A ratiometric aggregation-induced ECL / EC sensing platform, preparation method thereof and application thereof in detecting circulating tumor DNA

Abstract

The present invention discloses a ratiometric aggregation-induced ECL / EC sensing platform, a preparation method thereof, and an application thereof for detecting circulating tumor DNA. The sensing platform uses TPA-BT nanoparticles (TPA-BT NPs) prepared by a nanoprecipitation method as an ECL luminophore, which is dripped onto a treated gold electrode surface for incubation, and then H1 and MCH are dripped for incubation, and a target circulation reaction solution assisted by exonuclease III (Exo III) is dripped, and the signal ratio of ECL to EC is measured after incubation. During detection, based on the EC signal of ferrocene (Fc) and the ECL signal of TPA-BT NPs, as the concentration of target ctDNA increases, the ECL signal gradually increases, while the EC signal gradually decreases, and the ECL and EC signals are compared, and are linearly correlated within the range of 100 aM-100 pM. It can be used to detect ctDNA and has the characteristics of high sensitivity.

Description

Technical Field

[0001] The invention relates to an application of ultra-sensitive detection of circulating tumor DNA based on electrochemistry and electrochemiluminescence ratio sensors, belonging to the technical field of biological detection. Background Art

[0002] Tumor liquid biopsy obtains tumor-related molecular biological information, such as circulating tumor DNA (ctDNA), circulating tumor cells (CTC), and exosomes, through non-invasive blood sampling. It has been widely used in early diagnosis of cancer, residual lesions, recurrence monitoring, efficacy evaluation, and drug guidance. Among them, ctDNA is a genomic fragment released into the blood by tumor cells, and carries cancer-specific genetic and epigenetic abnormalities. It can reflect the burden, metastasis, and malignant progression of the tumor and is a very important liquid biopsy biomarker. Due to the extremely low abundance of ctDNA in the blood and its short half-life, traditional technologies such as polymerase chain reaction (PCR) and DNA sequencing have some limitations in detecting ctDNA, such as insufficient anti-interference ability, high cost, and long detection time. Therefore, it is still a huge challenge to develop a low-cost, rapid, highly sensitive, and highly specific ctDNA detection method.

[0003] Electrochemiluminescence (ECL) technology has many advantages, such as low background signal, high sensitivity, no need for additional light source, easy miniaturization and low cost. Therefore, it has received widespread attention in the field of constructing efficient biosensors and has the potential to become an important technical means for detecting ctDNA. Due to the extremely low abundance of ctDNA in blood, traditional ECL technology cannot meet the needs of ultra-sensitive detection of trace ctDNA. In recent years, aggregation-induced ECL (aggregation-induced electrochemiluminescence, AIECL) has been used as a new strategy to combine traditional ECL methods with aggregation-induced emission (aggregation-induced emission, AIE), which can provide a new technical path for sensitive detection of trace ctDNA. Aggregation-induced emission is an improved luminescence produced by molecules in an aggregated state due to the restriction of intramolecular vibration, rotation and motion. Compared with the luminophores of traditional ECL technology with aggregation-induced quenching (ACQ) characteristics, aggregation-induced ECL luminophores suppress non-radiative attenuation during aggregation, thereby improving the luminescence efficiency. Although aggregation-induced ECL technology improves luminophore performance and facilitates detection, its quantitative determination relying on single signal readout may be affected by coexisting interferents, operators, instruments, and nonstandard assay procedures.

[0004] In order to solve the above problems, this paper developed a ratiometric aggregation-induced ECL / EC sensing platform, which uses a dual-signal output ratio analysis method to effectively overcome the potential interference of complex environments, which will effectively improve the accuracy and reliability of detection. Summary of the invention

[0005] Purpose of the invention: Since the abundance of ctDNA in the blood is extremely low, its detection poses a considerable challenge. To solve the above problems, we invented a ratiometric aggregation-induced ECL / EC sensing platform, its preparation method, and its application in ultrasensitive detection of ctDNA. The sensing platform is a ratiometric biosensor for detecting ctDNA, which mainly combines the enzymatic cleavage cycle amplification technology of exonuclease III (Exo III), and uses the EC signal of Fc and the ECL signal of the aggregation-induced luminescent material TPA-BT NPs for detection, and achieves the purpose of sensitive detection of ctDNA based on the ratio signal of ECL and EC.

[0006] Technical solution: In order to achieve the above-mentioned invention object, the present invention adopts the following technical solution:

[0007] First, the inventors synthesized a TPA-BT small molecule material with a symmetrical donor-acceptor-donor (DAD) structure, which consists of an electron-withdrawing benzothiazole group (BT) and an electron-donating triphenylamine group (TPA), and has excellent AIECL properties. The AIECL properties of this material and its use in the construction of ratio biosensors have not been reported in the literature.

[0008] Furthermore, the synthesized TPA-BT small molecules were prepared into nanoparticles (TPA-BT NPs) by nanoprecipitation method.

[0009] The final aggregation-inducing ECL / EC (ECL （TPA-BT NPs） / EC （Fc） ) The ratio sensor can overcome the influence of complex environment and has good sensitivity and specificity, realizing sensitive detection of ctDNA KRAS gene in complex serum environment. When the target concentration is in the range of 100 aM-100 pM, the ratio biosensor shows a good linear range with a detection limit of 11.2 aM. The constructed sensor not only has good sensitivity and specificity, but also can detect other types of ctDNA by changing the sequence of the hairpin. It has good versatility and great application potential.

[0010] The specific contents are as follows:

[0011] A ratiometric aggregation-induced ECL / EC sensing platform is based on the EC signal of Fc (ferrocene, Fc, ferrocene) and the ECL signal of TPA-BT NPs for detection. As the target concentration increases, the ECL intensity gradually increases, while the EC signal gradually decreases. The ratio of ECL to EC signals is linearly correlated in the range of 100 aM-100 pM (R 2 =0.99).

[0012] The preparation method of the ratiometric aggregation-induced ECL / EC sensing platform is as follows:

[0013] Step 1, preparing TPA-BT material;

[0014] Step 2: Synthesize TPA-BT nanoparticles (NPs) by nanoprecipitation method; the obtained product is spherical, has good dispersibility, and has a diameter of about 35 nm; TPA-BT NPs have a donor-acceptor-donor (DAD) structure, which has aggregation-induced emission properties and a relatively efficient electron transfer reaction.

[0015] Step 3: Constructing a ratiometric analysis aggregation-induced ECL / EC sensing platform:

[0016] S1. Use Al2O3 powder with a particle size of 0.05 µm to polish the gold electrode and rinse it with ultrapure water. Then, clean the gold electrode in four solutions: 0.5 M NaOH, 0.5 M H2SO4, 0.1 M H2SO4+0.01 M KCl, and 0.05 M H2SO4. Then, ultrasonically clean the gold electrode in a mixed solution of anhydrous ethanol and ultrapure water, rinse it with ultrapure water, and finally use filter paper to absorb the moisture on the surface of the gold electrode for later use.

[0017] S2. TPA-BT NPs synthesized by nanoprecipitation method were modified on the working area of ​​gold electrode and dried at room temperature.

[0018] S3. Add 6 µL of H1 to the working area of ​​the gold electrode treated in S2, incubate for 90 minutes, and then wash with phosphate buffer for later use.

[0019] S4. Modify the working area of ​​the gold electrode treated in S3 with 6 µL of mercaptohexanol (MCH), incubate for 15 minutes, and then wash with phosphate buffer for later use.

[0020] S5. The target, H2 and Exo III were co-incubated at 37°C, and the target signal was amplified through Exo III-assisted enzymatic cleavage cycle amplification reaction to obtain a solution containing the W1 sequence.

[0021] S6. The solution containing W1 obtained in S4 is continued to be modified on the working area of ​​the gold electrode treated in S4, and after incubation for 60 minutes, it is washed with phosphate buffer for later use.

[0022] The sequence of H1 is: 5'-SH - (CH2)6 - TATGCTAGCTGCATA -Fc-3'

[0023] The H2 sequence is: 5'-GCAGCTAGCATACCACTAGCTCCAAC -3'

[0024] The sequence of W1 is: 5'-GCAGCTAGCATA -3'

[0025] The above sequence was synthesized and purified by Sangon Biotechnology (Shanghai) Co., Ltd.

[0026] The above-mentioned EC and ECL ratio sensor is used to detect ctDNA. When the target ctDNA exists, H1 will be opened, so that the modified Fc is away from the electrode surface, thereby restoring part of the quenched ECL signal of TPA-BT NPs. Correspondingly, the EC signal of Fc also gradually weakens. The ECL signal of TPA-BT NPs and the EC signal of Fc are related to the target concentration, and ECL is induced by aggregation. （TPA-BTNPs） / EC （Fc）Ratio value to detect ctDNA.

[0027] Beneficial effects:

[0028] (1) First, the small molecule TPA-BT was synthesized by Suzuki coupling reaction, and then TPA-BT NPs were prepared by nanoprecipitation method. They are uniformly dispersed and still have strong ECL emission in the aggregated state, with strong ECL efficiency. This ECL detection combines the aggregation-induced luminescence properties of TPA-BT materials. Aggregation-induced quenching effect is overcome.

[0029] (2) With the help of the enzymatic cleavage of Exo Ⅲ, the target signal is amplified through cyclic amplification, which improves the sensitivity of biosensor detection and helps to detect trace amounts of ctDNA.

[0030] (3) In this paper, a ratiometric aggregation-induced ECL / EC sensing platform was developed. The ratiometric analysis method with dual signal output can effectively overcome the potential interference of complex environments, which will effectively improve the accuracy and reliability of detection.

[0031] (4) Using TPA-BT NPs as the ECL luminophore and with the help of Exo III-assisted cyclic amplification, an AIECL / EC ratio biosensor for highly sensitive detection of trace ctDNA was constructed. The DAD structure of TPA-BT material is combined with the properties of ECL. It has excellent ECL performance and can achieve the purpose of efficient and sensitive detection of ctDNA, which has great guiding significance in the early diagnosis of related cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 . Synthesis route diagram of TPA-BT in the present invention.

[0033] Figure 2 . Schematic diagram of the synthesis of TPA-BT NPs in the present invention.

[0034] Figure 3 . Characterization diagram of TPA-BT NPs in the present invention.

[0035] Figure 4 . Fluorescence spectra of TPA-BT in different ratios of water and tetrahydrofuran.

[0036] Figure 5 . ECL intensity of TPA-BT in acetonitrile and TPA-BT NPs in water phase.

[0037] Figure 6 . Schematic diagram of the construction of the Exo III-assisted target cycling reaction and ratio sensor in the present invention.

[0038] Figure 7 . Polyacrylamide gel (PAGE) electrophoresis diagram of the present invention.

[0039] Figure 8 . Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) of the sensor constructed in the present invention.

[0040] Fig. 9 . Verification test of the construction principle of the ratio sensor in the present invention: ac represent TPA-BT NPs / Au, TPA-BT NPs+H1 / Au and TPA-BT NPs+H1+W1 / Au respectively.

[0041] Fig.10 . Linear relationship diagram of electrochemiluminescence (ECL) and electrochemical (EC) response of the ratio sensor in the present invention for detecting different concentrations of ctDNA: in Figure A, the target concentrations are 0, 50 aM, 100 aM, 1 fM, 10 fM, 100fM, 1 pM, 10 pM and 100 pM from bottom to top; in Figure C, the target concentrations are 0, 100 aM, 1 fM, 10 fM, 100 fM, 1pM and 10 pM from top to bottom. DETAILED DESCRIPTION

[0042] The technical solution of the present invention will be specifically described below in conjunction with the accompanying drawings and embodiments:

[0043] The reagents and instruments used in the following examples are: poly(styrene-maleic anhydride) copolymer (PSMA), tetrahydrofuran (THF), sodium chloride (NaCl), potassium ferrocyanide (K3[Fe(CN)6]), triethylamine (TEA), acetonitrile, ammonium persulfate, N,N,N',N'-tetramethylethylenediamine (TEMED), formaldehyde and ferrocene carboxylic acid were purchased from MacLean Biochemical Technology Co., Ltd. (Shanghai, China). Potassium persulfate (K2S2O8) and potassium nitrate (KNO3) were purchased from Sinopharm Chemical Reagent (Shanghai, China). Ethanol, nitric acid (HNO3), silver nitrate (AgNO3) and sulfuric acid (H2SO4, 98%) were purchased from Lingfeng Chemical Reagent Co., Ltd. (Shanghai, China). Acrylamide, N,N'-methylenebisacrylamide, N,N-dipropyl-1-propylamine (TPrA) and calcium chloride (CaCl2) were purchased from Aladdin Chemical Reagent Co., Ltd. (Shanghai, China). N-butyldiethanolamine (BDEA) and 2-N-dibutylaminoethanol (DBAE) were from Roan's Reagent (Shanghai, China). Sodium dihydrogen phosphate (NaH2PO4), potassium chloride (KCl), and sodium hydroxide (NaOH) were purchased from Titan Technology Co., Ltd. (Shanghai, China). Sodium dihydrogen phosphate (Na2HPO4) and magnesium chloride (MgCl2) were obtained from Bid Pharmaceutical Co., Ltd. 6X polysucrose gel loading buffer III (containing xylene cyanol, bromophenol blue, Tris-HCl, EDTA) and all oligonucleotides were purchased from Shanghai Shenggong Biotechnology Co., Ltd. (Shanghai, China). The phosphate buffer (PBS) used in the present invention consists of 0.1 M disodium hydrogen phosphate (Na2HPO4), 0.1 M sodium dihydrogen phosphate (NaH2PO4), and 0.1 M potassium nitrate (KNO3), in which ultrapure water (18.2 MΩ; Millipore Corporation, USA) was used in the experiments. The electrolyte solution in the nonaqueous reference electrode was purchased from ALS Co., Ltd. (Japan) and consisted of 0.01 M silver nitrate (AgNO3) and 0.1 M tetrabutylammonium perchlorate (Bu4NClO4) in acetonitrile.

[0044] The electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were measured using a CHI 660E electrochemical workstation (Shanghai, China) and an Autolab PGSTAT 302 N electrochemical workstation (Switzerland). In addition, the ECL response signal was measured using a weak chemiluminescence measurement instrument (BPCL).

[0045] Example 1: Preparation of TPA-BT materials (Reference 1: Xiaohujie Chen et al. "Non-doped Red Fluorophores with Hybridized Local and Charge-Transfer State for High-Performance Fluorescent White Organic Light-Emitting Diodes" "ACS Appl. Mater. Interfaces" 2019, 11, 39026- 39034):

[0046] like Figure 1 As shown, the specific synthesis steps of TPA-BT material are:

[0047] 4,7-Dibromobenzo[c][1,2,5]thiadiazole (0.20 g, 0.7 mmol) and 4-(diphenylamino)phenylboronic acid pinacol ester (0.67 g, 1.7 mmol) were dissolved in toluene (10 mL), and then 2 M K2CO3 solution (5.0 mL) was added. Then, catalytic Pd(PPh3)4 was added to the mixture under nitrogen atmosphere and stirred at 90˚C for 48 hours. The reaction mixture was then cooled to ambient temperature, washed with water, and then extracted with dichloromethane to remove K2CO3. The crude product was purified by silica gel column chromatography using dichloromethane / n-hexane (v / v=1:5) as eluent. TPA-BT was obtained as an orange powder with a yield of about 79% (0.33 g). Its synthesis process is shown in Figure 1 .

[0048] Example 2: Preparation and characterization of TPA-BT NPs:

[0049] The synthesized TPA-BT and the amphiphilic polymer PSMA were dissolved in tetrahydrofuran to obtain a stock solution with a concentration of 3 mg / mL. Then, 80 μL of TPA-BT and 20 μL of PSMA were added to 300 μL of THF. Under ultrasonic conditions, the above mixture was quickly added to 2 mL of ultrapure water, ultrasonicated for 10 to 20 seconds, and then blown under an air pump for 40 minutes to remove the organic solvent. Then, it was filtered with a 0.22 μm aqueous phase filter membrane, and the solution was collected after filtration. The preparation process is shown in Figure 2 Afterwards, the morphology and distribution of the synthesized TPA-BT NPs were characterized by transmission electron microscopy (TEM). Figure 3From the above, TPA-BT NPs are spherical and have good dispersion. The diameter is about 35 nm, which is consistent with the particle size results of dynamic light scattering (DLS) test ( Figure 3 illustration).

[0050] Example 3: Demonstration of the aggregation-induced emission properties of TPA-BT NPs:

[0051] In order to further verify the aggregation-induced emission properties of TPA-BT materials, the fluorescence emission spectra of different water / THF ratios were tested, and the relationship between the relative fluorescence intensity (I / I0) and the water / THF ratio was plotted, as shown in Figure 2. Figure 4 As shown. When the ratio of water and THF ( f w ) is less than 60%, the emission of TPA-BT material appears red-shifted, and the corresponding fluorescence intensity is also reduced. f w When the concentration of TPA-BT increases further (>60%), the fluorescence intensity of TPA-BT increases significantly due to the formation of aggregates. f w It is strongest when it is 95%, which shows that TPA-BT has good AIE properties.

[0052] In order to further verify the aggregation-induced ECL performance of TPA-BT materials, the ECL intensity of TPA-BT small molecules in 0.1 M tetrabutylammonium perchlorate and 25 mM BDEA in acetonitrile solution was measured ( Figure 5 a), and the ECL reaction in 25 mM BDEA phosphate solution after TPA-BT NPs were modified to the electrode ( Figure 5 b). When TPA-BT NPs aggregated on the electrode surface, the luminescence intensity was significantly enhanced in the presence of co-reactants, which was 445 times higher than that in the dispersed state, confirming the AIECL property of the material.

[0053] Example 4: Construction of ECL / EC ratio sensor with enzyme cleavage cycle amplification effect:

[0054] like Figure 6 As shown in Figure 1, a ratiometric aggregation-induced ECL / EC sensing platform for ctDNA detection was developed. First, in Example 1, we synthesized a TPA-BT small molecule material, which has a symmetrical donor-acceptor-donor (DAD) structure consisting of an electron-withdrawing benzothiazole group (BT) and an electron-donating triphenylamine group (TPA). It exhibits AIE characteristics and a relatively excellent electron transfer reaction, such as Figure 6As shown in A. In addition, in Example 2, TPA-BT NPs were prepared by nanoprecipitation method using the synthesized TPA-BT small molecule material.

[0055] In the Exo III (exonuclease III)-assisted target recycling amplification step, 10 μL H2 (1 μM), 20 μL Exo III (1 U / μL), and 10 μL ctDNA of different concentrations were first incubated at 37°C for 60 minutes. The solution was then heated at 80°C for 10 minutes to inactivate Exo III and block the enzyme cleavage reaction. Finally, the obtained Exo III-assisted target recycling reaction solution was stored in a refrigerator at 4°C for later use. The specific process of Exo III-assisted target recycling is described in Figure 6 Described in C.

[0056] exist Figure 6 In B, the exposed gold electrode was first polished with Al2O3 polishing powder to a smooth mirror level. After that, the gold electrode was cleaned with an Autolab instrument. Then, it was ultrasonically cleaned with a mixture of water and ethanol. After the electrode was treated, 6 μL of 20 μg / mL TPA-BT NPs was dropped onto the surface. After it dried, 1 μM H1 was added and incubated for 90 minutes. After the incubation, the gold electrode was washed with PBS. To prevent nonspecific binding, 1 μM MCH was added to the electrode and incubated for 20 minutes. After that, 6 μL of the above-mentioned Exo III-assisted target circulation reaction solution was added. After incubation for 60 minutes, the ECL and EC signals were measured.

[0057] Example 5: Verification of enzyme cleavage cycle amplification and verification of successful construction of the sensor:

[0058] like Figure 7 As shown, the polyacrylamide gel electrophoresis (PAGE) experiment characterized the target recycling process assisted by Exo III. As shown in the figure, lanes 1-4 represent the target, W1, H1 and H2, respectively. Lane 5 represents H1 and W1, and a new band appears at this time, indicating the successful binding of H1 and W1. In addition, compared with bands 1 and 4, a new band appears in lane 6, indicating that the target and H2 have been combined. Most importantly, lane 7 represents a mixture of H2, target and Exo III, and two bands appear, representing the target and W1 sequences, respectively, indicating the feasibility of cyclic amplification. In order to prove that Exo III does not react with H2, this is confirmed by lane 8. After incubating Exo III with H2, no new bands appear, proving that Exo III does not react with H2. The results of the PAGE experiment verified the successful implementation of the target recycling process assisted by Exo III.

[0059] like Figure 8As shown in A and B, the constructed ratiometric biosensor was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) experiments. 3- The CV curves of the stepwise modified biosensor were tested in solution and in 10 mM [Fe(CN)6] 3- / 4- EIS measurements were performed in solution. The Nyquist plot of EIS consists of a straight line segment in the low-frequency region and a semicircle diameter in the high-frequency region, which represents the electron transfer resistance (Ret). The bare gold electrode exhibits a symmetrical redox peak with a large peak current ( Figure 8 A, curve a), while the semicircular area is smaller ( Figure 8 B, curve a). Subsequently, when TPA-BT NPs (curve b), H1 (curve c), MCH (curve d), and W1 (curve e) with poor conductivity were sequentially decorated on the gold electrode surface, the electron transfer process was hindered, resulting in a gradual decrease in the peak current intensity and a gradual increase in the Ret value. In summary, the above results demonstrate the successful construction of the biosensor.

[0060] Example 6: Validation of the ECL / EC ratio method:

[0061] Since the present invention uses the electrochemical (EC) signal of Fc (ferrocene) to construct a ratiometric biosensor, it is necessary to ensure that the Fc signal and the EC signals of other EC active substances in the system have good potential resolution. Fig. 9 As shown in A, the DPV peak potential of Fc is around 0.25 V, which is very different from the initial potential and peak potential of TPA-BT NPs and co-reactant BDEA in the system. This shows that the EC signal of Fc and the above-mentioned EC active substances can be well distinguished, which also shows that it is feasible to use the EC signal of Fc to construct a ratiometric biosensor.

[0062] The ECL and EC signals of the ratiometric biosensor were then tested before and after the introduction of target ctDNA. Fig. 9As shown in B, when H1 was introduced into the system, the ECL signal of TPA-BT NPs was quenched by Fc. A large amount of W1 was produced during the enzymatic cycle amplification of Exo III. When W1 was introduced into the system, the quenched signal of TPA-BT NPs was restored. Afterwards, the EC signal of Fc was also measured after the introduction of the target ctDNA. When H1 was introduced, the EC signal of Fc was very strong because Fc was near the electrode. When W1 was introduced, the EC signal of Fc decreased because W1 could specifically bind to H1, keeping Fc away from the electrode surface. The above results confirmed that the ECL signal and EC signal of the ratiometric biosensor were closely related to the concentration of the target ctDNA, verifying the feasibility of the sensor construction.

[0063] Example 7: Detection of ctDNA:

[0064] Afterwards, we investigated the performance of the constructed ratiometric biosensor in detecting ctDNA. Fig.10 As shown in the figure, with the increase of target concentration, the ECL intensity gradually increased, while the EC signal gradually decreased. In the target concentration range of 50 aM-100 pM, the ECL intensity was linearly correlated, and the correlation coefficient (R 2 ) was 0.98; in the target concentration range of 100 aM-10 pM, the EC signal was linearly correlated, and the correlation coefficient (R 2 ) was 0.96. The results showed that the ECL method was superior to the EC method, with a wider detection range and lower detection limit. Then, by comparing the ECL and EC signals, it was found that there was a linear correlation (R 2 =0.99), with a detection limit of 11.2 aM (S / N=3). Compared with the individual ECL and EC signals, the detection limit is lower and less susceptible to background signal interference.

[0065] The ratio biosensor constructed in this application is based on the ratio of the EC signal of Fc and the ECL signal of TPA-BT NPs, and combines the AIE properties of TPA-BT materials, which is equivalent to constructing an AIECL / EC ratio sensor. When TPA-BT exists in the form of a small molecule, its ECL signal is very low, and only after it is made into TPA-BT NPs by nanoprecipitation, it has a strong ECL signal. The luminescent material TPA-BT NPs has AIE performance and a donor-acceptor-donor structure, and its luminescence performance is excellent, especially when detecting trace amounts of ctDNA. In addition, this patent uses an Exo Ⅲ-mediated cyclic amplification reaction, which can further improve the sensitivity of detection.

Claims

1. A ratiometric aggregation-induced ECL / EC sensing platform, characterized in that: It is based on the EC signal of ferrocene and the ECL signal detection of TPA-BT nanoparticles. As the concentration of target ctDNA increases, the ECL intensity gradually increases, while the EC signal gradually decreases. The ECL and EC signals are ratioed. In the range of 100 aM-100 pM, the ratio is linearly correlated with the logarithm of the ctDNA concentration.

2. The method for preparing the ratiometric aggregation-induced ECL / EC sensing platform according to claim 1, characterized in that: Here are the steps: Step 1, preparing TPA-BT material; Step 2: synthesizing TPA-BT NPs by nanoprecipitation method; the obtained product is spherical, has good dispersibility, and has a diameter of about 35 nm; Step 3: Constructing a ratiometric analysis aggregation-induced ECL / EC sensing platform: (1) Powder polishing, cleaning, and drying of the gold electrode for later use; (2) The TPA-BT NPs obtained in step 2 were modified on the working area of ​​the gold electrode and dried at room temperature; (3) Modify the working area of ​​the gold electrode treated in step 3 (2) with 6 µL H1, incubate for 90 minutes, and then wash with phosphate buffer for later use; (4) To prevent nonspecific binding, add 6 µL of MCH to the working area of ​​the gold electrode treated in step 3 (3), incubate for 15 minutes, and then wash with phosphate buffer for later use; (5) The target, H2 and Exo III are co-incubated at 37°C, and the target signal is amplified through an Exo III-assisted enzymatic cleavage cycle amplification reaction to obtain a solution containing W1; (6) The obtained solution containing W1 is used to modify the working area of ​​the gold electrode treated in steps (3) and (4). After incubation for 60 minutes, it is washed with phosphate buffer and set aside for use.

3. The method for preparing the ratiometric aggregation-induced ECL / EC sensing platform according to claim 2, characterized in that: The sequence of H1 is: 5'-SH - (CH2)6 - TATGCTAGCTGCATA -Fc-3' The H2 sequence is: 5'-GCAGCTAGCATACCACTAGCTCCAAC -3' The sequence of W1 is: 5'-GCAGCTAGCATA -3'.

4. Use of the ratiometric aggregation-induced ECL / EC sensing platform described in claim 1 for detecting ctDNA.

5. The use according to claim 4, characterized in that: When target ctDNA is present, H1 will be opened, causing its modified Fc to move away from the electrode surface, thereby restoring part of the quenched ECL signal of TPA-BT NPs; accordingly, the EC signal of Fc will gradually weaken; the ECL signal of TPA-BT NPs and the EC signal of Fc are related to the target concentration, and ctDNA is detected by the aggregation-induced ECL / EC ratio value.

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