judgment rule base.

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