Method and system for detecting biomarkers based on biomimetic phospholipid monolayer probe

Abstract

The present application relates to biomimetic phospholipid monolayer probe-based biomarker detection method and system, belongs to biomolecule detection field.The method of the present application is: the detection antibody for biomarker detection is coupled with the activated polyethylene glycol derivative capped with mercapto to obtain antibody conjugate; the obtained antibody conjugate is mixed with metal nanoparticles and incubated, then thiolated phospholipid is added, solid-liquid separation is carried out, the obtained solid phase is biomimetic phospholipid monolayer metal nanoparticle probe; the substrate is activated, aminated and aldehyde-treated, the capture antibody for biomarker is added dropwise and incubated to obtain a capture substrate; the obtained capture substrate is reacted with the sample to be measured, washed, and then the obtained biomimetic phospholipid monolayer metal nanoparticle probe is added and incubated, the obtained sandwich structure is analyzed by laser desorption ionization mass spectrometry, and high-sensitivity, stable and accurate detection of biomarkers is realized.

Description

Technical Field

[0001] This invention relates to the field of biomolecular detection, and in particular to a method and system for detecting biomarkers based on biomimetic phospholipid monolayer probes. Background Technology

[0002] The detection of cancer biomarkers is a primary means of achieving early cancer screening, efficacy monitoring, and prognostic assessment. Currently, the most widely used detection technologies in clinical and research fields are mainly divided into two major technical routes: conventional immunoassay and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS).

[0003] Conventional immunoassays are currently the mainstream technology for clinical cancer biomarker detection, with multiplex immunoassay systems based on fluorescently encoded microspheres as the core representative. Traditional enzyme-linked immunosorbent assays (ELISA) remain widely used as the basic detection protocol. The core hardware of a multiplex immunoassay system based on fluorescently encoded microspheres includes fluorescently encoded microspheres, a dual-color laser excitation system, a magnetic separation module, a charge-coupled device (CCD) detector, and a data processing terminal. The fluorescently encoded microspheres are doped with red / infrared fluorescent dyes, forming unique spectral identifiers to distinguish different detection targets. Traditional ELISA consists of an enzyme-linked immunosorbent assay (ELISA) plate, an ELISA reader integrating monochromatic light excitation and absorption detection modules, and pipettes. In terms of equipment connection, the sample is first mixed with the fluorescently encoded microspheres. Then, the magnetic separation module captures and immobilizes the microspheres containing the target molecules into a uniform monolayer. The laser excitation system and the CCD detector are coaxially aligned. When the excitation light penetrates the microspheres, the resulting fluorescence signal is acquired in real time by the detector and simultaneously transmitted to the data processing terminal for analysis. Its working principle is as follows: Fluorescently encoded microspheres are pre-coupled with specific capture antibodies. After binding to the target antigen in the sample, biotinylated detection antibodies are added to form a "sandwich" complex, which then binds to a streptavidin-phycoerythrin reporter molecule. A first laser excites the dye inside the microspheres, identifying its unique spectral signature to determine the target type. A second laser detects the fluorescence intensity produced by phycoerythrin, thereby achieving quantitative analysis of antigen concentration. In contrast, traditional enzyme-linked immunosorbent assays (ELISA) catalyze a colorimetric reaction between enzyme-labeled antibodies and antigens. The absorbance change of the reaction system is detected by an ELISA reader, ultimately achieving antigen quantification.

[0004] Furthermore, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MADS) based on traditional organic matrices possesses high mass resolution and unique advantages in the detection of protein biomarkers. The core equipment of this technology is a MADS instrument, primarily composed of a 355 nm ultraviolet laser system, a sample target plate, an ion source, a time-of-flight mass analyzer, an ion detector, and accompanying data analysis software. In terms of equipment connectivity, the ion source is tightly connected to the time-of-flight mass analyzer via a vacuum interface. The laser system is precisely focused onto the sample target plate surface. After sample ionization, the resulting ions are accelerated under the influence of an electric field and then enter the mass analyzer for separation. The separated ion signals are received by the detector and converted into electrical signals, ultimately transmitted to the data terminal for processing. Its working principle is as follows: During detection, the serum sample to be analyzed is first thoroughly mixed with an organic matrix (such as 2,5-dihydroxybenzoic acid), and the mixture is dropped onto a sample target plate. After drying, a co-crystallized film is formed. When the target plate is irradiated by ultraviolet laser, the matrix preferentially absorbs the laser energy and rapidly vaporizes, while simultaneously carrying the antigen molecules into the gas phase environment. The antigen molecules are ionized through proton transfer. After these charged ions enter the time-of-flight mass analyzer, they are separated according to the difference in the mass-to-charge ratio. The flight time of the ions is proportional to the square root of the mass-to-charge ratio. Finally, the signal intensity captured by the detector enables quantitative analysis of the antigen content.

[0005] Although improved MALDI-TOF MS technologies based on nanomaterials (such as graphene and metal-organic frameworks MOFs) have emerged in recent years, which improve the signal uniformity of small molecule metabolite detection through high specific surface area, they still have core limitations: they are only suitable for small molecule detection, and have limited improvement in ionization efficiency for large molecular weight cancer protein biomarkers (such as AFP and CEA). Furthermore, they have not solved the core pain points of "multiple detection" and "ultra-low abundance quantification"—single-target stepwise detection is still required, and the detection limit can only reach the picomolar level, which cannot meet the needs of early clinical screening.

[0006] The aforementioned conventional immunoassay methods, along with traditional and modified MALDI-TOF MS techniques, all have inherent limitations in the clinical application of ultra-high sensitivity multiplex screening for cancer biomarkers, making it difficult to meet the needs of ultra-high sensitivity multiplex screening. Specifically:

[0007] First, conventional immunoassays face the core problem of insufficient multiplex detection capability. This technology is limited by spectral overlap caused by cross-emission spectra of fluorescent probes, electrode signal interference from electrochemical immunosensors, and probe scarcity due to the limited variety of specific antibodies or fluorescent probes, making it unable to efficiently achieve simultaneous detection of multiple cancer biomarkers. Simultaneously, its sensitivity for low-abundance antigens is insufficient; for example, the naked-eye detection limit of traditional lateral flow immunoassay (LFIA) is only at the 1-10 ng / mL level, making it difficult to capture trace amounts of tumor markers in clinical samples. The sensitivity of fluorescent microsphere-based multiplex immunoassays also mostly remains at the ng / mL level, making it difficult to capture trace amounts of early tumor markers in clinical samples, easily leading to missed detections of early cancers.

[0008] Second, traditional matrix-assisted laser desorption / ionization time-of-flight mass spectrometry suffers from stability issues due to the randomness of co-crystallization. During detection, the co-crystallization process between the antigen and the organic matrix is ​​easily affected by factors such as solvent evaporation rate and surface tension, leading to uneven crystal distribution and the formation of "hot spot regions." This disordered crystallization causes large fluctuations and poor repeatability in the detection signal during laser irradiation.

[0009] Third, the signal intensity and detection sensitivity of traditional matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) are insufficient to meet the requirements for detecting ultra-low abundance antigens. This technology lacks an in-situ signal amplification mechanism, and the organic matrix generates background interference in the low molecular weight region. These two factors result in weak antigen detection signals, making it impossible to effectively detect ultra-low abundance antigens at the zeptomolar level (10⁻²¹ molar level). This makes it difficult to capture trace amounts of tumor markers in serum during clinical studies.

[0010] Fourth, traditional matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOFMS) has significant limitations in quantitative performance for multiplex detection. When simultaneously detecting multiple antigens, the binding affinity of different antigens to the matrix varies, easily leading to competition between antigens. Furthermore, its linear dynamic range is narrow, failing to cover the broad concentration distribution of antigens in clinical samples, ultimately resulting in large quantitative errors.

[0011] Fifth, the low ionization efficiency of traditional matrix-mediated ionization limits the signal response of traditional matrix-assisted laser desorption / ionization time-of-flight mass spectrometry. Organic matrices have poor chemical compatibility with high molecular weight antigens, resulting in low energy transfer efficiency during ionization and weak antigen signal response. Although mass tags such as ethylene glycol-terminated alkyl thiols can be used to assist detection, the signal enhancement effect is limited and still cannot meet the requirements for high-sensitivity detection.

[0012] Therefore, this invention urgently needs to provide a method for achieving highly sensitive, stable, and accurate detection of Zeptomore-level cancer biomarkers. Summary of the Invention

[0013] To address the aforementioned technical problems, this invention provides a method and system for detecting biomarkers based on biomimetic phospholipid monolayer probes. By constructing silicon-based trapping probes, biomimetic monolayer metal nanoprobes, and adaptive matrix-free laser desorption / ionization mass spectrometry (LDI-MS), this invention avoids multiple ionization competitions and achieves highly sensitive, stable, and accurate detection of zeptomolar-level cancer biomarkers.

[0014] This invention is achieved through the following technical solution:

[0015] The first objective of this invention is to provide a method for detecting biomarkers based on a biomimetic phospholipid monolayer probe, comprising the following steps:

[0016] (1) The detection antibody used for biomarker detection is coupled with a thiol-terminated activated polyethylene glycol derivative to obtain an antibody conjugate;

[0017] (2) After mixing and incubating the antibody conjugate obtained in step (1) with metal nanoparticles, thiolized phospholipids are added for reaction, and solid-liquid separation is performed. The resulting solid phase is a biomimetic phospholipid monolayer metal nanoprobe.

[0018] (3) The substrate is activated, aminated and aldehyde-treated, and then incubated with capture antibodies for biomarkers to obtain the capture substrate;

[0019] (4) The captured substrate obtained in step (3) is reacted with the sample to be tested, washed, and then the biomimetic phospholipid monolayer metal nanoprobe obtained in step (2) is added for incubation. The obtained sandwich structure is analyzed by laser desorption / ionization mass spectrometry. The metal nanoparticles are used to assist the ionization of the analyte without the addition of organic small molecule matrix, so as to achieve qualitative and quantitative analysis of biomarkers.

[0020] In one embodiment of the present invention, in step (1), the biomarker is selected from one or more of tumor markers, inflammatory factors, neurodegenerative disease markers and myocardial injury markers.

[0021] In one embodiment of the present invention, the biomarker is selected from one or more of AFP, CEA, CA19-9 and CA125.

[0022] In one embodiment of the present invention, the activated polyethylene glycol derivative is a polyethylene glycol derivative having both a thiol group and a reactive functional group; the reactive functional group is selected from succinimide ester group, maleimide group, carboxyl group or amino group;

[0023] And / or, the molecular weight of the polyethylene glycol derivative in the activated polyethylene glycol derivative is 500-5000 Da.

[0024] In one embodiment of the present invention, the activated polyethylene glycol derivative is used for covalent coupling with a protein or antibody; the activated polyethylene glycol derivative is selected from one or more of NHS-PEG-SH, MAL-PEG-SH, COOH-PEG-SH and NH2-PEG-SH; the molecular weight of the polyethylene glycol derivative is 1000-3000 Da, preferably 2000 Da.

[0025] In one embodiment of the present invention, in step (1), the coupling reaction conditions are: coupling at 0-30°C for 1-24 hours. Preferably, coupling at 4°C for 12 hours.

[0026] In one embodiment of the present invention, in step (2), the metal nanoparticles are selected from one or more of gold nanoparticles, silver nanoparticles, platinum nanoparticles and Fe3O4@Au core-shell nanoparticles;

[0027] And / or, the incubation is: incubation at 15-30°C for 2-24 h at a pH of 6.0-9.0; preferably, incubation at room temperature for 12 h at a pH of 8.0-8.5.

[0028] In one embodiment of the present invention, in step (2), the thiolated phospholipid is a phospholipid derivative with a terminal thiol group; preferably, the thiolated phospholipid includes phospholipid derivatives with different fatty acid chain lengths and / or different polyethylene glycol chain lengths; more preferably, the thiolated phospholipid is selected from DSPE-PEG. 2000 -SH and / or DPPE-PEG 2000 -SH.

[0029] In one embodiment of the present invention, in step (2), the molar ratio of the antibody conjugate to the metal nanoparticles is 1:(10) 2 -10 3 );

[0030] And / or, the molar ratio of the thiolized phospholipid to the metal nanoparticles is 0.1-10:1. Preferably, the molar ratio of the antibody conjugate to the metal nanoparticles is 1:(500-600), and the molar ratio of the thiolized phospholipid to the metal nanoparticles is 0.5-1:1; the ratio is used to form a low-density antibody distribution and a phospholipid monolayer structure on the surface of the metal nanoparticles.

[0031] In one embodiment of the present invention, in step (2), the reaction conditions are: room temperature reaction for 8-16 h;

[0032] And / or, the solid-liquid separation method is centrifugation; the centrifugation conditions are: 8000-12000 rpm for 10-20 min.

[0033] In one embodiment of the present invention, in step (3), the substrate is selected from one or more of monocrystalline silicon wafers, ITO conductive glass and monocrystalline silicon microarrays.

[0034] And / or, the incubation conditions are: incubation at 2-25℃ for 10-120 min; preferably, incubation at 4℃ for 30 min.

[0035] In one embodiment of the present invention, in step (4), the incubation conditions are: incubation at 20-40 ℃ for 10-120 min; preferably, incubation at 37 ℃ for 30 min.

[0036] In one embodiment of the present invention, the mass spectrometry analysis is in negative ion linear mode, and the laser energy is 30-90%, preferably 80%.

[0037] A second objective of this invention is to provide a system for detecting biomarkers using the method, the system comprising a biomimetic phospholipid monolayer metal nanoprobe, a trapping substrate, and laser desorption / ionization mass spectrometry;

[0038] The biomimetic phospholipid monolayer metal nanoprobe is obtained through the following steps: an antibody for biomarker detection is coupled with a thiol-terminated activated polyethylene glycol derivative to obtain an antibody conjugate; the obtained antibody conjugate is mixed with metal nanoparticles and incubated, then thiolized phospholipids are added to react, and the solid phase obtained by solid-liquid separation is the biomimetic phospholipid monolayer metal nanoprobe.

[0039] The capture substrate is obtained by activating, amylating and aldehyde-modifying the substrate, and then incubating it with capture antibodies for biomarkers.

[0040] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0041] (1) Compared with the shortcomings of traditional single-chain hydrophobic molecules (such as MHA) modified metal nanoprobes, which are prone to aggregation and near signal quenching, the multi-effect synergistic "biomimetic phospholipid monolayer" of this invention endows the probe with excellent monodispersity and efficient energy conduction network. This structural mutation not only prevents the probe from being deactivated, but also directly achieves an absolute enhancement of more than three orders of magnitude in the characteristic fragment signal. It achieves the technical effect of going from no signal to a super strong signal (amplified by 1000 times), providing the only material basis for the subsequent release of specific high-intensity mass spectrometry signals.

[0042] (2) In response to the problems of drastic signal fluctuations (RSD often exceeding 30%) and poor reproducibility caused by the randomness of organic matrix co-crystallization in traditional MALDI-TOF MS, this invention completely abandons the traditional matrix. Relying on the "electronic effect" excited by the gold nanoparticle core to dominate the specific thermal degradation and bond breaking of the monolayer, the disordered random fragmentation is transformed into extremely controllable deterministic dissociation, which reduces the relative standard deviation (RSD) of the detection signal to less than 10%, providing a solid quantitative basis for clinical practice.

[0043] (3) In traditional multiplex detection, different macromolecular antigens are prone to severe charge competition during ionization. This invention constructs a double-sandwich spatial decoupling system with enrichment at the bottom and signal emission at the top, cleverly transforming the detection of large and difficult-to-ionize proteins (AFP / CEA) into the detection of easily flying small molecule feature tags (PMT283 / PMT255). From a physical principle perspective, this completely avoids mutual interference and spectral overlap between markers, achieving truly synchronous and accurate reading.

[0044] (4) Thanks to the physical-level signal amplification engine that "identifies one target and releases a massive number (thousands) of feature tags", this invention successfully improves the detection sensitivity by 6-9 orders of magnitude compared to conventional immunoassay, and lowers the detection limit to a groundbreaking Zeptomore level (10). -21 (mol / L), possessing the rare ability to accurately capture trace amounts of tumor markers in clinical samples at a very early stage.

[0045] (5) With its extremely high specificity and stability, this invention not only possesses ultra-high sensitivity, but also unprecedentedly expands the linear dynamic range of quantitative analysis to nine orders of magnitude. In actual tests on complex clinical serum samples, the detection error was strictly controlled within 8% (of which 92% of the detection errors were ≤5%), perfectly meeting the stringent requirements for extremely high accuracy and reliability in cancer screening in real clinical environments. Attached Figure Description

[0046] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0047] Figure 1These are comparative images of the multi-effect synergistic assembly structure and morphology characterization of the biomimetic phospholipid monolayer probes prepared in the embodiments of this invention and the conventional probes of Comparative Example 1; wherein, (a) is the structure of the fatty acid single chains of DSPE-PEG-SH, DPPE-PEG-SH, and 18-MHA-SH; (b) is a comparative image of the scanning electron microscope (SEM) morphology of the probes obtained in Examples 1, 2 and Comparative Example 1; (c) is a dynamic light scattering (DLS) hydrodynamic particle size distribution diagram of each probe; and (d) is a quantitative image of the probe surface detection antibody and the mass spectrometry phospholipid tag modification density (concentration) measurement results.

[0048] Figure 2 This is a diagram illustrating the matrix-free in-situ dissociation mechanism and deep electronic effect mechanism of the probe generating "incomplete molecule" tags under ultraviolet laser in Example 3 of this invention. (a) is a schematic diagram of the microscopic dissociation mechanism where the probe, under 355nm ultraviolet laser irradiation, mediates energy transfer through a monolayer and specifically breaks specific chemical bonds; (b) is a high-resolution mass spectrum and structural analysis matching diagram of the probe dissociation products, confirming that the released mass-to-charge ratio peak is consistent with the theoretically derived fragment structure of the "incomplete molecule"; (c) is [BP]. + The survival yield (SY) of ions and the dissociation rate constant (k) exp (d) The curve of laser energy change confirms that there is a significant non-thermal desorption process in this dissociation mechanism; (d) is a bar chart showing the effect of different concentrations of electron scavenger juglone on the intensity of each characteristic ion in the system. The competitive blocking experiment confirms that this specific dissociation is dominated by electronic effects.

[0049] Figure 3 The figures show a comparison of the advantages of the mass spectrometry signal response of the probes obtained in Examples 1 and 2 of this invention with those of Comparative Example 1, and a verification of the repeatability after multiple excitations. Among them, (a) is a comparison of the absolute intensity of the mass spectrometry characteristic fragment signals of the two types of probes (AuNP-DPPE / AuNP-DSPE) of this invention with those of the traditional comparative probe (AuNP-18-MHA) and bare gold (AuNP) under the same test conditions; (b) is a line graph showing the absolute intensity fluctuation and relative standard deviation (RSD) of the characteristic fragment signals generated under multiple repeated laser excitations.

[0050] Figure 4The figures shown are from Example 5 of this invention, which describes the simultaneous ultrasensitive quantification of multiple tumor markers based on "incomplete molecule" tags and the detection results of actual clinical serum samples. Among them, (a) is the mass spectrum of multiple characteristic tag peaks (such as PMT255 and PMT283) clearly separated in the double sandwich spatial decoupled structure when simultaneously detecting multiple tumor markers (AFP and CEA); (b) is the standard curve of AFP and CEA antigen multiple quantification based on the relative signal intensity fitting of the corresponding characteristic peaks; (c) is the scatter regression diagram of the parallel quantification detection of actual clinical serum samples (AFP) using the sensor constructed in this invention and the clinical gold standard method; and (d) is the relative error box plot of the parallel quantification detection of actual clinical serum samples (AFP) using the sensor constructed in this invention and the clinical gold standard method. Detailed Implementation

[0051] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0052] This invention provides a method for detecting multiple cancer biomarkers based on biomimetic phospholipid monolayer metal nanoprobes, comprising the following steps:

[0053] (1) Interface capture: Provides silicon-based capture probes with specific capture antibodies on their surface, which are incubated with the biological sample to be tested to specifically enrich the target antigens in the sample (such as AFP in single-target detection, or AFP and CEA in dual-target simultaneous detection).

[0054] (2) Spatial decoupling and assembly: "Bionic phospholipid monolayer metal nanoprobes" with corresponding detection antibodies and specific mass spectrometry tags are added to the surface. The probes recognize the captured antigens and form a double-sandwich spatial decoupling structure on the substrate surface with "enrichment at the bottom and signal emission at the top";

[0055] (3) Matrix-free in-situ excitation and quantification: The assembled silicon substrate is placed directly in the MALDI-TOF mass spectrometer (without the need to add traditional organic matrix throughout the process). Under ultraviolet laser excitation at a specific wavelength, the metal nanoprobes release extremely abundant incomplete molecular feature tags based on electronic effects to specifically break bonds (e.g., PMT283 tag with m / z≈283 for AFP; PMT255 tag with m / z≈255 for CEA). Absolute quantitative analysis of the target antigen is achieved by analyzing the mass spectrometry peak signal intensity of the feature tags.

[0056] This invention overcomes the limitations of traditional metal nanoprobes, which rely on single-chain thiol molecules (such as MHA fatty acids) for modification, resulting in easy aggregation and insufficient steric hindrance. By synergistically integrating "gold-sulfur bond anchoring," "PEG chain steric hindrance," and "phospholipid double hydrophobic tail arrangement," a dense and highly ordered "biomimetic phospholipid monolayer" is constructed on the surface of the gold nanocore. This structure not only solves the problem of monodisperse stability of probes in complex matrices (compared to the severe aggregation of MHA), but more importantly, it constructs an extremely uniform interfacial environment, precisely controlling the energy transfer and electron distribution on the gold nanoparticle surface. This is the sole material basis for subsequently achieving the release of specific high-intensity mass spectrometry signals.

[0057] This invention completely overturns the traditional MALDI-TOF mass spectrometry model, which relies heavily on the random thermal desorption / ionization of organic small molecule matrices. Under 355 nm UV laser excitation, the gold nanocore of this invention generates high-energy electrons through localized surface plasmon resonance (SPR). The dense monolayer acts as a highly efficient conductive medium, specifically breaking certain chemical bonds through the "electron effect," stably releasing mass-specific "incomplete molecules" as characteristic tags (PMT283, m / z≈283; PMT255, m / z≈255). This mechanism completely eliminates signal fluctuations caused by matrix "hotspot effects," achieving extremely clean mass spectrometry baselines and extremely high signal repeatability (RSD≤10%).

[0058] In multiplex biomarker detection, traditional mass spectrometry faces the critical challenge of "large molecule ionization competition (charge grabbing)". This invention constructs a dual-sandwich spatial decoupling system consisting of a silicon-based trapping substrate (enriched at the bottom), dual antigens, and gold nanoparticle probes (signal emission at the top). During laser excitation, the mass spectrometer does not ionize the large AFP / CEA protein molecules; instead, it specifically ionizes the massive amounts of easily aspirated lipid tags (PMT283 / PMT255) released from the top of the gold nanoparticle probes. This mechanism avoids the ionization competition between multiple antigens, achieving truly interference-free simultaneous quantitative detection.

[0059] Compared to the direct 1:1 detection of traditional mass spectrometry, the monolayer structure of this invention provides extreme signal amplification. After a silicon wafer substrate captures a target antigen and connects it to a metal nanoprobe, each probe definitively releases approximately 3272 characteristic mass spectrometry tags at the instant of laser bombardment. Compared to traditional MHA single-chain probes (which have no effective signal response), this invention achieves an absolute signal boost of at least three orders of magnitude, thereby widening the detection linear range to nine orders of magnitude and lowering the detection limit to a groundbreaking Zeptomore level (10⁻⁶). -21 mol / L).

[0060] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0061] Example 1: Preparation of biomimetic phospholipid monolayer metal nanoprobes and silicon-based trapping substrates

[0062] This embodiment provides a method for preparing a mass spectrometry nanoprobe based on multi-effect synergistic assembly and its matching trapping substrate. The specific steps are as follows:

[0063] (1) Synthesis of gold nanoparticles (AuNPs): The classic sodium citrate reduction method was used. 50 mL of 0.01% chloroauric acid (HAuCl4) solution was heated to boiling, and 1.5 mL of 1% sodium citrate solution was quickly added while stirring vigorously. The mixture was kept boiling for 15 minutes and then cooled to room temperature to obtain dispersed AuNPs colloids, which were stored at 4℃ for later use.

[0064] (2) Pre-activation of the detection antibody: 30 µL of NHS-PEG with a concentration of 10 mg / mL was added. 2000 -SH and 500 µL of a specific detection antibody (AFP) at a concentration of 120 µg / mL det The mixture was stirred at 4°C for 12 hours to generate an antibody-conjugate. Among these, the specific detection antibody (AFP) was... det Purchased from Shanghai Lingchao Biotechnology Co., Ltd., product code L1C00302.

[0065] (3) Multi-effect synergistic assembly and purification of probes: 6 µL of the above antibody-conjugate was added to 200 µL of AuNPs solution, and the pH of the solution was adjusted to approximately 8.5 using potassium carbonate (K2CO3). The solution was incubated at room temperature in the dark for 12 hours. Subsequently, 18 µL of 100 µM thioglycolic acid phospholipid (DSPE-PEG) was added to the system. 2000 -SH). This thiolized phospholipid not only serves as a source of specific mass spectrometry tags, but its core role lies in constructing a biomimetic interface structure to regulate energy transfer and electron distribution on the surface of gold nanoparticles, thereby dominating the generation of specific characteristic fragments in the subsequent LDI process. Assembly continued at room temperature for 12 hours. After the reaction, the solution was centrifuged at 12,000 rpm for 15 minutes to allow the nanoprobe to settle, and the supernatant was discarded. The nanoprobe was resuspended in PBS and washed by centrifugation three times to remove free molecules. Finally, the precipitate was resuspended in PBS to a set volume to obtain a dense monolayer of biomimetic phospholipid monolayer metal nanoprobe (AuNP@AFP). det / DSPE), store at 4℃.

[0066] Example 2

[0067] This embodiment provides a method for preparing a mass spectrometry nanoprobe based on multi-effect synergistic assembly and its matching capture substrate, similar to Example 1, except that in step (2), the specific detection antibody AFP is... det Replace with CEA det In step (3), the thiolized phospholipid DSPE-PEG is... 2000 Replace -SH with DPPE-PEG 2000 -SH; the remaining steps are consistent with Example 1. A dense, monolayer biomimetic phospholipid monolayer metal nanoprobe AuNP@CEA was prepared. det / DPPE.

[0068] Comparative Example 1: Preparation of a traditional thiol fatty acid chain probe

[0069] This comparative example provides a conventional single-chain probe, similar to Example 1, except that in step (3), when adding the mass spectrometry tag, DSPE-PEG is used. 2000 -SH was replaced with the same amount (i.e., 18 µL, 100 µM) of the conventional single-chain hydrophobic molecule—18-mercaptooctadecanoic acid (18-MHA). The remaining steps were consistent with Example 1 to prepare the probe AuNP@AFP. dett / 18-MHA.

[0070] The probes prepared in Example 1, Example 2, and Comparative Example 1 were characterized by dynamic light scattering (DLS) and scanning electron microscopy (SEM). Figure 1 Combined with quantitative analysis of surface modification density, the results show that in the embodiments of the present invention, phospholipid molecules form a dense and uniform monolayer structure on the surface of gold nanoparticles, rather than a multilayer stacking.

[0071] Figure 1 (a) in the diagram is a schematic diagram of the structure of different surface-modified molecules; Figure 1 (b) in the figure shows the scanning electron microscope (SEM) image of the corresponding probe. The results show that the gold nanoparticles modified with 18-MHA have an obvious coating layer on the surface and relatively poor particle dispersion, suggesting that they form a relatively dense molecular stacking structure on the surface. In contrast, the gold nanoparticles modified with DSPE or DPPE are uniformly dispersed, and no obvious agglomeration or uneven coating phenomenon was observed, indicating that their interface modification is more uniform and stable.

[0072] Figure 1(c) shows the dynamic light scattering (DLS) hydrodynamic particle size distribution and quantitative results of surface modification density of the probes under different modification strategies. The DLS results show that when the detection antibody is modified on the surface of gold nanoparticles before the phospholipid tag is introduced, the hydrodynamic particle size gradually increases, indicating that both the detection antibody and phospholipid molecules are successfully immobilized on the surface of the nanoparticles. However, when the phospholipid tag is introduced first and then the detection antibody is modified, the particle size change is not significant, indicating that the phospholipid molecules form a highly covered interface layer on the surface of the gold nanoparticles, which has a significant steric hindrance effect on the subsequent binding of the antibody. This further shows that the order of surface modification has an important influence on probe construction.

[0073] also, Figure 1 (d) The modification amounts of detection antibodies and phospholipid tags on the surface of gold nanoparticles were quantitatively analyzed using the BCA and DTNB methods, respectively. The results showed that the modification amounts of detection antibodies and phospholipid tags remained consistent across different batches of samples, indicating that the method has high repeatability and stability. Further calculations showed that each gold nanoparticle surface was loaded with an average of approximately 10 detection antibody molecules and approximately 3000 phospholipid tag molecules. Combining the above particle size variations and quantitative results, it can be inferred that phospholipid molecules formed a highly covered and ordered monolayer structure on the surface of the gold nanoparticles.

[0074] Example 3: Verification of the microscopic dissociation mechanism of probe "incomplete molecule" tag generation (electronic effects dominant)

[0075] This embodiment explores in depth the bond breaking process and energy transfer pathway of the probe prepared in Example 1 under matrix-free conditions.

[0076] (1) LDI-MS parameters: A Bruker Autoflex Max MALDI-TOF mass spectrometer was used, operated in linear negative ion mode. A 355 nm Nd:YAG laser was used, with a delay time of 120 ns and laser energy optimized to 80% (to maximize the signal-to-noise ratio while avoiding excessive fragmentation). The mass scan range was set to m / z 0-1000. Figure 2 The mass spectrometry results in (b) show that the characteristic mass-to-charge ratio peak generated after probe dissociation has a good matching relationship with the theoretically derived fragment structure of the "incomplete molecule", indicating that the fragmentation process has clear structural selectivity rather than random fragmentation.

[0077] (2) Juglone competitive blocking experiment: To investigate the dissociation mechanism, juglone solutions with concentration gradients of 0-10 mmol / L were prepared. Gold nanoparticle solutions (concentrations of 1-10 nM) were mixed with DSPE, DPPE, 4-chlorobiphenyl (4-CL-BP) and juglone solutions (concentration of 1 mM) at a volume ratio of 1:1 to obtain initial mixed systems. Subsequently, these mixed systems were further mixed with equal volumes of 0, 0.1 mM, 0.5 mM, 1 mM, 2 mM, 5 mM and 10 mM juglone solutions, respectively. After thorough mixing, an appropriate amount of solution was dropped onto the surface of a mass spectrometry target plate, allowed to dry naturally, and then detected by laser desorption / ionization (LDI) mass spectrometry. The experimental results are as follows: Figure 2 As shown in (d), the intensity of the probe characteristic fragment signal decreases significantly with increasing juglone concentration in the system, exhibiting a clear competitive inhibition effect, indicating that this dissociation process is closely related to the electron capture process. Simultaneously, combined with... Figure 2 (c) of [BP] + ion survival yield (SY) and dissociation rate constant (k) exp The results showing the effect of laser energy variation indicate that SY remains at a high level under higher laser energy conditions, while k... exp The low order of magnitude indicates that no significant thermal decomposition process occurred in the system; combined with the strong characteristic ion signal, it can be inferred that this dissociation process does not conform to the traditional thermally driven mechanism, but is mainly dominated by electronic effects. Based on the above experimental results and... Figure 2 The mechanism illustrated in (a) shows that, under laser irradiation, the probe of this invention selectively breaks specific chemical bonds through energy transfer and electron transfer mediated by a monolayer interface, thereby generating stable and characteristic "incomplete molecule" mass spectrometry tags. This confirms that the dissociation mechanism of the probe of this invention in generating characteristic fragments is dominated by the electron effect excited by the gold nanoparticle core, fundamentally different from the traditional simple thermal decomposition process.

[0078] Example 4: Comparative Test of Mass Spectrometry Signal Response Performance

[0079] The probes of Examples 1, 2 and Comparative Example 1 were subjected to parallel control detection under the same conditions using the laser mass spectrometry instrument parameters described in Example 3.

[0080] Experimental results are as follows Figure 3 As shown, where Figure 3As shown in (a), under the same test conditions, the characteristic fragment signal intensity of the probe of the present invention is significantly higher than that of the comparative probe and bare gold nanoparticles, indicating that the probes obtained in Examples 1 and 2 can stably and efficiently release high-intensity characteristic fragment signals. This confirms that thanks to the unique electronic effect bond-breaking mechanism and the efficient energy conduction of the monolayer film in Examples 1 and 2, the probe of the present invention can stably and efficiently release high-intensity characteristic fragment signals. In contrast, the comparative example 1 (MHA probe) suffers from severe aggregation of gold nanoparticles due to the inability of traditional single-chain molecules to provide sufficient steric hindrance. Furthermore, this comparative example lacks the interface regulation capability and electronic effect enhancement mechanism provided by the biomimetic phospholipid structure, and has virtually no effective mass spectrometry signal response under the same laser excitation conditions. This stark contrast confirms that the structural design of the present invention directly achieves an absolute signal leap of at least three orders of magnitude. Meanwhile, as... Figure 3 As shown in (b), Example 1 has a relative standard deviation (RSD) of ≤10% under multiple excitations, overcoming the fatal flaw of traditional single-chain modification that easily leads to probe inactivation and signal fluctuation.

[0081] Example 5: Validation of Dual-Target Ultrasensitive Quantitative Analysis and Clinical Serum Sample Detection

[0082] This embodiment utilizes a double-sandwich immune assembly method to detect tumor markers (AFP and CEA) in clinical serum samples.

[0083] (1) Sandwich immune assembly procedure:

[0084] Construction of the silicon-based capture substrate: A single-crystal silicon wafer (500 µm thick) was activated at room temperature for 30 minutes in piranha wash solution (H2SO4 / H2O2, 7:3, v / v). After washing and drying, it was immersed in anhydrous ethanol containing 5% (v / v) APTES and reacted at 50°C for 5 hours for amination. Subsequently, it was treated with 2.5% glutaraldehyde (prepared in PBS) at room temperature for 30 minutes for aldehyde fixation. Finally, the AFP and CEA capture antibodies (Ab) were applied... cap The antibodies (100 µg / mL) were mixed in equal volumes and dropped onto the surface of an aldehyde-modified silicon wafer. The mixture was incubated at 4 °C for 30 min to achieve co-immobilization of the dual-target capture antibodies. The remaining sites were then blocked with 1% BSA to prepare the capture substrate (Si-Ab). cap The capture antibodies were purchased from Shanghai Leading Biotechnology Co., Ltd., with catalog number AFP. cap (L1C00301) and CEA cap (L1C00202).

[0085] Take the prepared Si-Ab capThe substrate was captured, and 10 µL of the antigen sample to be tested (i.e., a mixed sample containing two standard antigens, AFP and CEA, with AFP and CEA concentrations set at 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, 1 µg / mL, 10 µg / mL, and 100 µg / mL, a total of 10 concentration gradients covering 9 orders of magnitude) was added. The sample was incubated at 37 °C for 30 min. After thorough washing with PBS, 10 µL of the probe mixture prepared in Examples 1 and 2 (containing AuNP@AFP mixed in a 1:1 volume ratio) was added. det / DSPE and AuNP@CEA det / DPPE, and the gold nanoparticle concentration in both probe solutions was the same (1 mM), and incubation was continued at 37°C for 30 minutes to form a double-sandwich structure. After thorough rinsing with PBS, it was dried under a gentle nitrogen stream and fixed onto a MALDI target plate using conductive tape for mass spectrometry analysis. The results are as follows: Figure 4 As shown in (a) and (b), when simultaneously detecting multiple tumor markers (AFP and CEA), the double-sandwich spatial decoupled structure clearly separates multiple characteristic label peaks (such as PMT255 and PMT283) in the mass spectrometry. The AFP and CEA antigen multiplex quantitative standard curves fitted based on the relative signal intensities of the corresponding characteristic peaks exhibit good linearity and a wide dynamic range (100 fg / mL-100 µg / mL), with no significant signal interference between different markers. This confirms that the method successfully achieves interference-free target separation, with detection limits reaching the zeptomol level.

[0086] (2) Clinical serum detection: In practice, human serum samples obtained through ethical approval and frozen at -80℃ were thawed and diluted with PBS buffer containing 0.05% Tween-20 at a volume ratio of 1:10 to reduce matrix interference. Mass spectrometry analysis was then performed following the sandwich method described above. The scatter regression plot comparing the sensor constructed in this invention with the clinical gold standard method for parallel quantitative detection of actual clinical serum samples (AFP) is shown below. Figure 4 As shown in (c), it can be seen that the method still maintains extremely high clinical quantitative accuracy in complex serum matrices diluted 1:10, and exhibits a good linear correlation with the gold standard method. In practical tests on complex clinical serum samples, as shown... Figure 4As shown in (d), the detection errors were strictly controlled within 8% (of which 92% of the detection errors were ≤5%), indicating that the method has good detection stability and accuracy in complex matrices, and can meet the requirements of clinical testing for precision and reliability. The detection results of CEA and AFP in clinical serum samples by the sensor of this invention show a very strong linear positive correlation with the results of the clinical gold standard detection method, with high detection accuracy, and can replace the clinical gold standard for real sample testing.

[0087] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for detecting biomarkers based on a biomimetic phospholipid monolayer probe, characterized in that, Includes the following steps: (1) The detection antibody used for biomarker detection is coupled with a thiol-terminated activated polyethylene glycol derivative to obtain an antibody conjugate; (2) After mixing and incubating the antibody conjugate obtained in step (1) with metal nanoparticles, thiolized phospholipids are added for reaction, and solid-liquid separation is performed. The resulting solid phase is a biomimetic phospholipid monolayer metal nanoprobe. (3) The substrate is activated, aminated and aldehyde-treated, and then incubated with capture antibodies for biomarkers to obtain the capture substrate; (4) The captured substrate obtained in step (3) is reacted with the sample to be tested, washed, and then the biomimetic phospholipid monolayer metal nanoprobe obtained in step (2) is added for incubation. The obtained sandwich structure is analyzed by laser desorption / ionization mass spectrometry to achieve qualitative and quantitative analysis of biomarkers.

2. The method according to claim 1, characterized in that, In step (1), the biomarker is selected from one or more of tumor markers, inflammatory factors, neurodegenerative disease markers and myocardial injury markers.

3. The method according to claim 1, characterized in that, The activated polyethylene glycol derivative is a polyethylene glycol derivative that simultaneously possesses a thiol group and a reactive functional group; the reactive functional group is selected from succinimide ester group, maleimide group, carboxyl group or amino group; And / or, the molecular weight of the polyethylene glycol derivative in the activated polyethylene glycol derivative is 500-5000 Da.

4. The method according to claim 1, characterized in that, In step (1), the coupling reaction conditions are: coupling at 0-30℃ for 1-24 hours.

5. The method according to claim 1, characterized in that, In step (2), the metal nanoparticles are selected from one or more of gold nanoparticles, silver nanoparticles, platinum nanoparticles and Fe3O4@Au core-shell nanoparticles; And / or, the incubation is performed at 15-30°C for 2-24 hours under conditions of pH 6.0-9.

0.

6. The method according to claim 1, characterized in that, In step (2), the thiolized phospholipid is a phospholipid derivative with a terminal thiol group.

7. The method according to claim 1, characterized in that, In step (2), the molar ratio of the antibody conjugate to the metal nanoparticles is 1:(10) 2 -10 3 ); And / or, the molar ratio of the thiolated phospholipid to the metal nanoparticles is 0.1-10:

1.

8. The method according to claim 1, characterized in that, In step (3), the substrate is selected from one or more of the following: monocrystalline silicon wafer, ITO conductive glass, and monocrystalline silicon microarray. And / or, the incubation conditions are: incubation at 2-25℃ for 10-120 min.

9. The method according to claim 1, characterized in that, In step (4), the incubation conditions are: 20-40 ℃ for 10-120 min.

10. A system for detecting biomarkers using the method according to any one of claims 1-9, characterized in that, The system includes a biomimetic phospholipid monolayer metal nanoprobe, a capture substrate, and laser desorption / ionization mass spectrometry. The biomimetic phospholipid monolayer metal nanoprobe is obtained through the following steps: an antibody for biomarker detection is coupled with a thiol-terminated activated polyethylene glycol derivative to obtain an antibody conjugate; the obtained antibody conjugate is mixed with metal nanoparticles and incubated, then thiolized phospholipids are added to react, and the solid phase obtained by solid-liquid separation is the biomimetic phospholipid monolayer metal nanoprobe. The capture substrate is obtained by activating, amylating and aldehyde-modifying the substrate, and then incubating it with capture antibodies for biomarkers.

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