A multiplexed biomolecule tr-fret detection platform and methods of making and using the same

Abstract

The application relates to the technical field of biomedical detection, in particular to a multiple biomolecule TR-FRET detection platform and a preparation method and use method thereof. The multiple biomolecule TR-FRET detection platform comprises a solid-phase chip, a detection probe and a TR-FRET detection system; the solid-phase chip comprises a quartz substrate, a phosphorescent donor and a DARPin recognition molecule; the phosphorescent donor comprises a Zn-MOF encapsulated nitrogen-boron co-doped carbon dot composite material; the detection probe comprises a CdSe / ZnS core-shell quantum dot and a biorthogonal protein self-assembled dendrimer signal carrier. The multiple biomolecule TR-FRET detection platform provided by the application has the advantages of a detection sensitivity of 10-100 pg / mL, a dynamic range of 3-4 orders of magnitude, a single detection time of 20-40 min, a reagent cost of 15-25 yuan / once, and simultaneous support for multiple target parallel detection.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, specifically to a multiplex biomolecule TR-FRET detection platform and its preparation and usage methods. Background Technology

[0002] Precise and high-throughput detection of multiple biomolecules is a core technological support for early clinical disease diagnosis, basic biomedical research, and drug development. Among these technologies, time-resolved fluorescence resonance energy transfer (TR-FRET) has become the mainstream technique for quantitative detection of biomolecules due to its advantages of low background interference and high sensitivity. Currently, mainstream commercial detection platforms such as PerkinElmer DELFIA and Thermo Fisher Scientific HTRF mostly use rare earth chelates as fluorescence donors. Although they have certain detection performance, they suffer from problems such as dependence on rare earth resources, high reagent costs, and large investments in dedicated equipment, making it difficult to meet the application needs of primary healthcare and large-scale screening.

[0003] Chinese invention patent CN113720818B discloses a fluorescence immunoassay system based on a microfluidic chip. This system integrates a sample pool, reaction pool, washing pool, and detection area into a single chip structure, combined with an embedded control module, to automate multi-index detection, effectively simplifying the detection process and reducing device size. However, this technology uses traditional organic fluorescent dyes as markers, with fluorescence lifetimes only in the nanosecond range. It cannot eliminate interference from the autofluorescence of the biological matrix through time-gating technology, thus affecting detection sensitivity. Furthermore, the recognition element relies on monoclonal antibodies, resulting in a development cycle of 6-15 months, large batch-to-batch consistency variability, and poor thermal stability. Simultaneously, the system operates on a single-channel fluorescence detection mode, limiting its multiplex detection capabilities. When detecting more than three targets simultaneously, the spectral crosstalk rate increases significantly, and it requires dedicated detection equipment, making it incompatible with standard laboratory microplate readers, thus restricting its large-scale application. Therefore, developing a detection platform that combines high sensitivity, low cost, strong multiplex detection capabilities, and good equipment compatibility has become an urgent technical problem to be solved in this field. Summary of the Invention

[0004] The first aspect of this invention provides a multiplex biomolecular TR-FRET detection platform, comprising a solid-phase chip, a detection probe, and a TR-FRET detection system; the solid-phase chip comprises a quartz substrate, a phosphorescent donor, and a DARPin (ankylosing spondylogen) recognition molecule; the phosphorescent donor comprises a Zn-MOF (zinc-terephthalic acid coordination polymer) encapsulated nitrogen-boron co-doped carbon dot composite material; the detection probe comprises CdSe / ZnS (cadmium selenide / zinc sulfide) core-shell quantum dots and a dendritic polymer signal carrier self-assembled by biorthogonal proteins.

[0005] Existing TR-FRET detection technologies generally suffer from core defects such as short donor material lifetime, low quantum yield, poor batch-to-batch consistency of recognized molecules, and uncontrollable signal carrier assembly, resulting in insufficient detection sensitivity, high cost, and difficulty in achieving high-throughput multi-target parallel detection. The multi-biomolecule TR-FRET detection platform provided by this invention achieves a performance breakthrough through innovative synergistic mechanisms of material systems, recognition units, and signal carriers: the rigid three-dimensional porous framework of Zn-MOF confines and encapsulates nitrogen-boron co-doped carbon dots (CDs), utilizing the dense crystal structure of MOF to suppress vibrational and non-radiative transitions of carbon dot molecules, reduce oxygen permeation rates to avoid triplet exciton quenching, and simultaneously leverages Zn... 2+ The heavy atom effect of the metal center enhances spin-orbit coupling and improves intersystem crossing efficiency. Combined with the regulation of the electronic structure of carbon dots by nitrogen and boron co-doping, the triplet energy level is stabilized, ultimately resulting in a high-performance phosphorescent donor with a phosphorescence lifetime of >700ms and a quantum yield of >50%. The detection sensitivity is 10-100pg / mL, the dynamic range is 3-4 orders of magnitude, the single detection time is 20-40min, the reagent cost is 15-25 yuan / test, and it also supports parallel detection of multiple targets.

[0006] The DARPin recognition molecule is used to generate target-specific DARPin recognition molecules with KD (dissociation constant) < 100 nM and Tm (melting temperature) > 70 °C.

[0007] The dendritic polymer signal vector that is self-assembled by biorthogonal proteins adopts two orthogonal sequence systems, SpyTag003 / SpyCatcher003 and SnoopTag / SnoopCatcher, and includes a main backbone, a secondary backbone, and terminal functional units.

[0008] Further research revealed that a tertiary dendritic polymer signal carrier constructed based on two orthogonal protein interaction systems achieves spatially ordered arrangement of DARPin recognition molecules and CdSe / ZnS core-shell quantum dots through stepwise hierarchical assembly. This completely avoids the recognition site masking and local quantum dot aggregation quenching problems caused by traditional chemical coupling. Each carrier can stably load 3-5 quantum dot units, resulting in an overall signal intensity increase of 3-8 times compared to single-point loading methods. Furthermore, the particle size distribution index is <0.3 and the assembly efficiency is >90%, providing a precise and controllable molecular scaffold for signal amplification and modular expansion. Simultaneously, the AI-driven DARPin recognition molecule, through targeted optimization of non-conserved regions, achieves excellent performance with binding affinity KD <100 nM, thermal stability Tm >70℃, and batch consistency coefficient of variation <15%, solving the problems of long development cycles, poor thermal stability, and large batch fluctuations associated with traditional monoclonal antibodies. This provides a rapidly customizable, highly specific recognition element for multi-target detection. When target molecules are present in the sample, they simultaneously bind to DARPin recognition molecules on both the solid-phase chip and the detection probe, forming a "donor-target-acceptor" ternary complex. Under 365nm UV excitation, the ultra-long-lifetime phosphorescence generated by the phosphorescent donor excites the CdSe / ZnS quantum dot acceptor via resonant energy transfer. By employing a 100ns time-gated acquisition strategy, short-lifetime autofluorescence background in the sample can be completely filtered out, improving background suppression capability by more than 10 times compared to traditional steady-state detection. Ultimately, a high-sensitivity detection of 10-100 pg / mL is achieved. Furthermore, the combination of quantum dot multicolor encoding and solid-phase chip spatial encoding mechanisms naturally supports parallel multi-target detection. The solid-phase chip mode also allows for the reusability of donor materials, reducing the cost of a single detection and shortening the detection time.

[0009] The dendritic polymer signaling vector, which is a biorthogonal protein self-assembly, uses ProteinA (Staphylococcus aureus protein A) as its backbone and alternately inserts SpyTag003 sequences between its five domains, enabling the backbone to covalently bind to any protein module carrying SpyCatcher003. SnoopTag is further co-expressed at the end of the backbone to introduce a secondary backbone carrying SnoopCatcher, thereby achieving hierarchical expansion of the vector.

[0010] The CdSe / ZnS core-shell quantum dots have a size of 2.7-4.8 nm.

[0011] The size of the dendritic polymer signal carrier that the biorthogonal protein self-assembles is 10-100 nm.

[0012] The Zn-MOF-encapsulated nitrogen-boron co-doped carbon dot composite material is a phosphorescent donor material obtained by encapsulating nitrogen-boron co-doped carbon dots with Zn-MOF as a confined framework.

[0013] The detection platform is compatible with standard ELISA readers, with a detection sensitivity of 10-100 pg / mL and a single detection time of 20-40 min.

[0014] The phosphorescence lifetime of the Zn-MOF-encapsulated nitrogen-boron co-doped carbon dot composite material is >700ms, and the absolute quantum yield is >50%; after being stored at room temperature in PBS buffer for 30 days, the phosphorescence intensity retention rate is >90%.

[0015] A second aspect of this invention provides a method for preparing a multiplex biomolecule detection platform, comprising the following steps: S1: Obtain the DARPin recognition molecule through target structure analysis, variable site definition, initial mutation data construction, multi-model AI training, sequence design and experimental verification; S2: The main backbone, secondary backbone and terminal functional units are expressed and purified respectively, and a dendritic polymer signal carrier for biorthogonal protein self-assembly is formed through stepwise assembly. S3: Zn-MOF encapsulated nitrogen-boron co-doped carbon dot composite material was synthesized by hydrothermal in-situ reaction using zinc nitrate hexahydrate and terephthalic acid as MOF precursors, and 4-carbamoylphenylboronic acid and urea as carbon dot precursors. S4: Activate the quartz substrate, spray Zn-MOF encapsulated nitrogen-boron co-doped carbon dot composite material to form a coating, covalently fix DARPin recognition molecules, and integrate solid-phase chip; S5: CdSe / ZnS core-shell quantum dots are coupled with a dendritic polymer signal carrier self-assembled by biorthogonal proteins to obtain a detection probe.

[0016] The DARPin engineered identification molecule preparation unit uses single-point mutation scanning and random multi-point mutation to construct an initial training dataset. It generates candidate DARPin sequences through integrated prediction of a linear baseline model and a deep learning sequence model. Experimental verification shows that identification molecules with a batch consistency coefficient of variation of <15% are obtained.

[0017] The coating is prepared by spraying and has a thickness of 5-20 μm.

[0018] The DARPin recognition molecule is covalently immobilized via an EDC / NHS activation-amidation strategy.

[0019] The conditions for the hydrothermal in-situ reaction in step S3 are: reaction temperature 180-220℃, reaction time 4-8h; after the reaction, the composite powder is obtained by washing with DMF and anhydrous ethanol and vacuum low-temperature drying.

[0020] The quartz substrate activation treatment in step S4 includes: sequential ultrasonic cleaning with acetone and isopropanol for 15 minutes each, followed by plasma treatment for 5-10 minutes to introduce hydroxyl groups; during the spraying process, the spray gun nozzle diameter is controlled at 0.3-1 mm, the working air pressure at 0.2-0.3 MPa, the substrate temperature at 70-90℃, and the spraying is performed 3-5 times.

[0021] The third aspect of this invention provides a method for using a multiplex biomolecular detection platform, comprising the following steps: mixing the sample to be tested with a detection probe to bind the target molecules in the sample to the detection probe, and adding the sample to a chip pool for incubation to form a ternary complex of the target molecules on a solid-phase chip; after removing unbound components, performing detection using a TR-FRET detection system, acquiring the FRET (Foster resonance energy transfer) signal intensity at each detection site, and obtaining the target molecule concentration through data analysis.

[0022] The detection system parameters are set as follows: excitation by irradiation with 365nm light for 5-10s, acquisition of FRET signal starting after a delay of 100-200ns, and integration time of 1.5-2.5s.

[0023] Beneficial effects 1. The multiplex biomolecule TR-FRET detection platform provided by this invention has a detection sensitivity of 10-100 pg / mL, a dynamic range of 3-4 orders of magnitude, a single detection time of 20-40 min, a reagent cost of 15-25 yuan / test, and supports parallel detection of multiple targets.

[0024] 2. The dendritic polymer signal carrier of the present invention, which is a biorthogonal protein self-assembly, achieves the orderly integration of recognition molecules, protein scaffolds and quantum dot signal units through a stepwise self-assembly method, fundamentally solving the problems of configuration disorder, spatial occlusion and signal attenuation in traditional random chemical coupling methods.

[0025] 3. The carrier size of this invention can be precisely controlled within the range of 10-100 nm. Each carrier can stably load 3-5 quantum dot signal units. The overall signal strength is 3-8 times higher than that of the traditional single-point loading method, and the signal variation coefficient between batches is controlled within 15%.

[0026] 4. The DARPin recognition molecule of this invention is used to generate target-specific DARPin recognition molecules with KD (dissociation constant) <100nM and Tm (melting temperature) >70℃.

[0027] 5. The CdSe / ZnS core-shell quantum dots of this invention achieve multicolor spectral encoding with emission wavelengths of 510-610nm by controlling the quantum dot size to 2.7-4.8nm, providing clear and distinguishable spectral identifiers for different detection targets, and solving the core defect of severe spectral overlap in the multiplex detection of traditional organic fluorescent dyes.

[0028] 6. The Zn-MOF-encapsulated nitrogen-boron co-doped carbon dot composite material used in this invention has ultra-long phosphorescence properties with a phosphorescence lifetime of over 700 ms and a quantum yield of over 50%. It can maintain a stable and strong signal output under solid-state conditions, providing a reliable donor material for time-gated background suppression and high-sensitivity FRET detection.

[0029] 7. The detection platform of this invention can be used in clinical diagnosis (multi-target cytokine detection, combined detection of tumor markers), biomedical research (high-throughput screening of protein interactions, drug target validation), in vitro diagnostic product (IVD) development, and rapid multiplex screening in primary healthcare institutions. Detailed Implementation

[0030] Example 1 A multi-biomolecule TR-FRET detection platform comprises a solid-phase chip, detection probes, and a TR-FRET detection system; the solid-phase chip includes a quartz substrate, a phosphorescent donor (Zn-MOF-encapsulated nitrogen-boron co-doped carbon dot composite material), and a DARPin recognition molecule; the detection probe includes CdSe / ZnS core-shell quantum dots and a dendritic polymer signal carrier self-assembled by biorthogonal proteins.

[0031] A method for preparing a multiplex biomolecular detection platform includes the following steps: S1: Through target structure analysis, variable site definition (engineering the non-conserved regions of DARPin, focusing on the loop region in the repeat unit as the main target recognition interface, variable sites in the N-terminal and C-terminal cap regions that have little impact on overall stability, and key sites in the repeat unit connection region that can adjust binding dynamics; DARPin with 3 repeat units can form a designable space of approximately 20-30 variable sites), initial mutation data construction (using a combination of single-point mutation scanning and random multi-point mutation), multi-model AI training (scoring and ranking the functional feasibility of DARPin sequences: one is a baseline model based on the linear assumption, used to capture simple additive effects; the other is a sequence model based on deep learning, used to learn the nonlinear relationship between mutation combinations), sequence design and experimental verification (gradually screening recognition molecules that meet the project requirements through a combination of high-throughput screening and small-scale expression, displaying and screening candidate sequences or performing in vitro binding verification to evaluate target recognition capabilities; expressing and purifying the preferred sequences in E. coli to verify manufacturability and batch consistency), DARPin recognition molecules are obtained. S2: Separate expression and purification of the main backbone, secondary backbone, and terminal functional unit. Specific methods: The first step is component preparation and purification. The main backbone, secondary backbone, and terminal functional unit are expressed in *E. coli* and purified by Ni-NTA affinity chromatography using a His tag, with purity controlled above 95%. The second step is secondary structure pre-assembly. The terminal functional unit carrying SpyCatcher003 is mixed with the secondary backbone containing SpyTag003 at a predetermined molar ratio and reacted at 37°C for 30 min to form a complex. The third step is main backbone integration and assembly. The above complex is further mixed with the main backbone (C-terminus carrying SnoopTag) and reacted at the same temperature for 30 min to construct a dendritic polymer signal vector of biorthogonal protein self-assembly. S3: The first step is the preparation of the MOF growth precursor system: Zinc nitrate hexahydrate is dissolved in DMF to form a metal source solution, and terephthalic acid is added as an organic ligand. The solution is then sonicated to ensure complete dissolution. Subsequently, carbon dot precursors 4-carbamoylphenylboronic acid and urea are added, and trace amounts of water are introduced to promote MOF crystal nucleation and growth. The second step is hydrothermal in-situ reaction synthesis: The homogeneous mixed solution is transferred to a polytetrafluoroethylene-lined high-pressure reactor and reacted at 200℃ for 6 hours to achieve simultaneous in-situ generation and doping of the MOF framework and carbon dots. The third step is product purification and drying: After the reaction, the product is naturally cooled and repeatedly washed with DMF and anhydrous ethanol to remove unreacted precursors and weakly bound components. It is then dried at low temperature under vacuum to obtain a Zn-MOF encapsulated nitrogen-boron co-doped carbon dot composite material. S4: Activate the quartz substrate, spray Zn-MOF encapsulated nitrogen-boron co-doped carbon dot composite material to form a coating, use EDC / NHS activation-amidation coupling covalent fixation of DARPin recognition molecules, and integrate to obtain a solid-phase chip; S5: CdSe / ZnS core-shell quantum dots (Suzhou Xingshuo Nanotechnology Co., Ltd.) were coupled with a dendritic polymer signal carrier self-assembled by biorthogonal proteins to obtain a detection probe.

[0032] The specific method of the activation treatment is as follows: a high-purity quartz chip (8mm×8mm×1mm) is selected as the solid phase carrier substrate; after the quartz substrate is precision cut, ground and polished in sequence, it is ultrasonically cleaned with acetone and isopropanol (15min each) to remove organic contaminants; then it is thoroughly rinsed with deionized water and dried with nitrogen; finally, it is cleaned with O2 plasma (10min).

[0033] The specific spraying method is as follows: First, the Zn-MOF encapsulated nitrogen-boron co-doped carbon dot composite material is dispersed in a mixed solvent of DMF / ethanol (volume ratio 7:3), and 10wt% PVP is added as a binder. After ultrasonic treatment for 30 minutes, a stable spraying solution is formed. During the spraying process, the nozzle diameter of the spray gun is controlled at 0.5 mm, the working air pressure is 0.2 MPa, the quartz substrate is heated to 80°C, the distance between the spray gun and the substrate is maintained at 15 cm, and the spraying is performed in a uniform scanning manner. After each spraying, the mixture is allowed to stand for about 30 seconds to promote solvent evaporation. This process is repeated 3-5 times to obtain a uniform functional coating with a controllable thickness (10 μm). After the spraying is completed, the temperature is raised to 150°C at a heating rate of 5°C / min under a nitrogen protective atmosphere and held at that temperature for 2 hours.

[0034] The specific method for fixation is as follows: The surface of the above-mentioned coating is immersed in EDC / NHS activation solution (50mM EDC + 100mM NHS, pH 5.5 MES buffer) and reacted at room temperature for 45 min to convert the surface carboxyl groups into active esters; then it is quickly washed with deionized water and dried with nitrogen gas. The activated quartz fragments are immersed in the target protein solution (0.1-1 mg / mL, PBS pH 7.4 or sodium bicarbonate buffer pH 8.0) and reacted at 4℃ or room temperature for 2-4 h to complete covalent coupling; then unreacted sites are blocked with ethanolamine (100mM, pH 8.5). After multiple rounds of washing with PBS / PBS-T, it is finally stored at 4℃ in PBS containing 0.02% sodium azide.

[0035] The specific integration method is as follows: A chip pool structure compatible with a standard 96-well plate is constructed. The pool body is made of black polypropylene material (127.76mm × 85.48mm × 10mm). Quartz wafers are precisely arranged in an 8×12 array inside, with a row and column spacing of 9mm. A 1mm optical isolation band is maintained between the wafers to reduce crosstalk. The quartz wafers are fixed at four points using optical-grade silicone, assembled using a precision positioning fixture and a low-pressure pneumatic head (0.1kPa), and cured at room temperature for 24 hours. Each detection site is assigned a unique number through laser etching, and a correspondence between site-protein-batch information is established in the database to achieve traceability management.

[0036] A method for using a multiplex biomolecular detection platform includes the following steps: mixing the sample to be tested with the detection probe to bind the target molecules in the sample to the detection probe, and adding the sample to a chip cell for incubation. The target molecules are added to the chip cell for incubation, with the liquid level controlled at 2 mm, and incubated at 4°C for 2 hours. This allows the target molecules to form a ternary complex on the solid-phase chip. Unbound components are removed by washing four times with PBS-T, and finally replaced with PBS. Detection is performed using a TR-FRET detection system (Zn-MOF-encapsulated nitrogen-boron co-doped carbon dot donors are excited by 365 nm light for 10 s; FRET signal acquisition begins 100 ns after the excitation light is turned off, with an integration time of 2 s). The FRET (Foster resonance energy transfer) signal intensity of each detection site is acquired, and the target molecule concentration is obtained through automated data analysis using a software system.

[0037] Comparative Example 1 The specific implementation method is the same as in Example 1; the difference is that the Zn-MOF-encapsulated nitrogen-boron co-doped carbon dot composite material is replaced with a rare earth chelate donor (Eu). 3+ / Tb 3+ Chelates).

[0038] Comparative Example 2 The specific implementation method is the same as in Example 1; the difference is that the DARPin recognition molecule is replaced with a monoclonal antibody.

[0039] Comparative Example 3 The specific implementation method is the same as in Example 1; the difference is that the biorthogonal protein self-assembly technology is replaced by the traditional chemical coupling (EDC) method.

[0040] Performance testing methods The following performance tests were performed on the examples and comparative examples.

[0041] (I) DARPin Recognition of Molecular Function Characterization Methods Affinity assays were performed using surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC), with nanomolar levels (KD < 100 nM) as the core performance indicator. The binding rate constant (kon) and dissociation rate constant (koff) of DARPin to the target protein were measured to comprehensively evaluate the affinity and kinetic properties of the recognition molecule. Thermal stability was characterized by determining the Tm of DARPin using differential scanning fluorescence (DSF) or differential scanning calorimetry (DSC), with a target value of Tm > 70 °C, to demonstrate its ability to maintain structural integrity under routine detection conditions and during transportation and storage. Batch consistency verification was performed through comparative analysis of at least three independent expression batches, using SDS-PAGE purity analysis, SEC-HPLC molecular weight distribution detection, and SPR activity assays to evaluate the coefficient of variation (CV < 15%) of key indicators between batches.

[0042] (II) Characterization methods for the structure and function of protein self-assembly vectors The size and uniformity of the carrier were characterized by determining the hydrodynamic particle size and particle size distribution index (PDI) using dynamic light scattering (DLS), with a target PDI < 0.3. The morphology and size range of the dendritic structure (tunable from < 10 nm to tens of nanometers) were verified using transmission electron microscopy (TEM) with negative staining imaging. Molecular weight distribution was precisely determined using SEC-HPLC, with a target product peak percentage higher than 90% and batch-to-batch molecular weight distribution CV < 15%, to assess assembly consistency and scalable production capability. Quantum dot loading efficiency was determined by ICP-MS (inductively coupled plasma mass spectrometry) to determine the number of quantum dots on each protein carrier, with a target of 3-5 stable quantum dot units per carrier. The thermal stability of the carrier was confirmed by DSC measurement of thermal denaturation curves within the range of 20-100℃, confirming the structural stability of the carrier under conventional storage and testing conditions (4℃, more than 6 months). Functional validation was achieved by measuring the FRET signal of the ternary composite material. The quantum dot-labeled carrier was combined with a solid-phase phosphorescent donor chip in an experiment. The actual signal transmission efficiency of the carrier was evaluated using the FRET signal to background ratio (S / B ratio) as the core indicator (target S / B > 5).

[0043] (III) Characterization method of optical properties of Zn-MOF encapsulated nitrogen-boron co-doped carbon dot composite materials Phosphorescence lifetime was determined using a time-resolved spectrometer under ambient temperature and atmospheric conditions to measure the phosphorescence decay curve of the composite material. The phosphorescence lifetime τ (target τ > 700 ms) was obtained by fitting the curve and compared with that of traditional rare-earth chelates (0.5-2 ms) to quantify the technological advantage of the ultra-long lifetime. Quantum yield was determined using an integrating sphere method (absolute quantum yield analyzer) under 365 nm excitation to measure the absolute photoluminescence quantum yield (PLQY) of the material, with a target value higher than 50%. The PLQY variation under different nitrogen and boron doping ratios, reaction temperatures, and time conditions was compared to provide data for process parameter optimization. Material structure characterization was performed by confirming the integrity of the MOF crystal structure using X-ray diffraction (XRD), verifying the distribution of carbon dots in the MOF channels and the nitrogen and boron doping status using transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDX), and precisely analyzing the chemical bonding state of nitrogen and boron using X-ray photoelectron spectroscopy (XPS) to prove the realization of co-doping. Stability evaluation was performed by characterizing the environmental stability of the material by observing the changes in phosphorescence intensity over time in aqueous buffer (PBS, pH 7.4), at different temperatures (4℃, 25℃, 37℃), and under light conditions. The goal was to achieve a phosphorescence intensity retention rate of over 90% after 30 days of storage in PBS at room temperature.

[0044] (iv) Performance evaluation method for solid-state chip testing platform Sensitivity testing was performed by constructing a standard curve using serially diluted standards of the target protein (concentration range 0.001-10000 pg / mL). The limit of detection (LOD) and limit of quantitation (LOQ) of the platform were evaluated based on the fitting results of TR-FRET signal intensity against concentration. The target LOD reached 10 pg / mL. Dynamic range validation was performed by calculating the R-squared value of the logarithmic concentration of the response signal within the linear interval. 2A value ≥0.99 confirms the platform's linear detection capability within a 3-4 order of magnitude range, meeting the mainstream needs of clinical testing and high-throughput screening. Background suppression effectiveness was evaluated by comparing the signal-to-background ratio (S / B ratio) in time-gated (100 ns delay) and non-time-gated modes, as well as its detection performance in complex matrix samples containing 1% serum or 0.5 mg / mL BSA. The suppression effect on short-lived background fluorescence was quantitatively assessed, with the target background suppression effect being more than 10 times better than traditional steady-state detection methods. Multiplex detection performance was evaluated by simultaneously detecting three or more biomarkers (such as cytokines, tumor markers, or infectious disease-related proteins). The feasibility of the quantum dot multicolor coding scheme was verified using the inter-channel crosstalk rate (<5% is acceptable), the accuracy and reproducibility of multi-target detection (CV <10%) as core criteria. The reagent reusability test assesses the signal intensity decay trend by performing more than 10 consecutive tests on the same batch of chips, with a target signal retention rate of over 85% after 10 tests, thus demonstrating the practical effectiveness of the solid-phase chip mode in reducing detection costs. Chip uniformity and batch stability evaluation involves performing signal normalization tests on all sites of the same batch of 96-bit array chips, calculating the signal variation coefficient between sites (target CV < 8%), and comparing the results among more than three independently prepared batches to verify the reproducibility of the solid-phase chip fabrication process.

[0045] The phosphorescence lifetime of the rare earth chelate in Comparative Example 1 is only 0.5-2 ms, far lower than the donor material of this invention (over 700 ms), significantly reducing the freedom of setting the time gating window. Furthermore, rare earth elements are strategic resources, posing supply chain risks to raw material sourcing, and the material preparation process is highly dependent on laboratory conditions, resulting in high costs for mass production. The donor quantum yield of rare earth systems is typically below 20%, while the system of this invention can reach over 50%, directly leading to a systematic loss in sensitivity and signal stability.

[0046] The development cycle of the multi-target detection system in Comparative Example 2 will be extended from 2-4 months to 6-15 months, the material production cost will increase by 70-85%, the thermal stability will decrease (the melting temperature of monoclonal antibodies is usually below 60℃), and the batch consistency variation coefficient will exceed 20%. Therefore, it cannot support the high requirements of the consistency of the large-scale preparation of recognition elements for multi-target parallel detection. Moreover, the recognition activity will be significantly lost under high temperature storage and transportation conditions, which will seriously restrict the practical application and promotion of the platform.

[0047] In Comparative Example 3, the spatial arrangement of quantum dots and recognition molecules on the carrier will be random and disordered, resulting in partial masking of recognition sites, local aggregation and quenching of quantum dots, a 30%-50% decrease in signal transmission efficiency, and a batch-to-batch signal intensity variation coefficient exceeding 25%, making it impossible to achieve a highly consistent signal carrier with controllable structure and modular expansion.

Claims

1. A multiplexed biomolecule TR-FRET detection platform characterized in that, The invention includes a solid-phase chip, a detection probe, and a TR-FRET detection system; the solid-phase chip includes a quartz substrate, a phosphorescent donor, and a DARPin recognition molecule; the phosphorescent donor includes a Zn-MOF-encapsulated nitrogen-boron co-doped carbon dot composite material; the detection probe includes CdSe / ZnS core-shell quantum dots and a dendritic polymer signal carrier self-assembled by biorthogonal proteins.

2. The detection platform of claim 1, wherein, The dendritic polymer signal vector that is self-assembled by biorthogonal proteins adopts two orthogonal sequence systems: SpyTag003 / SpyCatcher003 and SnoopTag / SnoopCatcher. The vector contains a main backbone, a secondary backbone, and terminal functional units.

3. The detection platform according to claim 2, characterized in that, The dendritic polymer signaling vector, which is a biorthogonal protein self-assembly, uses ProteinA as its backbone and alternately inserts SpyTag003 sequences between its five domains, enabling the backbone to covalently bind to any protein module carrying SpyCatcher003. SnoopTag is further co-expressed at the end of the backbone to introduce a secondary backbone carrying SnoopCatcher, thereby achieving hierarchical expansion of the vector.

4. The detection platform according to claim 3, characterized in that, The CdSe / ZnS core-shell quantum dots have a size of 2.7-4.8 nm.

5. The detection platform according to claim 4, characterized in that, The size of the dendritic polymer signal carrier that the biorthogonal protein self-assembles is 10-100 nm.

6. The detection platform according to claim 5, characterized in that, The Zn-MOF-encapsulated nitrogen-boron co-doped carbon dot composite material has a phosphorescence lifetime of >700ms and an absolute quantum yield of >50%.

7. A method for preparing a detection platform according to any one of claims 2-6, comprising the following steps: S1: Obtain the DARPin recognition molecule through target structure analysis, variable site definition, initial mutation data construction, multi-model AI training, sequence design and experimental verification; S2: The main backbone, secondary backbone and terminal functional units are expressed and purified respectively, and a dendritic polymer signal carrier for biorthogonal protein self-assembly is formed through stepwise assembly. S3: Zn-MOF encapsulated nitrogen-boron co-doped carbon dot composite material was synthesized by hydrothermal in-situ reaction using zinc nitrate hexahydrate and terephthalic acid as MOF precursors, and 4-carbamoylphenylboronic acid and urea as carbon dot precursors. S4: Activate the quartz substrate, spray Zn-MOF encapsulated nitrogen-boron co-doped carbon dot composite material to form a coating, covalently fix DARPin recognition molecules, and integrate solid-phase chip; S5: CdSe / ZnS core-shell quantum dots are coupled with a dendritic polymer signal carrier self-assembled by biorthogonal proteins to obtain a detection probe.

8. The method for preparing the detection platform according to claim 7, characterized in that, The conditions for the hydrothermal in-situ reaction in step S3 are: reaction temperature 180-220℃, reaction time 4-8h.

9. A method of using the detection platform according to any one of claims 1-6, characterized in that, Includes the following steps: The sample to be tested is mixed with the detection probe to allow the target molecules in the sample to bind to the detection probe. The mixture is then added to a chip pool for incubation, allowing the target molecules to form a ternary complex on the solid-phase chip. After removing unbound components, the TR-FRET detection system is used for detection, and the FRET signal intensity at each detection site is collected. The concentration of the target molecules is obtained through data analysis.

10. The method of using the detection platform according to claim 9, characterized in that, The detection system parameters are set as follows: excitation by irradiation with 365nm light for 5-10s, acquisition of FRET signal starting after a delay of 100-200ns, and integration time of 1.5-2.5s.