Colorimetric-fluorescent dual-mode carcino-embryonic antigen immunoassay method based on dual-ligand iron metal organic framework nano-enzyme

Abstract

The invention discloses a colorimetric-fluorescent dual-mode immunoassay method based on a dual-ligand iron metal organic framework (Fe-MOF) nano-enzyme, which is used for detecting protein tumor markers, taking carcinoembryonic antigen (CEA) as an example. The double-ligand Fe-MOF has both peroxidase simulation activity and intrinsic fluorescence, and is coupled with a labeled antibody Ab2 to form Fe-MOF-Ab2; a to-be-detected substance in a sample is captured by the solid-phase coated antibody Ab1 and then forms a sandwich compound with Fe-MOF-Ab2. After H2O2 / TMB is added, Fe-MOF is catalyzed to generate a blue oxidation product (oxTMB) to generate a 652 nm absorbance signal, and meanwhile, oxTMB generates internal filtration / energy transfer quenching on Fe-MOF fluorescence, so that absorbance enhancement-fluorescence weakening dual-channel quantification and mutual verification are realized. The method does not need a natural enzyme or an exogenous fluorescent probe, and is simple, convenient, rapid and reliable to operate.

Description

Technical Field

[0001] This invention belongs to the field of biosensing and clinical analysis and detection technology, specifically involving an immunoassay method and detection platform for protein tumor markers that utilizes dual-ligand iron metal-organic framework nanozymes to achieve colorimetric-fluorescence dual-mode output. The method construction and performance evaluation are carried out using CEA as a typical example. Background Technology

[0002] CEA is a commonly used broad-spectrum biomarker for various malignant tumors. Changes in its serum level can provide important evidence for early screening, efficacy evaluation, and recurrence monitoring of diseases such as colorectal cancer, gastric cancer, pancreatic cancer, lung cancer, and breast cancer. At present, clinical detection of CEA mostly relies on traditional enzyme-linked immunosorbent assay (ELISA) or chemiluminescent immunoassay. Although these methods have high sensitivity and reliability, they usually require natural enzyme labeling, have long incubation times, and complex procedures, which limits their ability to enable rapid detection and multiplex validation.

[0003] Metal-organic frameworks (MOFs) possess high specific surface area, regular channels, and designable organic ligand frameworks. By introducing luminescent groups or coordinating unsaturated metal sites, they can be endowed with functions such as fluorescence emission and enzyme-like catalysis. In recent years, MOF-based nanozyme sensing systems have gradually become an important direction for constructing novel immunoassay platforms. However, existing MOF systems often only utilize one function: either as nanozymes for colorimetric detection or using their fluorescence for single-signal readout. It is difficult to achieve both high-efficiency catalysis and stable fluorescence on the same material, thus failing to realize a truly dual-mode synergistic output.

[0004] On the other hand, dual-mode immunoassay, by measuring the same sample with two independent but related signals, helps improve the reliability of results and reduce the risk of false positives / false negatives. An ideal dual-mode system should be as simple in structure as possible, avoiding the synthetic complexity and batch-to-batch variability caused by the superposition of multiple nanomaterials or multiple labels. Therefore, how to design a single MOF material that simultaneously possesses nanozyme activity and fluorescence properties, capable of generating a reversed "colorimetric enhancement-fluorescence attenuation" dual-mode signal in a simple substrate system, and introduce it into CEA sandwich immunoassay, is a pressing problem that needs to be solved. Summary of the Invention

[0005] This invention aims to overcome the problems of single signal, time-consuming detection process, and complex structure of existing MOF sensing system in traditional tumor marker detection methods. It proposes a dual-material strategy based on dual-ligand Fe-MOF nanozymes, which can simultaneously obtain two optical signals with opposite directions of colorimetry and fluorescence in the same immune recognition event, and realize highly sensitive and cross-validable dual-mode quantitative analysis of protein tumor markers (represented by CEA).

[0006] To achieve the above objectives, the technical solution provided by this invention includes the following core elements: (1) Design of dual-ligand Fe-MOF signal unit: Aromatic dicarboxylic acid ligands containing amino groups and aromatic dicarboxylic acid ligands containing polyhydroxy groups were selected, and ferric ions were used as metal nodes to construct dual-ligand Fe-MOF. By adjusting the molar ratio of the two ligands, the resulting material maintained a regular morphology and good dispersibility, while exhibiting high intrinsic fluorescence emission intensity and excellent peroxidase mimicry activity, providing a material basis for subsequent dual-mode signal output; (2) Construction of Fe-MOF-Ab2 immunoprobe: Using EDC / NHS crosslinking chemistry, the active groups on the surface of the dual-ligand Fe-MOF were covalently coupled with Ab2 to obtain Fe-MOF-Ab2 immunoprobe with uniform Ab2 loading on the surface. This probe can not only specifically recognize the target CEA, but also carry a large number of signal units that can participate in catalysis and luminescence, realizing the integration of "recognition-amplification"; (3) H2O2 / TMB triggered reverse dual-mode amplification mechanism: After the sandwich immune structure is formed, H2O2 / TMB substrate is added. Fe-MOF nanozyme catalyzes H2O2 to oxidize colorless TMB to blue oxTMB, which significantly increases the absorbance at 652 nm and forms a colorimetric channel. At the same time, the broad absorption spectrum of oxTMB overlaps with the fluorescence emission region of Fe-MOF. The fluorescence signal of Fe-MOF is quenched through processes such as internal filtration effect / energy transfer, thereby producing a fluorescence change in the opposite direction to the colorimetric signal, realizing the synergistic amplification of dual modes in one material. (4) Construction of a dual-mode immunoassay platform compatible with ELISA plates: The above-mentioned Fe-MOF-Ab2 immunoprobes are combined with a solid-phase carrier pre-coated with Ab1 to construct a sandwich immunoassay system in the typical ELISA style. Colorimetric and fluorescence detection can be performed sequentially in the same well without changing the carrier or aliquoting the samples. This facilitates compatibility with existing laboratory ELISA equipment and is beneficial for promotion and practical application.

[0007] The colorimetric-fluorescence dual-mode immunoassay method of this invention uses CEA as a model protein tumor marker. Its core principle is as follows: CEA in the sample first specifically binds to Ab1 on the surface of a solid support. Subsequently, a Fe-MOF-Ab2 immunoprobe is added, forming an "Ab1–CEA–Fe-MOF-Ab2" sandwich complex within the wells of the ELISA plate. The amount of Fe-MOF bound in the sandwich structure is directly proportional to the CEA concentration. Based on this, an H2O2 / TMB substrate solution is added, and Fe-MOF catalyzes the oxidation of TMB by H2O2 to generate oxTMB. On one hand, oxTMB has a significant absorption peak at 652 nm, changing the solution from colorless to blue, and the colorimetric signal increases with increasing CEA concentration. On the other hand, the broad absorption band of oxTMB covers part of the emission band of Fe-MOF, effectively quenching the fluorescence of Fe-MOF through an internal filtration effect or energy transfer pathway, resulting in a decrease in fluorescence intensity with increasing CEA concentration. When the CEA concentration is low, the sandwich structure carries a small amount of Fe-MOF-Ab2, resulting in a limited amount of oxTMB and low absorbance at 652 nm. Simultaneously, the quenching effect on Fe-MOF fluorescence is weak, and the fluorescence intensity remains at a high level. Conversely, when the CEA concentration increases, the amount of Fe-MOF-Ab2 binding increases, the catalytic product oxTMB accumulates, the system shows deeper color development and a significant increase in absorbance, while the fluorescence signal weakens considerably, forming a distinct inverse dual-mode response curve. By recording two calibration curves—"CEA concentration-absorbance" and "CEA concentration-fluorescence intensity"—quantitative analysis in both colorimetric and fluorescence channels can be achieved, and a ratio signal of absorbance to fluorescence intensity can be further constructed to improve analytical robustness.

[0008] The advantages of this invention are as follows: (1) Dual-purpose material with simple structure: The dual-ligand Fe-MOF simultaneously undertakes the functions of nanozyme catalysis and fluorescence emission, without the need for additional modification of natural enzymes or exogenous fluorescent dyes, thus avoiding the synthesis complexity and batch-to-batch differences caused by multi-component systems. (2) Reverse dual-mode synergistic amplification improves the reliability of results: In a single H2O2 / TMB substrate system, two optical signals with opposite directions, namely "increased absorbance and decreased fluorescence", are obtained. Both originate from the same immune recognition and catalytic process, which can cross-validate the measurement results and help reduce false positives / false negatives caused by matrix interference. (3) High sensitivity and wide linear range: thanks to the efficient catalytic ability and abundant signal carrier sites of Fe-MOF, both the colorimetric channel and the fluorescence channel can achieve detection limits at the pg / mL level, and the linear range covers the CEA concentration range from low ng / mL to several ng / mL, which is suitable for early clinical screening scenarios; (4) Simple operation, short time consumption, easy to promote: The method of this invention follows the conventional enzyme plate operation process, the sample processing is simple, the color development and fluorescence response are completed in a short time, and the reading can be directly read with the help of existing enzyme readers and fluorescence enzyme readers, which is convenient for promotion and application in ordinary biological laboratories or testing departments. (5) High versatility and can be extended to other biomarkers: By changing the recognition unit (antibody, aptamer, etc.), the dual-mode Fe-MOF immunoassay platform of the present invention can be extended to the detection of other tumor biomarkers or biomolecules, and has good versatility and application prospects. Attached Figure Description

[0009] Figure 1 This is a schematic diagram illustrating the principle of colorimetric-fluorescence dual-mode CEA immunoassay based on dual-ligand Fe-MOF nanozymes. Figure 2 The following are diagrams for a feasibility test of colorimetric analysis (a: dual-ligand Fe-MOF + H₂O₂ + TMB; b: dual-ligand Fe-MOF + TMB; c: H₂O₂ + TMB; d: dual-ligand Fe-MOF; e: TMB). (The insets are corresponding solution photographs.) Figure 3 Figure 1 shows a feasibility test diagram for fluorescence analysis (a: dual-ligand Fe-MOF; b: dual-ligand Fe-MOF + H2O2 + TMB). Figure 4 This is a standard working curve for the colorimetric immunoassay of CEA based on dual-ligand Fe-MOF. Figure 5 This is a standard working curve for detecting CEA using fluorescence immunoassay based on dual-ligand Fe-MOF. Detailed Implementation

[0010] The present invention will be further described below with reference to embodiments, but the present invention is not limited to the embodiments described below. Those skilled in the art will understand that although this specification uses CEA as a typical target to optimize and verify the method of the present invention, by simply changing the corresponding coating antibody and labeling antibody according to different protein tumor markers, while keeping the dual-ligand Fe-MOF as the signal amplification unit and the H2O2 / TMB substrate system unchanged, the same operating procedure can be used to achieve colorimetric-fluorescence dual-mode detection of other protein or peptide tumor markers (such as alpha-fetoprotein AFP, prostate-specific antigen PSA, etc.). Such equivalent substitutions all fall within the protection scope of the present invention. Example

[0011] 1. Preparation of antibody immobilization on ELISA plates A 96-well polystyrene ELISA plate with high binding strength was selected as the solid-phase carrier. Ab1 was diluted to 20 μg / mL with pH 7.4 phosphate-buffered saline (PBS), and 200 μL was added to each well. The plate was incubated overnight at 4 °C to immobilize the antibody on the bottom surface of the wells via physical adsorption. After incubation, the supernatant was discarded, and the plate was washed three times with pH 7.4 PBS buffer to remove unbound antibodies. Then, 300 μL of pH 7.4 PBS solution containing 0.1% bovine serum albumin (BSA) was added to each well as a blocking buffer, and the plate was incubated at 37 °C for 1 h to block the hydrophobic sites on the ELISA plate surface not occupied by antibodies, reducing subsequent non-specific adsorption. After blocking, the blocking buffer was discarded, and the plate was gently washed three times with PBS to obtain a ready-to-use solid-phase immunomodulator.

[0012] 2. Preparation of dual-ligand Fe-MOF-Ab2 immunoprobe (1) Synthesis of dual-ligand Fe-MOF (Example conditions) A specific molar ratio of amino-containing aromatic dicarboxylic acid ligands (such as 2-aminoterephthalic acid) and polyhydroxy aromatic dicarboxylic acid ligands was dissolved in a mixed solvent of N,N-dimethylformamide / ethanol. An appropriate amount of FeCl3·6H2O was added, and the mixture was solubilized under ultrasonication before being transferred to a high-pressure reactor. The reaction was then carried out at 120 °C for 24 h using a solvothermal method. After the reaction was completed and cooled to room temperature, the precipitate was collected by centrifugation. The precipitate was repeatedly washed with DMF and ethanol to remove unreacted ligands and small molecule impurities, and then vacuum dried at 60 °C to obtain dual-ligand Fe-MOF powder. The fluorescence intensity and peroxidase-like catalytic activity of the material were investigated by adjusting the molar ratio of the two ligands. The ratio that showed the best overall performance in both aspects was selected as the Fe-MOF material for subsequent immunoassays.

[0013] (2) Construction of Fe-MOF-Ab2 immunoprobe A certain amount of dual-ligand Fe-MOF powder was weighed and dispersed in pH 7.4 PBS buffer. After sonication, a homogeneous nanosuspension with a concentration of approximately 1.0 mg / mL was obtained. EDC and NHS solutions were added to bring the final concentration to 20 mM. The mixture was then gently shaken at room temperature for 30 min to activate the carboxyl or amino groups on the Fe-MOF surface.

[0014] Subsequently, Ab2 (1.7 mg / mL) was added, and the mixture was incubated at 37 °C for 3 h to allow the antibody to condense with the Fe-MOF surface active groups, forming a stable Fe-MOF-Ab2 immunoprobe. After the reaction, the precipitate was collected by centrifugation (10000 rpm, 5 min), resuspended in PBS containing a small amount of BSA, and washed repeatedly to remove uncoupled free antibody and small molecule byproducts. Finally, the obtained Fe-MOF-Ab2 was resuspended in an appropriate amount of pH 7.4 PBS (1 mL) and stored at 4 °C for later use.

[0015] 3. Feasibility verification of dual-mode signal 1) Feasibility test of colorimetric channels The dual-ligand Fe-MOF nanosuspension was added to PBS buffer, and five control systems were set up. Each group was incubated for the same time under the same conditions, and the absorption spectra in the 300-800 nm range were recorded using a spectrophotometer. As shown in Figure 2, only when Fe-MOF, H2O2, and TMB were present simultaneously did the solution exhibit a significant absorption peak at 652 nm and a visible blue color, indicating that Fe-MOF has significant peroxidase-mimicking activity and can catalyze the oxidation of TMB by H2O2 to generate a colorimetric signal.

[0016] 2) Feasibility test of fluorescence channel In another set of parallel experiments, Fe-MOF was dispersed in PBS and its intrinsic fluorescence emission spectrum was recorded. Then, under the same conditions, a mixed solution of H₂O₂ / TMB was added, and after incubation for 10 min, the emission spectrum was recorded again. As shown in Figure 3, after adding H₂O₂ / TMB, the fluorescence emission peak intensity of Fe-MOF decreased significantly, while the change in the blank group was smaller. This indicates that the oxTMB generated in the reaction quenches the fluorescence signal of Fe-MOF, thereby constructing a second fluorescence mode opposite to the colorimetric signal.

[0017] 4. Dual-mode immunoassay for CEA detection Figure 1 is a schematic diagram illustrating the principle of the colorimetric-fluorescence dual-mode CEA immunoassay based on dual-ligand Fe-MOF nanozymes of this invention. The specific detection steps are as follows: (1) Add different concentrations of CEA standard solution or actual sample (100 μL) to the wells of the ELISA plate that have been pre-immobilized with Ab1 and completed the blocking treatment, and incubate at 37 °C for 1 h to allow CEA to specifically bind to Ab1; after incubation, wash three times with pH 7.4 PBS (PBST) solution containing 0.05% Tween-20 to remove unbound components.

[0018] (2) Add an appropriate amount of Fe-MOF-Ab2 immunoprobe working solution (75 μL) to each well and continue incubation at 37 °C for 1 h to allow the probe to bind with the captured CEA to form an “Ab1–CEA–Fe-MOF-Ab2” sandwich structure; after incubation, wash three times with PBST to remove unbound probes.

[0019] (3) Add 100 μL of a colorimetric substrate solution containing H2O2 and TMB to each well and react at room temperature for 10 min to allow Fe-MOF to catalyze the generation of oxTMB.

[0020] (4) Colorimetric detection: The absorbance of each well was recorded at 652 nm using an ELISA reader to obtain a standard working curve between CEA concentration and absorbance, as shown in Figure 4.

[0021] (5) Fluorescence detection: In the same well, the fluorescence emission spectrum of Fe-MOF was recorded at the set excitation wavelength. The intensity of the main emission peak was used as the response signal to plot the standard working curve between CEA concentration and fluorescence intensity, as shown in Figure 5.

[0022] Under optimized conditions, in colorimetric mode, CEA concentration and absorbance at 652 nm showed a good linear relationship in the range of 0.01–5.0 ng / mL, with a detection limit of 1.08 pg / mL. In fluorescence mode, CEA concentration and fluorescence intensity showed a negative linear correlation in the range of 0.01–5.0 ng / mL, with detection limits reaching the same order of magnitude. Both modes can be used for quantitative determination of unknown samples, and cross-validation of the two signals can improve the accuracy and stability of the results.

[0023] Compared with traditional single-enzyme catalytic colorimetric methods, this embodiment integrates "nanozyme catalytic color development" and "fluorescence quenching response" into one through dual-ligand Fe-MOF, realizing colorimetric-fluorescence dual-mode amplification detection of CEA without the need for natural enzymes and exogenous fluorescent probes. This significantly improves detection sensitivity and reduces the difficulty of system construction, providing a new technical path for the efficient detection of tumor markers.

[0024] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the present invention; any equivalent substitutions or modifications made within the spirit and scope of the claims of the present invention shall be deemed to fall within the scope of protection of the present invention.

Claims

1. A colorimetric-fluorescence dual-mode immunoassay method for detecting protein tumor markers, characterized in that, The method includes the following steps: (1) Preparation and coating of solid-phase carrier: Ab1, which is used to identify target protein tumor markers, is coated on the surface of a solid-phase carrier, which is a 96-well microplate, magnetic microspheres or other substrates that can adsorb proteins; after coating, blocking solution is added to block unoccupied adsorption sites, and the solid-phase immunocarrier is obtained after washing. (2) Synthesis of dual-ligand Fe-MOF nanomaterials: Using amino-containing aromatic dicarboxylic acid ligands and polyhydroxy aromatic dicarboxylic acid ligands as organic ligand sources, and ferric salts as metal nodes, dual-ligand Fe-MOF nanomaterials containing two organic ligands are prepared by solvothermal or other wet chemical methods in N,N-dimethylformamide, ethanol or their mixed solvents. The preferred molar ratio of the dual ligands is such that the material simultaneously has high fluorescence emission intensity and peroxidase-mimicking activity. (3) Construction of Fe-MOF-Ab2 immunoprobe: Fe-MOF with dual ligands was dispersed in a buffer solution. The carboxyl or amino active sites on the surface of Fe-MOF were activated by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS). Then, Ab2, which can specifically recognize the protein tumor markers, was added for incubation and coupling. After centrifugation and washing, unbound antibodies were removed to obtain Fe-MOF-Ab2 immunoprobe with a high surface load of Ab2. (4) Formation of sandwich immune complex: The sample or standard solution containing the target protein tumor marker is added to the solid-phase immune carrier obtained in step (1) and incubated to allow the protein tumor marker to specifically bind to Ab1; after washing to remove unbound components, the Fe-MOF-Ab2 immune probe is added and incubated for a further period of time to allow Fe-MOF-Ab2 to bind to the protein tumor marker on the solid-phase carrier to form an "Ab1-target-Fe-MOF-Ab2" sandwich structure; (5) Nanozyme-catalyzed colorimetric reaction: After washing away unbound immune probes, a colorimetric substrate solution containing H2O2 and TMB was added to each reaction system and incubated under mild conditions so that the dual-ligand Fe-MOF acts as a peroxidase-mimicking catalyst to catalyze the oxidation of TMB by H2O2 to generate the blue oxidation product oxTMB. (6) Colorimetric and fluorescence dual-mode detection: In colorimetric mode, the absorbance of each reaction system at 652 nm is measured using a spectrophotometer or ELISA reader, and the change in absorbance is used as the first signal readout of the concentration of the protein tumor marker; in fluorescence mode, the fluorescence intensity of the Fe-MOF emission peak is recorded at a suitable excitation wavelength, and the degree of fluorescence quenching caused by the change in the concentration of the protein tumor marker is used as the second signal readout; wherein, as the concentration of the protein tumor marker increases, the binding amount of Fe-MOF-Ab2 increases and the amount of oxTMB generated increases, the absorbance at 652 nm increases, while the fluorescence intensity of Fe-MOF decreases, realizing the reverse dual-mode response of "increased absorbance-decreased fluorescence", thereby establishing the quantitative relationship between the concentration of the protein tumor marker and the colorimetric / fluorescence signal.

2. The colorimetric-fluorescence dual-mode immunoassay method according to claim 1, characterized in that, The protein-based tumor marker is CEA.

3. The colorimetric-fluorescence dual-mode immunoassay method according to claim 1 or 2, characterized in that, The dual-ligand Fe-MOF simultaneously serves as a peroxidase-mimicking catalyst and a fluorescence emission source. By adjusting the molar ratio of the two organic ligands, the material possesses both high catalytic activity and strong fluorescence emission capability. The oxTMB generated in the H2O2 / TMB system exhibits spectral overlap with the emission spectrum of Fe-MOF, causing the fluorescence signal to be quenched through an internal filtering effect or energy transfer. This achieves synergistic output of colorimetric signal amplification and fluorescence signal inverse response in a single nanomaterial, enabling dual-mode sensitive detection of the protein-based tumor markers without the need for additional natural enzymes or exogenous fluorescent probes. Furthermore, the intrinsic correlation between the two signals enhances the accuracy and anti-interference capability of the detection results.

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