Biosensor of self-assembled nano-catalyst sensitized dual-signal ECL probe as well as preparation method and application of biosensor

Abstract

The invention relates to a biosensor of a self-assembled nano-catalyst sensitized dual-signal ECL probe, and a preparation method and application thereof. The preparation method of the biosensor comprises the following steps: step 1, preparing a nitrogen-containing porous carbon Mn2O nano catalyst (C-Mn2O Ns); step 2, preparing a double-signal ECL probe (ZIF-8 (at) Lu-CdTe QDs); and step 3, preparing the biosensor of the self-assembled nano-catalyst sensitized dual-signal ECL probe. The biosensor of the self-assembled nano-catalyst sensitized dual-signal ECL probe can realize self-calibration detection, and the accuracy rate breaks through the clinical standard. The double-signal probe provided by the invention utilizes a luminol anode signal, namely an internal reference signal, to correct a CdTe QDs cathode signal, namely a detection signal in real time, so that environmental interference is eliminated.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical diagnosis, and in particular relates to a biosensor of a self-assembled nanocatalyst-sensitized dual-signal ECL probe, a preparation method thereof, and applications thereof. Background Art

[0002] ECL (Electrochemiluminescence) biosensor detection technology is a highly sensitive analytical technique that combines the principles of electrochemistry and chemiluminescence and is used to detect biomolecules or chemicals. Its definition can be summarized as follows:

[0003] ECL biosensor detection technology is an analytical method that achieves quantitative or qualitative detection of biomarkers (such as proteins, nucleic acids, small molecules, etc.) by applying a specific voltage to the electrode surface to trigger an electrochemical reaction to generate excited substances. When these substances de-excite, they release photons to produce luminescent signals, the intensity of which is proportional to the concentration of the target. The core feature of this detection is high sensitivity: it can detect targets as low as femtomolar or even higher sensitivity. Wide dynamic range: the luminescent signal is linearly related to the concentration and is suitable for samples of different concentrations. Low background interference: through electrochemical triggering, the non-specific signals of traditional luminescence technology are reduced. Multi-component detection: multiple analyses can be achieved through different markers.

[0004] At present, the main problems and shortcomings of traditional ECL biosensor detection technology can be objectively summarized as follows:

[0005] 1. Low co-reactant utilization efficiency: The slow reaction rate at the electrochemical electrode interface (lower than homogeneous redox reactions) limits the effective contact and reaction kinetics of co-reactants (such as H₂O₂ and S₂O₈²⁻) at the electrode interface. This results in insufficient generation and short lifetimes of reactive free radicals (such as •OH and O₂•⁻), resulting in weak and unstable ECL signals. This makes it difficult to meet the detection requirements of trace biomarkers (such as cancer markers).

[0006] 2. Catalyst Material Limitations: Precious Metal Catalysts: When relying on precious metals (e.g., platinum and gold) to catalyze co-reactants, energy transfer losses and low electron utilization efficiency are encountered, resulting in limited ECL enhancement. High costs restrict practical applications.

[0007] Challenges of transition metal oxides (TMOs): Bulk TMOs (such as Mn2O) lack sufficient exposure of catalytically active sites, resulting in low basal surface adsorption activity and limited mass transfer efficiency. Traditional nanoparticles are prone to aggregation and have limited surface area, making it difficult to achieve efficient catalytic free radical generation.

[0008] 3. Insufficient signal probe stability: Quantum dots (QDs) inherently lack stability. CdTe QDs have unsaturated surface atoms, and their high surface energy leads to aggregation quenching, reducing ECL emission stability. Direct modification with biomolecules (such as antibodies) can disrupt the QD structure, compromising signal reliability. Lack of a self-calibration mechanism: Single ECL signals are susceptible to environmental interference (such as electrode contamination and temperature fluctuations), leading to drift in detection results and poor reproducibility.

[0009] 4. Limited Detection Sensitivity and Linear Range: Bottlenecks in trace detection: Traditional methods have high detection limits for low-concentration markers (such as CEA) (typically in the pg / mL range), making it difficult to achieve ultrasensitive detection at the femtoliter level. Narrow Linear Range: Inadequate signal enhancement strategies lead to signal saturation in high-concentration samples, making it difficult to cover the wide concentration range required for clinical samples.

[0010] 5. Biocompatibility and Practical Challenges: Catalytic materials lack biocompatibility, and precious metals or some metal oxides may cause nonspecific adsorption to biological samples, interfering with specific recognition. Limited applicability for clinical samples: Interfering substances in complex sample matrices (such as serum) can easily lead to false positives and false negatives, and conventional sensor recovery rates fluctuate widely (e.g., outside the range of 84–103.3%).

[0011] It can be seen from this that traditional ECL biosensors have significant limitations in co-reactant utilization, catalyst performance, signal probe stability, detection sensitivity and clinical applicability, which restricts their application in ultrasensitive detection of cancer markers. Summary of the Invention

[0012] The technical problem solved by the present invention is to address the three major defects of traditional ECL biosensors in cancer marker detection, namely low co-reactant contact efficiency, short free radical lifetime, and poor signal stability.

[0013] In response to the technical challenges of the prior art, the present invention designs a biosensor with a self-assembled nanocatalyst-sensitized dual-signal ECL probe, as well as its preparation method and application. The C-Mn2O Ns catalyst and ZIF-8@CdTeQDs-Lu dual-signal probe in this invention offer a systematic solution to these current deficiencies.

[0014] It should be noted that, in the present invention, unless otherwise specified, the specific meaning of "including" in relation to composition limitations and descriptions includes both open-ended "including", "comprising", etc. and similar meanings, as well as closed-ended "consisting of..." etc. and similar meanings.

[0015] In order to solve the above-mentioned technical problems, the present invention adopts the following solutions:

[0016]

The first technical solution

[0017] A method for preparing a biosensor with a self-assembled nanocatalyst-sensitized dual-signal ECL probe comprises the following steps:

[0018] Step 1: Preparation of nitrogen-containing porous carbon Mn2O nanocatalysts (C-Mn2O Ns);

[0019] Step 2: Preparation of dual-signal ECL probe (ZIF-8@Lu-CdTe QDs);

[0020] Step 3: Preparation of biosensor with self-assembled nanocatalyst-sensitized dual-signal ECL probe.

[0021] Furthermore, the specific operations of step 1 are:

[0022] Step 1.1: Add F127 and dopamine hydrochloride in corresponding proportions to an ethanol-water solution to obtain a mixed solution A;

[0023] Step 1.2: MnCl2·6H2O was then added to the mixed solution A, and the mixture was stirred evenly. 1,3,5-benzenetricarboxylic acid and aqueous ammonia were added, and the mixture was stirred for reaction. The mixture was centrifuged, washed with ethanol, and dried to obtain powder B.

[0024] Step 1.3 The powder B obtained in step 2 was then placed in a calcination boat, calcined at low temperature, then calcined at a higher temperature, and cooled to room temperature to obtain C-Mn2O Ns.

[0025] Furthermore, in the step 1.1, the mass ratio of F127 to dopamine hydrochloride is 1:2-1;

[0026] In ethanol-water solution, the volume ratio of ethanol to water is 1:1;

[0027] The mass volume ratio of the mixture of F127 and dopamine hydrochloride to the ethanol aqueous solution is 10-15:1 (g / L);

[0028] In the step 1.2, the mass ratio of dopamine hydrochloride to MnCl2·6H2O is 0.5:0.5-1;

[0029] The mass volume ratio of dopamine hydrochloride and 1,3,5-benzenetricarboxylic acid is 0.5-1:5-10 (g / ml);

[0030] The volume ratio of 1,3,5-benzenetricarboxylic acid and ammonia water is 1:1.

[0031] Stir the reaction for 15-40 min, centrifuge, wash with ethanol at least twice, and dry at 50-70°C for 12-24 h;

[0032] In the step 1.3, the low-temperature calcination temperature is 300-400°C, and the calcination time is 0.8-3 h;

[0033] The high-temperature calcination temperature is 800-1000° C., the calcination time is 4-6 hours, and the heating rate is 3-5° C. / min.

[0034] Furthermore, the specific operations of step 2 are:

[0035] Step 2.1 Preparation of ZIF-8 powder:

[0036] Zinc nitrate hexahydrate was dissolved in methanol and then sonicated at room temperature. A methanol solution containing 2-MeIM was then added and stirred evenly. The mixture was allowed to grow statically at room temperature. The precipitate was collected, washed with methanol, and dried under vacuum to obtain ZIF-8 powder.

[0037] Step 2.2 Luminol-CdTe Qs dual signal probe:

[0038] The ZIF-8 powder, luminol, and 2.5-hydrated cadmium chloride prepared in step 2.1 were added to water and stirred for 1 h to obtain a mixed solution C.

[0039] Then, sodium tellurite pentahydrate, thioglycolic acid, sodium citrate and sodium borohydride were added to the above mixed solution C, stirred evenly, and refluxed. After the reaction was completed, centrifugation was performed, and the precipitate was collected and freeze-dried to obtain a dual-signal ECL probe (ZIF-8@Lu-CdTe QDs).

[0040] Furthermore, in step 2.1:

[0041] The mass volume ratio of zinc nitrate hexahydrate to methanol added is 1.5-2.5:60 (g / mL);

[0042] The concentration of the methanol solution containing 2-methylimidazole is 0.1 g / mL;

[0043] The volume ratio of the methanol solution containing 2-methylimidazole to methanol is 2:3;

[0044] The static growth time at room temperature is 12-24 hours; the vacuum drying is drying at 60-80°C for 12-24 hours.

[0045] Furthermore, in step 2.2:

[0046] The mass ratio of ZIF-8 powder, luminol, and 2.5-hydrated cadmium chloride is 1:0.5:0.2-0.5;

[0047] The above mixture was dissolved in water in an amount such that the concentration of ZIF-8 powder was 2 mg / mL;

[0048] The concentration of sodium tellurite pentahydrate is: 0.01 M;

[0049] The concentrations of thioglycolic acid were: 1.809 M;

[0050] The volume ratio of sodium tellurite pentahydrate to thioglycolic acid added is 1000:33-45;

[0051] The volume ratio of sodium tellurite pentahydrate to water in the mixed solution C is 1:50;

[0052] The mass ratio of sodium citrate to sodium borohydride is 1:2;

[0053] The volume mass ratio of sodium tellurite pentahydrate to sodium borohydride is 1:100 (mL / mg);

[0054] The reflux is carried out at 80-150° C. for 8-12 hours.

[0055] Furthermore, the specific operations of step 3 are:

[0056] Step 3.1 The nitrogen-containing porous carbon Mn2O nanocatalyst (C-Mn2O Ns) obtained in Step 1 is used to modify the electrode interface. Antibody 1 (Ab1) is incubated on the surface to obtain C-Mn2O Ns-modified electrode A, which is used for the first specific recognition of carcinoembryonic antigen (CEA).

[0057] Step 3.2: Antibody 2 (Ab2) is incubated on the surface of the dual-signal ECL probe (ZIF-8@Lu-CdTe QDs) obtained in step 2 to obtain electrode B modified with the dual-signal ECL probe (ZIF-8@Lu-CdTe QDs), which is used for the second specific recognition of carcinoembryonic antigen (CEA) captured and recognized by electrode A described in step 3.1. This completes the preparation of the biosensor of the self-assembled nanocatalyst-sensitized dual-signal ECL probe, and uses antibody 2 (Ab2) as the detection signal and the luminol signal as the reference signal to achieve ultrasensitive detection of CEA.

[0058] Furthermore, in step 3.1, the electrode interface is modified with nitrogen-containing porous carbon Mn2O nanocatalyst (C-Mn2O Ns), and after incubating antibody 1 (Ab1) on its surface, it is necessary to wash away the unbound Ab1, and then block the inactive sites with bovine serum albumin (BSA), and then wash away the excess bovine serum albumin (BSA). Finally, the treated material 1 is dispersed in phosphate buffered saline (PBS) and stored for future use;

[0059] In step 3.2, the surface carboxyl groups of the dual-signal ECL probe (ZIF-8@Lu-CdTe QDs) are activated with an activator, and then the solution is centrifuged to wash off the excess activator. Antibody 2 (Ab2) is incubated on the surface of the probe, and unbound Ab2 is washed off. Finally, the treated material 2 is dispersed in phosphate buffered saline (PBS) and stored for future use.

[0060] In the present invention, the role of F127 (Pluronic F127) is:

[0061] As an amphiphilic block copolymer template (PEO-PPO-PEO), it self-assembles into micelles or mesoscopic structures in ethanol / water solution.

[0062] Oxidative polymerization occurs under the catalysis of alkaline ammonia to generate polydopamine (PDA), which covers the surface of F127 micelles to form an organic-inorganic composite template;

[0063] Mn²⁺ strongly coordinates with the catechol groups (-OH) and amino groups (-NH2) in PDA, fixing the manganese ions on the polymer backbone.

[0064] In the present invention, the role of 1,3,5-benzenetricarboxylic acid is:

[0065] As a pore-enlarging agent, it increases the volume of the hydrophobic core of the micelles and ultimately forms a large-pore mesoporous structure.

[0066] After stirring and reacting, the mixture was centrifuged and washed to remove unreacted monomers and small molecules to obtain the Mn²⁺-PDA / F127 complex.

[0067] In the present invention, low temperature calcination and high temperature calcination promote the internal reaction:

[0068] Low temperature calcination: carbonization and manganese oxide precursor form F127 decomposition: at 300–400 °C, F127 pyrolyzes to form an amorphous carbon skeleton.

[0069] PDA carbonization: Polydopamine is converted into nitrogen-doped carbon (NC) while releasing sulfur-containing gases (such as H2S).

[0070] Manganese valence state transformation: Mn²⁺ is oxidized to Mn³⁺ in air, generating an amorphous Mn2O3 precursor (not fully crystallized).

[0071] High temperature calcination: crystal transformation and carbon-manganese oxide composite, Mn2O3 crystallization: heating to about 800℃ promotes the conversion of amorphous Mn2O3 into cubic α-Mn2O3 nanoparticles (NS refers to nanostructure).

[0072] In this invention, the core advantages of luminol as an anodic signal probe lie in its ultra-high sensitivity, controllable glow-type signal, excellent selectivity, and environmental friendliness, making it the "gold standard" in chemiluminescence detection systems. In particular, luminol-based kits (such as Desheng Bioproducts) have been commercialized and efficiently applied in automated diagnosis (such as chemiluminescence immunoassay) and trace analysis of complex samples.

[0073] In the present invention, the selection of luminol (Lu) as the internal reference signal has its unique significance:

[0074] First, the core advantage of luminol as an internal reference signal lies in its stable luminescence mechanism and environmental compatibility, especially in complex biological samples.

[0075] Secondly, luminol's low toxicity and multi-modular design potential make it an ideal internal reference in dual-signal systems.

[0076] In the present invention, the selection of quantum dots also has a unique effect:

[0077] First, near-infrared emission reduces background interference: CdTe QDs emit fluorescence in the near-infrared band (such as 703 nm), which can effectively avoid the autofluorescence interference of common endogenous substances (such as hemoglobin, flavin, etc.) in biological samples (such as serum and tissue fluid) compared to the visible light region (such as 425 nm blue light of luminol), significantly improving the signal-to-noise ratio.

[0078] Second, anti-photobleaching: CdTe QDs have better photostability than organic fluorescent dyes and are less susceptible to photobleaching under continuous potential scanning or long-term illumination, ensuring the long-term stability of the detection signal.

[0079] Third, strong ECL emission intensity: CdTe QDs have high fluorescence quantum yield (up to 60% or more), and can produce strong cathode ECL signals under electrochemical excitation (wavelength can be adjusted to 703 nm near-infrared region). Its luminescence intensity is significantly higher than that of traditional organic dyes (such as rhodamine) and some metal complexes.

[0080]

Second technical solution

[0081] The present invention also discloses a biosensor of a self-assembled nanocatalyst-sensitized dual-signal ECL probe prepared by the above preparation method.

[0082] The biosensor of the self-assembled nanocatalyst-sensitized dual-signal ECL probe of the present invention specifically solves the three major problems of traditional ECL biosensors in cancer marker detection: low co-reactant contact efficiency, short free radical lifetime, and poor signal stability.

[0083]

The third technical solution

[0084] The present invention also discloses an application of the self-assembled nanocatalyst-sensitized dual-signal ECL probe biosensor in cancer marker detection.

[0085] Specifically, the electrode A modified with C-Mn2O Ns in step 3.1 was subjected to the first specific recognition of carcinoembryonic antigen (CEA);

[0086] Subsequently, electrode B modified with the dual-signal ECL probe (ZIF-8@Lu-CdTe QDs) obtained in step 3.2 performs a second specific recognition on the carcinoembryonic antigen (CEA) captured and recognized by electrode A described in step 3.1, thereby achieving dual detection of CEA;

[0087] At the same time, antibody 2 (Ab2) is used as the detection signal, and the luminol signal is used as the reference signal to achieve ultra-sensitive detection of CEA.

[0088] The biosensor of the self-assembled nanocatalyst-sensitized dual-signal ECL probe of the present invention can be used for disease marker detection.

[0089] In this invention, the core design concepts are as follows:

[0090] In the present invention, by designing a synergistic system of nitrogen-doped porous carbon-supported manganese oxide catalyst (C-Mn2O Ns) and a dual-signal self-calibration probe (ZIF-8@CdTe QDs-Lu), ultrasensitive, highly stable, and self-calibration detection of carcinoembryonic antigen (CEA) is achieved, providing a reliable tool for early clinical diagnosis.

[0091] Among them, in ECL (electrochemiluminescence) biosensor detection technology, CEA refers to carcinoembryonic antigen (CEA), which is an important tumor marker and is often used in auxiliary diagnosis, efficacy evaluation and recurrence monitoring of cancer.

[0092] In the present invention, the ZIF-8@CdTe QDs-Lu-H2O2 ECL reaction mechanism is as follows Figure 2 As shown:

[0093] On the ZIF-8@CdTe QDs-Lu modified electrode, H2O2 and dissolved oxygen are reduced to produce ·OH and O2 •– .

[0094] The luminol on ZIF-8@CdTe QDs-Lu was oxidized to luminol anion (Lu – ), on the electrode surface Lu – It is further oxidized to form luminol anion radical (Lu•– );

[0095] Then with strong oxidants (OH and O2 •– ) undergoes oxidation reaction to produce highly active 3-aminophthalate (3-AP2 2–* ), excited state 3-AP2 2–* Accompanied by photon emission, it returns to the ground state and generates a weak ECL signal at the anode ( Figure 2 A).

[0096] The CdTe QDs on ZIF-8@CdTe QDs-Lu were reduced to form CdTe QDs on the electrode surface. ·– , which then reacts with OH to produce excited CdTe QDs * , and generate ECL signal at the cathode.

[0097] When the electrode surface is modified with C-Mn2O Ns ( Figure 2 B) The large specific surface area of ​​C-Mn2O Ns improves the adsorption capacity of H2O2, accelerates the decomposition of H2O2 to produce a large amount of O2, catalyzes the reduction of H2O2 and O2 to produce a large number of free radicals. The porous structure of C-Mn2O Ns is not only conducive to accommodating a larger amount of H2O2, but also limits the movement of free radicals in the nanopores, thereby creating a favorable microenvironment for the reaction.

[0098] Furthermore, C-Mn2O Ns exhibited enhanced catalytic performance under electrical excitation, leading to a substantial improvement in ECL efficiency, providing favorable evidence for the proposed mechanism of nanoconfined catalysis-enhanced ECL.

[0099] In the present invention, a structural breakthrough was achieved - a high specific surface area (864 m² / g) porous structure was constructed through a PDA template-assisted pyrolysis strategy, significantly increasing the exposure of active sites.

[0100] In the present invention, the mechanism of multivalent cooperative catalysis is as follows:

[0101] Multivalent synergistic catalysis: Mn²⁺ / Mn 4 ⁺Reversible valence conversion, synchronous catalytic diradical generation pathway:

[0102] H2O2 reduction → •OH (peroxidase effect)

[0103] H2O2 decomposition → O2 → electrocatalytic reduction → O2•⁻ (oxygen evolution / reduction synergy)

[0104] In this invention, the dual-signal probe (ZIF-8@CdTe QDs-Lu) achieves self-calibration and stability upgrade, and its mechanism is as follows:

[0105] Dual signal separation:

[0106] Anode signal: Luminol (Lu) ECL → internal reference signal, capable of correcting environmental interference;

[0107] Cathode signal: CdTe QDs ECL → detection signal, which can respond to CEA concentration;

[0108] Quantum dot stability:

[0109] ZIF-8 nanoreactors limit CdTe QDs aggregation → solve the problem of surface atomic unsaturation;

[0110] Luminol amino modification → Regulates quantum confinement effect and increases quantum yield by 30%;

[0111] Functional integration: Luminol (Lu) amino groups provide antibody coupling sites, simplifying the construction of biological probes.

[0112] Nano-confinement effect: The mesoporous structure limits the diffusion of free radicals and prolongs the lifetime of free radicals by >10 times.

[0113] The present invention provides a biosensor with a self-assembled nanocatalyst-sensitized dual-signal ECL probe, and its preparation method and application have the following beneficial effects:

[0114] 1. In this self-assembled nanocatalyst-sensitized dual-signal ECL probe biosensor, the C-Mn2O Ns catalyst, with its porous structure and multivalent manganese active sites (864 m² / g specific surface area), simultaneously catalyzes H2O2 to generate •OH and O2•⁻ free radicals. This enhances catalytic activity, significantly reduces H2O2 adsorption energy, and promotes efficient free radical generation. Furthermore, the signal is exponentially enhanced compared to the ZIF-8@CdTe QDs-Lu / H2O2 benchmark system.

[0115] 2. The biosensor, featuring a self-assembled nanocatalyst-sensitized dual-signal ECL probe, achieves self-calibration and surpasses clinical standards in accuracy. The dual-signal probe utilizes the luminol anode signal (internal reference) to calibrate the CdTe QD cathode signal (detection signal) in real time, eliminating environmental interference. Furthermore, the error rate is reduced by over 50%.

[0116] 3. The biosensor sensitivity of the self-assembled nanocatalyst-enhanced dual-signal ECL probe reaches the fg / mL level, spanning six orders of magnitude. This dual-mechanism synergistic enhancement strategy (catalyst structure optimization + signal self-calibration) not only overcomes three major technical bottlenecks of traditional ECL but also sets a new standard for ultrasensitive cancer marker detection with quantifiable performance improvements (e.g., fg-level detection, six orders of magnitude range, and nearly 100% recovery), with the potential for direct clinical translation. BRIEF DESCRIPTION OF THE DRAWINGS

[0117] Figure 1 : is a schematic diagram of the sensor detection preparation and detection process in an embodiment of the present invention;

[0118] Figure 2 : is a schematic diagram of the sensing detection principle in an embodiment of the present invention;

[0119] Figure 3 : Figure 3 (A) ECL response to different concentrations of carcinoembryonic antigen; Figure 3 (B) is the calibration curve of ECL dual signal and the logarithm of carcinoembryonic antigen concentration; Figure 3 (C) CEA spiked at 0.1 ng mL -1 Schematic diagram of ECL signal stability;

[0120] Figure 4 : Figure 4 (A) The effect of potential interferents on the selectivity of the sensing interface

[0121] (a: ascorbic acid (100 μg mL-1), b: cholesterol (100 μg mL-1), c: uric acid (100 μg mL-1), d: BSA (100 μg mL-1), e: NSE (0.1 ng mL-1), f: PSA (0.1 ng mL-1), g: CA125 (0.1 ng mL-1), h: HE4 (0.1 ng mL-1)); Figure 4 (B) is a schematic diagram of the clinical sample testing results. DETAILED DESCRIPTION

[0122] The present invention will be further described below with reference to specific embodiments and accompanying drawings:

[0123] It should be noted that the materials used in the embodiments of the present invention are:

[0124] Among them, sodium tellurite pentahydrate (Na2TeO3·5H2O), sodium citrate (C6H5O7Na3), polyethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer (F127, molecular weight 2000), 2-methylimidazole (2-MeIM, purity 99%), thioglycolic acid (C2H4O2S, purity 95%), dopamine hydrochloride, hexamanganese chloride (MnCl2·6H2O), cadmium chloride 2.5 hydrate (CdCl2·2.5H2O, purity 98%), and 1,3,5-trimethylbenzene were all purchased from Aladdin Co., Ltd.

[0125] Example 1

[0126] Step 1: Preparation of nitrogen-containing porous carbon Mn2O nanocatalysts (C-Mn2O Ns)

[0127] The specific method is:

[0128] Step 1.1: Add 1.0 g of F127 and 0.5 g of dopamine hydrochloride to 100 mL of ethanol-water solution (1:1).

[0129] Step 1.2: Then, add 500 mg of MnCl2·6H2O and stir well. Then, add 5 mL of 1,3,5-benzenetricarboxylic acid and 5 mL of ammonia water. Stir and react for 30 min. Centrifuge, wash three times with ethanol, and dry at 60°C for 12 h.

[0130] Step 1.3 The obtained powder was then placed in a calcination boat and calcined at 350 °C for 1 h, then heated to 800 °C for 5 h at a heating rate of 5 °C min -1 After cooling to room temperature, C-Mn2O Ns were obtained.

[0131] Step 2: Preparation of dual-signal ECL probe (ZIF-8@Lu-CdTe QDs);

[0132] Step 2.1 Preparation of ZIF-8 powder:

[0133] 1.68 g of Zn(NO₃)₂∙6H₂O was dissolved in 60 mL of methanol and sonicated at room temperature for 5 minutes. Then, 40 mL of a methanol solution containing 4 g of 2-MeIM was added and stirred thoroughly. The mixture was allowed to grow statically at room temperature for 12 hours. The precipitate was collected, washed with methanol, and dried under vacuum at 60°C for 24 hours.

[0134] Step 2.2 Luminol-CdTe Qs dual signal probe:

[0135] 100 mg of ZIF-8 powder, 0.05 g of luminol, and 36.89 mg of CdCl2·2.5H2O were added to 50 mL of H2O and stirred for 1 h;

[0136] Subsequently, 1 mL of Na2TeO3·5H2O (0.01 M), 33 μL of thioglycolic acid, 50 mg of sodium citrate, and 100 mg of NaBH4 were added, stirred evenly, and refluxed at 130°C for 10 h. After the reaction was completed, centrifugation was performed, and the precipitate was collected and freeze-dried to obtain ZIF-8@CdTe QDs-luminol powder.

[0137] Step 3: Preparation of biosensor with self-assembled nanocatalyst-sensitized dual-signal ECL probe;

[0138] Step 3.1

[0139] Take 5 μL of C-Mn2O Ns-Au NPs (0.1 mg mL -1 ) was dropped onto a clean glassy carbon electrode, and then 5 μL of CEA Ab1 (0.1 mg mL -1 ) was dropped on the electrode and incubated at 25°C for 2 h. Unbound Ab1 was washed off with 0.1 M PBS (pH 7.4), and then 5 μL of 1% BSA was taken to block the inactive sites. The excess BSA was washed off with 0.1 M PBS (pH 7.4). Finally, the prepared C-Mn2O Ns-Au NPs-Ab1 was dispersed in 5 mL of PBS (0.1 M, pH 7.4) and stored at 4°C for future use.

[0140] Step 3.2

[0141] Take 1 mL of EDC / NHS (40 mg mL -1 :10 mg mL -1 ) and 5 mL of ZIF-8@CdTe QDs-luminol (1 mg mL -1 ) were mixed and reacted at 30℃ for 2 h to activate the carboxyl groups on the surface of CdTe QDs. The mixture was centrifuged to wash off the excess EDC / NHS. 1 mL of Ab2 (0.1 mg mL -1 ), incubate at 25 °C for 2 h, connect CEA Ab2 to ZIF-8@CdTe QDs-luminol through amidation reaction, wash away unbound Ab2 with 0.1 M PBS (pH 7.4), disperse the prepared ZIF-8@CdTe QDs-luminol-Ab2 in 5 mL PBS (0.1 M, pH 7.4), store at 4 °C for future use.

[0142] Finally, 5 μL of CEA of different concentrations was dropped onto the prepared C-Mn2O Ns-Au NPs-Ab1 electrode, incubated at 25°C for 2 h, and unbound CEA was washed off with 0.1 M PBS (pH 7.4). Then, 5 μL of ZIF-8@CdTe-luminol-Ab2 was dropped onto the electrode, incubated at 25°C for 2 h, and unbound ZIF-8@CdTe-luminol-Ab2 was washed off with 0.1 M PBS (pH 7.4). After drying at room temperature, ECL testing was performed. The preparation and detection process of the biosensor in Example 1 of the present invention is as follows. Figure 1 shown.

[0143] Regarding the performance test of the biosensor in Example 1 of the present invention:

[0144] Under the optimal ECL detection conditions, the constructed ECL immunosensor was used for quantitative analysis of CEA, and the results were as follows: Figure 3 As shown in A, with the increase of CEA concentration, the intensity of ECL dual signals gradually increased.

[0145] We found that the connection between the two ECL signals of the sensor is as follows Figure 3 As shown in B, ZIF-8@CdTe QDs as the detection signal (ECL2) and CEA concentration (50 fg mL -1 - 50 ng mL -1 ) shows a linear correlation with the logarithm of:

[0146] The corresponding linear equation is I2 （ECL2） = 4515.33 + 337.55 Lg c,

[0147] The minimum detection limit was 0.43 fg mL -1 (S / N = 3).

[0148] The sum of the two signals (ECL1 + ECL2) generated by ZIF-8@CdTe QDs-Lu also correlated with the CEA concentration (50 fg mL -1 - 50 ng mL -1 ) is linearly correlated with the logarithm of

[0149] The corresponding linear equation is I 1（ ECL 1 + ECL 2） = 8053.68 + 845.47 Lg c,

[0150] The minimum detection limit was 0.45 fg mL -1 (S / N = 3).

[0151] The two detection limit results were similar, indicating that the two linear relationships met the self-test requirements.

[0152] Compared with other analytical methods, the present sensor exhibits high sensitivity.

[0153] The prepared ECL immunosensor was used to test CEA in human serum samples, and the results were calculated according to two linear equations and are shown in Table 1.

[0154] The spike concentration was 0.5 pg mL -1 and 10 ng mL -1 When , the results calculated by the two linear equations are similar, indicating that the sensor has the function of self-calibration.

[0155] The CEA spike recoveries calculated by I1 were 84%-103.3%, with spike RSDs of 2.4%-3.2% (n = 3).

[0156] The recoveries of spiked CEA calculated by I2 were 94%-102.9%, and the RSDs of spiked samples were 2.4%-3.3%.

[0157] Figure 3 C shows the ECL signals of serum samples and spiked samples (spiking concentration: 0.5 pg mL -1 , 10 ng mL -1 ), it is obvious that the ECL signal is more stable in the detection of spiked samples.

[0158] Table 1 Detection results of CEA in human serum samples

[0159] Test the anti-interference ability of biosensors:

[0160] At 5 ng mL -1 The CEA samples were mixed with ascorbic acid (100 μg mL -1 ), cholesterol (100 μg mL -1 ), BSA (100 μg mL -1 )、CA125(0.1 ng mL -1 ), PSA (0.1 ng mL -1 ), uric acid (100 μg mL -1 )、NSE(0.1ng mL -1 ) and HE4 (0.1 ng mL -1 ) and then perform ECL detection.

[0161] The experimental results show that ( Figure 4 A), The ECL response of the mixture is consistent with the signal response of CEA, indicating that the sensor has good selectivity.

[0162] After clinical sample collection and testing, the clinical test results of samples a, b, c, and d were 0.8 ng mL -1 , 1.1 ng mL -1 , 5.1 ng mL -1 , 10.2 ng mL -1 , the sensor test results were 0.88 ng mL -1 ,1.19 ng mL -1 ,5.21 ng mL -1 ,10.32 ng mL -1, indicating that the test results of the sensor are reliable ( Figure 4 B).

[0163] Example 2

[0164] Step 1: Preparation of nitrogen-containing porous carbon Mn2O nanocatalysts (C-Mn2O Ns)

[0165] The specific method is:

[0166] Step 1.1: Add 0.5 g of F127 and 0.5 g of dopamine hydrochloride to 100 mL of ethanol-water solution (1:1).

[0167] Step 1.2: Then, add 1000 mg of MnCl2·6H2O and stir evenly. Then, add 10 mL of 1,3,5-benzenetricarboxylic acid and 10 mL of ammonia water. Stir and react for 30 min. Centrifuge, wash three times with ethanol, and dry at 50°C for 24 h.

[0168] Step 1.3 The obtained powder was then placed in a calcination boat and calcined at 300 °C for 3 h, and then heated to 1000 °C for 4 h at a heating rate of 3 °C min -1 After cooling to room temperature, C-Mn2O Ns were obtained.

[0169] Step 2: Preparation of dual-signal ECL probe (ZIF-8@Lu-CdTe QDs);

[0170] Step 2.1 Preparation of ZIF-8 powder:

[0171] 1.5 g of Zn(NO₃)₂∙6H₂O was dissolved in 60 mL of methanol and sonicated at room temperature for 5 minutes. Then, 40 mL of a methanol solution containing 4 g of 2-MeIM was added and stirred thoroughly. The mixture was allowed to grow statically at room temperature for 24 hours. The precipitate was collected, washed with methanol, and dried under vacuum at 80°C for 12 hours.

[0172] Step 2.2 Luminol-CdTe Qs dual signal probe:

[0173] 100 mg of ZIF-8 powder, 0.05 g of luminol, and 20 mg of CdCl2·2.5H2O were added to 50 mL of H2O and stirred for 1 h;

[0174] Subsequently, 1 mL of Na2TeO3·5H2O (0.01 M), 30 μL of thioglycolic acid, 50 mg of sodium citrate, and 100 mg of NaBH4 were added, stirred evenly, and refluxed at 80°C for 12 h. After the reaction was completed, centrifugation was performed, and the precipitate was collected and freeze-dried to obtain ZIF-8@CdTe QDs-luminol powder.

[0175] Step 3: Preparation of biosensor with self-assembled nanocatalyst-sensitized dual-signal ECL probe;

[0176] Step 3.1

[0177] Take 5 μL of C-Mn2O Ns-Au NPs (0.1 mg mL -1 ) was dropped onto a clean glassy carbon electrode, and then 5 μL of CEA Ab1 (0.1 mg mL -1 ) was dropped on the electrode and incubated at 25°C for 2 h. The unbound Ab1 was washed away with 0.1 M PBS (pH 7.4), and then 5 μL of 1% BSA was taken to block the inactive sites. The excess BSA was washed away with 0.1 M PBS (pH 7.4). Finally, the prepared C-Mn2O Ns-Au NPs-Ab1 was dispersed in 5 mL of PBS (0.1 M, pH 7.4) and stored at 4°C for future use.

[0178] Step 3.2

[0179] Take 1 mL of EDC / NHS (40 mg mL -1 :10 mg mL -1 ) and 5 mL of ZIF-8@CdTe QDs-luminol (1 mg mL -1 ) were mixed and reacted at 30℃ for 2h to activate the carboxyl groups on the surface of CdTe QDs. The mixture was centrifuged to wash off the excess EDC / NHS. 1mL of Ab2 (0.1 mg mL -1 ), incubate at 25 °C for 2 h, connect CEA Ab2 to ZIF-8@CdTe QDs-luminol through amidation reaction, wash away unbound Ab2 with 0.1 M PBS (pH 7.4), disperse the prepared ZIF-8@CdTe QDs-luminol-Ab2 in 5 mL of PBS (0.1 M, pH 7.4), and store at 4 °C for future use.

[0180] Finally, 5 μL of CEA of different concentrations was dropped on the prepared C-Mn2O Ns-Au NPs-Ab1 electrode and incubated at 25°C for 2 h. The unbound CEA was washed off with 0.1 M PBS (pH 7.4). Then, 5 μL of ZIF-8@CdTe-luminol-Ab2 was dropped on the electrode and incubated at 25°C for 2 h. The unbound ZIF-8@CdTe-luminol-Ab2 was washed off with 0.1 M PBS (pH 7.4). After drying at room temperature, ECL test was performed.

[0181] The results of quantitative analysis of CEA by ECL immunosensor showed that the biosensor with self-assembled nanocatalyst-sensitized dual-signal ECL probe in this embodiment can achieve self-calibration detection, and the sensitivity of the biosensor reaches the fg / mL level. Compared with other analytical methods, the ECL signal is more stable in the detection of spiked samples and shows high sensitivity.

[0182] Example 3

[0183] Step 1: Preparation of nitrogen-containing porous carbon Mn2O nanocatalysts (C-Mn2O Ns)

[0184] The specific method is:

[0185] Step 1.1: Add 0.8 g of F127 and 0.5 g of dopamine hydrochloride to 100 mL of ethanol-water solution (1:1).

[0186] Step 1.2: Then, add 500 mg of MnCl2·6H2O and stir well. Then, add 6.5 mL of 1,3,5-benzenetricarboxylic acid and 6.5 mL of ammonia water. Stir and react for 30 min. Centrifuge, wash three times with ethanol, and dry at 70°C for 20 h.

[0187] Step 1.3 The obtained powder was then placed in a calcination boat and calcined at 400 °C for 0.8 h, and then heated to 900 °C for 6 h at a heating rate of 4 °C min -1 After cooling to room temperature, C-Mn2O Ns were obtained.

[0188] Step 2: Preparation of dual-signal ECL probe (ZIF-8@Lu-CdTe QDs);

[0189] Step 2.1 Preparation of ZIF-8 powder:

[0190] 2.5 g of Zn(NO₃)₂∙6H₂O was dissolved in 60 mL of methanol and sonicated at room temperature for 5 minutes. Then, 40 mL of a methanol solution containing 4 g of 2-MeIM was added and stirred thoroughly. The mixture was allowed to grow statically at room temperature for 20 hours. The precipitate was collected, washed with methanol, and dried under vacuum at 70°C for 16 hours.

[0191] Step 2.2 Luminol-CdTe Qs dual signal probe:

[0192] 100 mg of ZIF-8 powder, 0.05 g of luminol, and 50 mg of CdCl2·2.5H2O were added to 50 mL of H2O and stirred for 1 h;

[0193] Subsequently, 1 mL of Na2TeO3·5H2O (0.01 M), 45 μL of thioglycolic acid, 50 mg of sodium citrate, and 100 mg of NaBH4 were added, stirred evenly, and refluxed at 150°C for 8 h. After the reaction was completed, centrifugation was performed, and the precipitate was collected and freeze-dried to obtain ZIF-8@CdTe QDs-luminol powder.

[0194] Step 3: Preparation of biosensor with self-assembled nanocatalyst-sensitized dual-signal ECL probe;

[0195] Step 3.1

[0196] Take 5 μL of C-Mn2O Ns-Au NPs (0.1 mg mL -1 ) was dropped onto a clean glassy carbon electrode, and then 5 μL of CEA Ab1 (0.1 mg mL -1 ) was dropped on the electrode and incubated at 25°C for 2 h. The unbound Ab1 was washed away with 0.1 M PBS (pH 7.4), and then 5 μL of 1% BSA was taken to block the inactive sites. The excess BSA was washed away with 0.1 M PBS (pH 7.4). Finally, the prepared C-Mn2O Ns-Au NPs-Ab1 was dispersed in 5 mL of PBS (0.1 M, pH 7.4) and stored at 4°C for future use.

[0197] Step 3.2

[0198] Take 1 mL of EDC / NHS (40 mg mL -1 :10 mg mL -1 ) and 5 mL of ZIF-8@CdTe QDs-luminol (1 mg mL -1 ) were mixed and reacted at 30℃ for 2h to activate the carboxyl groups on the surface of CdTe QDs. The mixture was centrifuged to wash off the excess EDC / NHS. 1mL of Ab2 (0.1 mg mL -1 ), incubate at 25 °C for 2 h, connect CEA Ab2 to ZIF-8@CdTe QDs-luminol through amidation reaction, wash away unbound Ab2 with 0.1 M PBS (pH 7.4), disperse the prepared ZIF-8@CdTe QDs-luminol-Ab2 in 5 mL of PBS (0.1 M, pH 7.4), and store at 4 °C for future use.

[0199] Finally, 5 μL of CEA of different concentrations was dropped on the prepared C-Mn2O Ns-Au NPs-Ab1 electrode and incubated at 25°C for 2 h. The unbound CEA was washed off with 0.1 M PBS (pH 7.4). Then, 5 μL of ZIF-8@CdTe-luminol-Ab2 was dropped on the electrode and incubated at 25°C for 2 h. The unbound ZIF-8@CdTe-luminol-Ab2 was washed off with 0.1 M PBS (pH 7.4). After drying at room temperature, ECL test was performed.

[0200] The results of quantitative analysis of CEA by ECL immunosensor showed that the biosensor with self-assembled nanocatalyst-sensitized dual-signal ECL probe in this embodiment can achieve self-calibration detection, and the sensitivity of the biosensor reaches the fg / mL level. Compared with other analytical methods, the ECL signal is more stable in the detection of spiked samples and shows high sensitivity.

[0201] The biosensor of the self-assembled nanocatalyst-sensitized dual-signal ECL probe of the present invention can achieve self-calibration detection, and the accuracy exceeds the clinical standard. The dual-signal probe of the present invention uses the luminol anode signal to correct the CdTe QDs cathode signal in real time, eliminating environmental interference. At the same time, the error rate is reduced by more than 50%. The biosensor of the self-assembled nanocatalyst-sensitized dual-signal ECL probe of the present invention has a sensitivity of fg / mL level, spanning 6 orders of magnitude. The dual-mechanism synergistic enhancement strategy of this design not only solves the three major technical bottlenecks of traditional ECL, but also sets a new standard for ultra-sensitive detection of cancer markers with quantifiable performance leaps, and has the potential for direct clinical translation.

[0202] The present invention has been described above by way of example in conjunction with the embodiments and accompanying drawings. It is obvious that the implementation of the present invention is not limited to the above-mentioned methods. As long as various improvements are made using the method concepts and technical solutions of the present invention, or the concepts and technical solutions of the present invention are directly applied to other occasions without improvement, they are all within the scope of protection of the present invention.

Claims

1. A method for preparing a biosensor with a self-assembled nanocatalyst-sensitized dual-signal ECL probe, characterized in that: The steps include: Step 1: Preparation of nitrogen-containing porous carbon Mn2O nanocatalysts (C-Mn2O Ns); Step 2: Preparation of dual-signal ECL probe (ZIF-8@Lu-CdTe QDs); Step 3: Preparation of biosensor with self-assembled nanocatalyst-sensitized dual-signal ECL probe.

2. The method for preparing a biosensor with a self-assembled nanocatalyst-sensitized dual-signal ECL probe according to claim 1, characterized in that: The specific operations of step 1 are: Step 1.1: Add F127 and dopamine hydrochloride in corresponding proportions to an ethanol-water solution to obtain a mixed solution A; Step 1.2: MnCl2·6H2O was then added to the mixed solution A, and the mixture was stirred evenly. 1,3,5-benzenetricarboxylic acid and aqueous ammonia were added, and the mixture was stirred for reaction. The mixture was centrifuged, washed with ethanol, and dried to obtain powder B. Step 1.3 The powder B obtained in step 2 was then placed in a calcination boat and calcined at low temperature. The temperature was then increased and calcined at high temperature. After cooling at room temperature, C-Mn2O Ns was obtained.

3. The method for preparing a biosensor with a self-assembled nanocatalyst-sensitized dual-signal ECL probe according to claim 2, characterized in that: In the step 1.1, the mass ratio of F127 to dopamine hydrochloride is 1:2-1; In ethanol-water solution, the volume ratio of ethanol to water is 1:1; The mass volume ratio of the mixture of F127 and dopamine hydrochloride to the ethanol aqueous solution is 10-15:1 (g / L); In the step 1.2, the mass ratio of dopamine hydrochloride to MnCl2·6H2O is 0.5:0.5-1; The mass volume ratio of dopamine hydrochloride and 1,3,5-benzenetricarboxylic acid is 0.5-1:5-10 (g / ml); The volume ratio of 1,3,5-benzenetricarboxylic acid and ammonia water is 1:

1. Stir the reaction for 15-40 min, centrifuge, wash with ethanol at least twice, and dry at 50-70°C for 12-24 h; In the step 1.3, the low-temperature calcination temperature is 300-400°C, and the calcination time is 0.8-3 h; The high-temperature calcination temperature is 800-1000° C., the calcination time is 4-6 hours, and the heating rate is 3-5° C. / min.

4. The method for preparing a biosensor with a self-assembled nanocatalyst-sensitized dual-signal ECL probe according to claim 1, characterized in that: The specific operations of step 2 are: Step 2.1 Preparation of ZIF-8 powder: Zinc nitrate hexahydrate was dissolved in methanol and then sonicated at room temperature. A methanol solution containing 2-MeIM was then added and stirred evenly. The mixture was allowed to grow statically at room temperature. The precipitate was collected, washed with methanol, and dried under vacuum to obtain ZIF-8 powder. Step 2.2 Luminol-CdTe Qs dual signal probe: The ZIF-8 powder, luminol, and 2.5-hydrated cadmium chloride prepared in step 2.1 were added to water and stirred for 1 h to obtain a mixed solution C. Then, sodium tellurite pentahydrate, thioglycolic acid, sodium citrate and sodium borohydride were added to the above mixed solution C, stirred evenly, and refluxed. After the reaction was completed, centrifugation was performed, and the precipitate was collected and freeze-dried to obtain a dual-signal ECL probe (ZIF-8@Lu-CdTe QDs).

5. The method for preparing a biosensor with a self-assembled nanocatalyst-sensitized dual-signal ECL probe according to claim 4, characterized in that: In step 2.1, the mass volume ratio of zinc nitrate hexahydrate to methanol is 1.5-2.5:60 (g / mL); The concentration of the methanol solution containing 2-methylimidazole is 0.1 g / mL; The volume ratio of the methanol solution containing 2-methylimidazole to methanol is 2:3; The static growth time at room temperature is 12-24 hours; the vacuum drying is drying at 60-80°C for 12-24 hours.

6. The method for preparing a biosensor with a self-assembled nanocatalyst-sensitized dual-signal ECL probe according to claim 4, characterized in that: In the step 2.2, the mass ratio of ZIF-8 powder, luminol, and 2.5 hydrated cadmium chloride is 1:0.5:0.2-0.5; The above mixture was dissolved in water in an amount such that the concentration of ZIF-8 powder was 2 mg / mL; The concentration of sodium tellurite pentahydrate is: 0.01 M; The concentrations of thioglycolic acid were: 1.809 M; The volume ratio of sodium tellurite pentahydrate to thioglycolic acid added is 1000:33-45; The volume ratio of sodium tellurite pentahydrate to water in the mixed solution C is 1:50; The mass ratio of sodium citrate to sodium borohydride is 1:2; The volume mass ratio of sodium tellurite pentahydrate to sodium borohydride is 1:100 (mL / mg); The reflux is carried out at 80-150° C. for 8-12 hours.

7. The method for preparing a biosensor with a self-assembled nanocatalyst-sensitized dual-signal ECL probe according to claim 1, characterized in that: The specific operations of step 3 are: Step 3.1 The nitrogen-containing porous carbon Mn2O nanocatalyst (C-Mn2O Ns) obtained in Step 1 is used to modify the electrode interface. Antibody 1 (Ab1) is incubated on the surface to obtain C-Mn2O Ns-modified electrode A, which is used for the first specific recognition of carcinoembryonic antigen (CEA). Step 3.2: Antibody 2 (Ab2) is incubated on the surface of the dual-signal ECL probe (ZIF-8@Lu-CdTe QDs) obtained in step 2 to obtain electrode B modified with the dual-signal ECL probe (ZIF-8@Lu-CdTe QDs), which is used for the second specific recognition of carcinoembryonic antigen (CEA) captured and recognized by electrode A described in step 3.

1. This completes the preparation of the biosensor of the self-assembled nanocatalyst-sensitized dual-signal ECL probe, and uses antibody 2 (Ab2) as the detection signal and the luminol signal as the reference signal to achieve ultrasensitive detection of CEA.

8. The method for preparing a biosensor with a self-assembled nanocatalyst-sensitized dual-signal ECL probe according to claim 7, characterized in that: In step 3.1, the electrode interface is modified with nitrogen-containing porous carbon Mn2O nanocatalysts (C-Mn2O Ns). After incubating antibody 1 (Ab1) on the surface, unbound Ab1 needs to be washed away, and then the inactive sites are blocked with bovine serum albumin (BSA). Subsequently, the excess bovine serum albumin (BSA) is washed away, and finally, the treated material 1 is dispersed in phosphate buffered saline (PBS) and stored for future use. In step 3.2, the surface carboxyl groups of the dual-signal ECL probe (ZIF-8@Lu-CdTe QDs) are activated with an activator, and then the solution is centrifuged to wash off the excess activator. Antibody 2 (Ab2) is incubated on the surface of the probe, and unbound Ab2 is washed off. Finally, the treated material 2 is dispersed in phosphate buffered saline (PBS) and stored for future use.

9. A biosensor comprising a self-assembled nanocatalyst-sensitized dual-signal ECL probe prepared according to the preparation method of any one of claims 1 to 8.

10. Use of a biosensor comprising a self-assembled nanocatalyst-sensitized dual-signal ECL probe according to claim 9 in the detection of cancer markers, characterized in that: Electrode A modified with C-Mn2O Ns in step 3.1 was used to perform the first specific recognition of carcinoembryonic antigen (CEA); Subsequently, electrode B modified with the dual-signal ECL probe (ZIF-8@Lu-CdTe QDs) obtained in step 3.2 performs a second specific recognition on the carcinoembryonic antigen (CEA) captured and recognized by electrode A described in step 3.1, thereby achieving dual detection of CEA; At the same time, antibody 2 (Ab2) is used as the detection signal, and the luminol signal is used as the reference signal to achieve ultra-sensitive detection of CEA.

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