Preparation method and biological application of chemiluminescence-electrochemical dual-mode sensor based on magnetic biological aggregate

Abstract

According to the invention, the chemiluminescence and electrochemical dual-mode sensor based on the magnetic biological aggregate is innovated through the electrostatic interaction between DNA and Fe3O4-NH2 nanoparticles and the catalytic reduction capability of the Fe3O4-NH2 nanoparticles to H2O2. The method comprises the following steps: firstly, synthesizing Fe3O4-NH2 by adopting a hydrothermal method, and then combining the Fe3O4-NH2 with TdT extended DNA to form a sensor core; the sensor can generate chemiluminiscence and electrochemical signals through catalytic reaction when H2O2 exists, and the intensity of the chemiluminiscence and electrochemical signals changes along with the change of the concentration of H2O2 or TdT. The TdT inhibitor PP is added to limit the TdT activity and reduce the signal strength. The technology realizes efficient detection of H2O2, TdT and PP, is applied to quantitative analysis of TdT in Jurkat cells, and shows the application potential in biomedicine and clinical diagnosis. The method is simple to operate, low in cost and accurate in detection.

Description

Technical Field

[0001] The present invention relates to a method for preparing a dual-mode sensor based on magnetic bioaggregates and its application in Jurkat cells, particularly to a magnetic bioaggregate formed based on the electrostatic adsorption of Fe3O4-NH2 by DNA, a chemiluminescence-electrochemical sensor constructed and applied to TdT activity analysis in Jurkat cells, and belongs to the field of chemical biosensing in analytical chemistry. Background Art

[0002] Artificial nanozymes have gradually become a focus of scientific research due to their numerous advantages, including low cost, simple preparation, and tunable catalytic activity. With the rapid development of nanotechnology and its unique properties, the development of nanoenzyme-mimicking nanomaterials is facing new opportunities. In particular, ferroferric oxide (Fe₃O₄) nanoparticles, a traditional catalyst support, are particularly important due to their superparamagnetic properties, which allow them to be easily separated from the reaction system by an external magnetic field. Furthermore, surface modification of Fe₃O₄ nanoparticles can effectively adjust key properties such as their size and surface electronegativity, further broadening their application range. DNA-functionalized nanomaterials are particularly attractive and are considered promising candidates for biosensor switches. In this process, the binding between DNA and nanomaterials is a crucial step, not only maintaining the peroxidase-like activity but also enhancing the sensor's specificity and sensitivity through precise molecular recognition. This binding not only demonstrates the enormous potential of nanotechnology in the biomedical field but also points to possible new directions for future research. Through this approach, researchers can explore a wider range of nanomaterial-based applications, thereby promoting progress in fields such as biomedicine, environmental monitoring, and even energy conversion.

[0003] Acute lymphoblastic leukemia (ALL) is the most common malignancy in children under 15 years of age, accounting for approximately 25% of all childhood cancers. In the United States, approximately 35-40 new cases of ALL occur per million children. Among all childhood leukemias, B-cell ALL (B-ALL) is the most prevalent form, while T-cell leukemia (T-ALL) is more common in adults. Research has shown that terminal deoxynucleotidyl transferase (TdT) increases the diversity of immunoglobulins and T-cell receptors (TCRs) by adding nucleotides to DNA ends during V(D)J recombination, making it an ideal target for the treatment of B-ALL and T-ALL. TdT is overexpressed in over 80% of lymphoblastic tumors and has been widely used as a diagnostic marker. In particular, the Jurkat cell line, which expresses TdT, has become an important cell model for studying T-cell lymphomas. Although cell-type-specific antigens are often uniformly expressed in malignant cells, targeting broadly representative lineage markers can be toxic. Therefore, selective targeting of TdT on specific cell types that are transiently expressed during differentiation provides a therapeutic avenue. For T-ALL patients who have relapsed after stem cell transplantation and have no current cellular immunotherapy options, specific recognition of TdT provides an attractive treatment option. The pathological process of ALL involves the uncontrolled proliferation of tumor lymphocytes, which leads to a decrease in the number of normal functional cells, thereby triggering a series of fatal symptoms such as anemia, bleeding, infection and organ failure. Studies have shown that the expression of TdT is associated with high-risk subtypes of ALL, which exhibit faster disease progression, shorter overall survival and poor treatment outcomes. Therefore, the development of a simple and rapid TdT detection method is extremely important for further exploring the function and mechanism of action of TdT in T-ALL.

[0004] This invention utilizes the electrostatic adsorption of single-stranded DNA to nanomaterials and the peroxidase-like activity of Fe₃O₄-NH₂ nanoparticles to propose a method for preparing a chemiluminescent-electrochemical dual-mode sensor based on a magnetic bioaggregate and its biological application. First, a magnetic bioaggregate is formed by the induced adsorption of Fe₃O₄-NH₂ (positively charged) by T-rich DNA (negatively charged). This property provides a foundation for the design and application of biosensors. In this method, the longer the T-rich DNA strands, the more Fe₃O₄-NH₂ nanoparticles they can adsorb, and the stronger their catalytic activity. This technology utilizes both chemiluminescent and electrochemical detection methods. For chemiluminescent detection, under alkaline conditions, in the presence of peroxidase-like enzymes, particularly iron ions, H₂O₂ undergoes oxidation and reduction. Due to the presence of the substrate luminol, a series of reactions generate an excited state of 3-aminophthalate (AP*). However, this excited state is unstable, so AP* returns to the ground state and emits 425nm of radiation. For electrochemical detection, under neutral conditions, the aggregate electrochemically reduces H₂O₂, generating a corresponding electrochemical signal. Utilizing these principles, the present invention not only enables qualitative and quantitative analysis of H₂O₂, but also effectively detects TdT activity and its inhibitor, sodium pyrophosphate (PP), ultimately enabling the analysis of TdT content in Jurkat cells. To our knowledge, there are currently no reports demonstrating the detection of these substances using this method, demonstrating the significant significance and promising application prospects of this technology for modern medicine and even social development. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a rapid, simple, low-cost and novel method for preparing a chemiluminescence-electrochemical dual-mode sensor based on magnetic bioaggregates and its biological application.

[0006] The technical solution adopted by the present invention to solve the above technical problems is: a method for preparing a chemiluminescence-electrochemical dual-mode sensor based on magnetic bioaggregates and its biological application, the specific steps are as follows:

[0007] 1. Synthesis of Fe3O4-NH2

[0008] Soak a 100mL round-bottom flask and a stirrer in aqua regia for 2-3 hours, then rinse thoroughly with distilled water and air-dry for later use. Add the stirrer, 40-50mL of ethylene glycol (C₂H₂O₂), 1.0-1.5g of ferric chloride hexahydrate (FeCl₃·6H₂O), and 3.0-4.0g of sodium acetate (NaAc) to the 100mL round-bottom flask in sequence. Seal the flask with parafilm and place it in a 50°C oil bath, stirring until a yellow mixture forms. Add 0.5-1.0g of sodium hydroxide (NaOH) and continue stirring until a black, transparent liquid forms. Transfer the solution to a polytetrafluoroethylene reactor and heat at 190°C for 10-15 hours. After the reactor cools to room temperature, transfer the product to a 50mL centrifuge tube. Use a magnet to attract the powder below, discard the supernatant, and then wash the resulting black powder precipitate with anhydrous ethanol and then distilled water 2-3 times, then disperse it in 10mL of anhydrous ethanol. Finally, 300-500 μL of aminopropyltriethoxysilane (APTES) was added to the dispersion, mixed, and ultrasonically treated for 30-50 min. The mixture was placed in a shaker at room temperature for 5-10 h, and then dried in a vacuum drying oven at 55°C for 5-10 h. The mixture was then dispersed into a 0.1 mg / mL dispersion for later use.

[0009] 2. Preparation of Sensor

[0010] 1. Preparation of Chemiluminescent Sensor

[0011] (1) Synthesis of T-rich DNA chains

[0012] Reaction system (20 μL): Add 1–3 μL of 3000 nM single-stranded DNA (ssDNA, SH-ATCAGA), 1–3 μL of 1 mM deoxythymidine triphosphate (dTTP), 1–3 μL of 1000 U / L TdT, 2–5 μL of 10× TdT buffer, and 5–10 μL of distilled water to a 200 μL PCR tube. Incubate the mixture in a 37°C water bath for 150–200 min, then terminate the reaction at 75°C for 10–20 min. After completion, dilute the mixture 5-fold for later use.

[0013] (2) Preparation of chemiluminescent sensors

[0014] a. Add 100-300 μL of 2% bovine serum albumin (BSA) to the enzyme-labeled wells, cover the wells, and incubate overnight in a 4°C refrigerator.

[0015] b. Add 100-300 μL of 0.05% Tris-HCl buffer to the wells and wash three times to remove unbound BSA. Then, invert the plate onto filter paper and allow it to completely dry.

[0016] c. Add 100-300 μL of a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDS) / N-hydroxysuccinimide (NHS) (75 mM / 30 mM, v / v = 1:1) to the ELISA plate and shake it for 10-20 minutes to activate the carboxyl groups. Then, rinse the ELISA plate 2-4 times with distilled water. Immediately add the T-rich DNA chain synthesized in step (1) above and let it react at room temperature for 20-30 minutes to allow the sulfhydryl and carboxyl groups to react.

[0017] d. Add 100-300 μL of 0.05% Tris-HCl buffer to the wells and wash 2-4 times to remove unbound DNA. Then, invert the plate onto filter paper to completely absorb the water.

[0018] e. Add 100-300 μL of 0.1 mg / mL Fe3O4-NH2 dispersion to the enzyme-labeled wells and incubate at room temperature for 20-30 minutes;

[0019] f. Add 100-300 μL of 0.05% Tris-HCl buffer to the wells and wash 2-4 times to remove unbound DNA. Then, invert the plate onto filter paper to completely absorb the moisture.

[0020] g. Add 80-100 μL of Na2CO3-NaHCO3 (0.1 M pH 11.0) buffer, 5-10 μL of Luminol, and 5-10 μL of H2O2 to the ELISA wells. Measure the chemiluminescence intensity using a microplate reader, setting the test interval to 1 second.

[0021] Detection and analysis of H2O2:

[0022] Based on the above steps (1) and (2), the concentration of H2O2 in step (2)-g was changed (final concentration: 0-8000 μM), and the other steps remained unchanged. The luminescence intensity of the measurement system changed with the increase of the H2O2 concentration, based on which the detection and analysis of H2O2 was achieved.

[0023] Detection and analysis of TdT:

[0024] Based on the above steps (1) and (2), the concentration of TdT in step (1) is changed (final concentration: 0 to 6000 U / L), and the other steps remain unchanged. The luminescence intensity of the measurement system changes with the increase of TdT concentration, based on which the detection and analysis of TdT is achieved.

[0025] Screening of inhibitor PP:

[0026] Based on the above experimental steps, different concentrations of PP (final concentration: 0-5 mM) were added in step (1) of extending the T-rich DNA chain, and other steps remained unchanged. The changes in the luminescence intensity of the measurement system with the increase of PP concentration were used to screen the inhibitor PP.

[0027] 2. Preparation of Electrochemical Sensors

[0028] (1) A gold electrode (Au, 2 mm in diameter) was polished on suede with 1.0, 0.3, and 0.05 μm alumina powder for 10–30 s, respectively. The electrode was then ultrasonically cleaned with water for 2–4 times for 1–2 min, ultrasonically cleaned with ethanol for 10–30 s, and then dried with nitrogen.

[0029] (2) 1500 nM 1-5 μL ssDNA (SH-ATCAGA) solution was dropped onto the surface of the gold electrode and then incubated in a 4°C refrigerator overnight. The electrode was then immersed in a 1.0 mM 6-mercapto-1-hexanol (MCH) solution for 20-50 min to remove unbound DNA. The electrode was then slowly rinsed with phosphate buffer solution (PBS) (0.1 M pH 7.0).

[0030] (3) 4000U / L 1-5μL TdT, 1mM 1-3μL dTTP, 5×TdT reaction buffer 1-3μL, and 1-5μL distilled water were sequentially dripped onto the surface of the electrode (2), and then placed in a biological incubator at 37°C for 150-200 minutes, and then the electrode was slowly rinsed with PBS (0.1M pH 7.0);

[0031] (4) 1-5 μL of Fe3O4-NH2 prepared in step 1 above was dripped onto the electrode in step (3) above, and kept in the dark at room temperature for 20-40 min. After slowly rinsing with distilled water, a series of electrochemical experiments were performed;

[0032] (5) The prepared electrode was placed in a total volume of 1-3 mL of PSB buffer (0.1 M pH 7.0), which contained 400 mM 5-10 μL H2O2.

[0033] Detection and analysis of H2O2:

[0034] Based on the above step 2, the concentration of H2O2 in step (5) is changed (final concentration: 0-10mM), and the other steps remain unchanged. The electrochemical signal of the measurement system changes with the increase of the concentration of H2O2, based on which the detection and analysis of H2O2 is achieved.

[0035] Detection and analysis of TdT:

[0036] Based on the above step 2, the concentration of TdT in step (3) was changed (final concentration: 0-2000 U / L), and the other steps remained unchanged. The electrochemical signal of the measurement system changed with the increase of TdT concentration, based on which the detection and analysis of TdT was achieved.

[0037] Screening of inhibitor PP:

[0038] Based on the above experimental steps, different concentrations of PP (final concentration: 0-5 mM) were added in step (3), while other steps remained unchanged. The changes in the electrochemical signal of the measurement system with the increase of PP concentration were measured, based on which the inhibitor PP was screened.

[0039] 3. Detection of TdT in Jurkat Cells

[0040] In the present invention, Jurkat cells were subcultured and lysed to facilitate subsequent detection and analysis of samples (see Example 6 for specific steps).

[0041] Principle of the Invention: By leveraging the electrostatic interaction between DNA and nanomaterials and their catalytic reduction ability for hydrogen peroxide (H2O2), this paper develops a method for preparing a chemiluminescent-electrochemical dual-mode sensor based on magnetic bioaggregates and its biological applications. The specific steps include first synthesizing Fe3O4-NH2 nanoparticles with peroxidase-like activity using a one-step hydrothermal method. Subsequently, the core reaction component of the sensor is constructed by combining T-strand-rich DNA generated by TdT enzyme extension with the synthesized Fe3O4-NH2. In the presence of H2O2, the sensor undergoes a redox reaction of H2O2 through the catalytic action of the Fe3O4-NH2 nanoparticles, generating chemiluminescence and electrochemical signals. Under conditions of fixed TdT concentration and DNA chain length, the chemiluminescence intensity and electrochemical signal increase synchronously with increasing H2O2 concentration. When the H2O2 concentration is fixed and the TdT concentration is varied, the DNA chain length increases with increasing TdT concentration, resulting in more Fe3O4-NH2 adsorption, which in turn increases the chemiluminescence intensity and electrochemical signal. The introduction of the TdT inhibitor PP limits TdT activity, leading to a decrease in TdT activity with increasing PP concentration. This weakens the adsorption capacity of Fe3O4-NH2 and its catalytic reduction ability for H2O2, resulting in a decrease in both chemiluminescence intensity and electrochemical signal. This method, through dual chemiluminescence and electrochemical signal detection, achieves efficient analysis and detection of H2O2, TdT, and its inhibitor PP, and has been successfully applied to the quantitative analysis of TdT in Jurkat cells. This method boasts simple synthesis, convenient operation, and high detection reliability, demonstrating significant potential for application in biomedical research and clinical diagnostics.

[0042] Compared with existing technologies, the present invention offers advantages in that it combines magnetic bioaggregates with DNA for the first time, enabling the analysis and detection of H2O2, TdT, and its inhibitor PP through dual chemiluminescence and electrochemical signals. This approach was then applied to the analysis and detection of TdT in Jurkat cells. Experimental results demonstrated a linear relationship between the luminescence intensity and electrochemical signal values ​​of the system and the logarithmic concentrations of H2O2, TdT, and its inhibitor PP within a certain range, successfully constructing a biosensor. Its advantages include:

[0043] (1) Novel biosensing method. This patent introduces an innovative biosensing technology that, for the first time, uses ultra-long DNA to induce magnetic nanomaterials to adsorb on the substrate surface to form magnetic bioaggregates. By using H2O2 as a signal output medium, this technology realizes chemiluminescence and electrochemical analysis of terminal deoxyribonucleotidyl transferase (TdT) activity. To the best of our knowledge, this is the first report of a chemiluminescence sensor for TdT activity, demonstrating the innovation of this technology. In addition, by combining chemiluminescence analysis and electrochemical analysis on the same material, this technology constructs a dual-mode analysis method. This method helps to reduce false positive or false negative signals, provides a reliable basis for analyzing and detecting TdT activity in Jurkat cells, and ensures high accuracy of the data.

[0044] (2) High sensitivity. The present invention has successfully developed a highly sensitive chemiluminescence and electrochemical dual-signal biosensor. The sensor exhibits excellent catalytic performance for H2O2 and achieves accurate detection of H2O2 concentration through chemiluminescence intensity and electrochemical cyclic voltammetry (CV) signal output. Specifically, the linear equation of chemiluminescence intensity versus H2O2 logarithmic concentration is: y = 5.06x + 4.73, R 2 =0.9970, the detection limit is 0.028μM; the linear equation of the current response to the logarithmic concentration of H2O2 is: y=-24.03x-114.18, R 2 =0.9940, the detection limit is 3.24nM; the linear equation of chemiluminescence intensity versus TdT logarithmic concentration is: y=5.53x+4.35, R 2 =0.9942, the detection limit is 0.067U / L; the linear equation of the current response to the logarithmic concentration of TdT is: y=-45.44x+6.09, R 2 =0.9928, the detection limit is 0.35U / L; IC of PP 50 The results show that the sensor has high efficiency and sensitivity in detecting TdT activity, which is 1.16mM and 0.87mM respectively. These results highlight the innovation and application potential of the present invention in the field of biosensing, especially its superior performance in low concentration detection.

[0045] (3) High selectivity and strong anti-interference ability. In this patent, chemiluminescence and electrochemical dual signal technology are used to analyze and detect the target, which significantly improves the selectivity and anti-interference ability of the detection. When detecting H2O2, even if other common substances such as ascorbic acid (AA), uric acid (UA), lysine (Lys), cysteine ​​(Cys), citric acid (CA), and dopamine (DA) are present, they will not interfere with the detection results of H2O2. Similarly, when detecting the activity of terminal deoxyribonucleotidyl transferase (TdT), even if other control substances such as alkaline phosphatase (ALP), lysozyme (LZM), acetylcholinesterase (AChE), choline oxidase (ChOx), glucose oxidase (GOx), and thrombin (TB) are added, they will not interfere with the detection of TdT. This achievement not only proves the high selectivity of the sensor in identifying specific biomarkers, but also demonstrates its excellent anti-interference ability, ensuring the reliability of accurate detection in complex biological samples. This has important implications for biomedical research and clinical diagnostics, especially in applications where it is necessary to distinguish between highly similar biomolecules.

[0046] (4) Low cost and strong practicality. The dual-signal detection method proposed in this patent requires only a small amount of reagents when analyzing and detecting H2O2, TdT and its inhibitor PP, which reflects the advantage of realizing low-cost production concepts. Through the carefully designed dual-signal output mechanism, this technology not only successfully achieved accurate detection of TdT, but also has been used to conduct preliminary analysis and detection of TdT in Jurkat cells, demonstrating its excellent practicality and application potential. This cost-effectiveness and easy operation give it significant advantages in industrial applications.

[0047] In summary, the present invention provides a method for preparing a chemiluminescent-electrochemical dual-mode sensor based on magnetic bioaggregates and its biological applications. This method stands out for its simplicity, low cost, high sensitivity, excellent selectivity, and robust anti-interference capabilities, enabling accurate detection and analysis of H2O2, TdT, and its inhibitor PP. In particular, the present invention demonstrates significant practicality and application potential in the analysis and detection of Jurkat cell samples. This specific detection method is unprecedented in the art and holds great promise for its development in biomedical research and clinical diagnostics. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Feasibility analysis of the chemiluminescent and electrochemical sensors of the present invention;

[0049] Figure 2 The standard curve diagram of the luminescence intensity and current response of the chemiluminescence and electrochemical sensors of the present invention to different concentrations of H2O2;

[0050] Figure 3 The standard curve diagram of the luminescence intensity and current response of the chemiluminescence and electrochemical sensors of the present invention to different concentrations of TdT;

[0051] Figure 4 The standard curve diagram of the luminescence intensity and current response of the chemiluminescence and electrochemical sensors of the present invention to different concentrations of PP;

[0052] Figure 5 This is an experimental diagram showing the specificity of the chemiluminescence and electrochemical sensors of the present invention for H2O2;

[0053] Figure 6 This is an experimental diagram showing the specificity of the chemiluminescent and electrochemical sensors of the present invention for TdT;

[0054] Figure 7 This is a diagram of the present invention's analysis of TdT activity in proteins extracted from Jurkat cells. DETAILED DESCRIPTION

[0055] The present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

[0056] Example 1 Synthesis of Fe3O4-NH2

[0057] Soak a 100mL round-bottom flask and a stirrer in aqua regia for 3 hours, then rinse thoroughly with distilled water and air-dry. Add the stirrer, 40mL of ethylene glycol, 1.08g of FeCl₃·6H₂O, and 3.0g of NaAc to the 100mL round-bottom flask. Seal the flask with parafilm and place it in a 50°C oil bath, stirring until a yellow mixture forms. Add 0.6g of NaOH and continue stirring until a black, transparent liquid forms. The solution is then quickly transferred to a polytetrafluoroethylene reactor and heated at 190°C for 10 hours. After the reactor cools to room temperature, transfer the product to a 50mL centrifuge tube. Magnetically attract the powder below, discard the supernatant, and rinse the resulting black powder precipitate with anhydrous ethanol and then distilled water three times, then disperse it in 10mL of anhydrous ethanol. Finally, 500 μL of APTES was added to the dispersion, mixed, and ultrasonicated for 40 min. The mixture was placed in a shaker at room temperature for 8 h, then dried in a vacuum drying oven at 55°C for 6 h, and dispersed into a 0.1 mg / mL dispersion for later use.

[0058] Example 2 Preparation of sensor

[0059] 1. Preparation of Chemiluminescent Sensor

[0060] (1) Synthesis of T-rich DNA chains

[0061] Reaction system (20 μL): Add 2 μL of 3000 nM ssDNA (300 nM), 2 μL of 1 mM dTTP (0.1 mM), 2 μL of 1000 U / L TdT (1000 U / L), 5 μL of 10× TdT buffer, and 9 μL of distilled water to a 200 μL PCR tube. Incubate the mixture in a 37°C water bath for 200 min, then terminate the reaction at 75°C for 20 min. After completion, dilute the mixture 5-fold to 100 μL for later use.

[0062] (2) Preparation of chemiluminescent sensors

[0063] a. Add 200 μL of 2% BSA to the enzyme-labeled wells, cover the wells, and incubate overnight in a 4°C refrigerator.

[0064] b. Add 200 μL of 0.05% Tris-HCl buffer to the microplate wells and wash three times to remove unbound BSA. Then, invert the microplate onto filter paper and completely dry it.

[0065] c. Add 100 μL of EDS / NHS mixture (75 mM / 30 mM, v / v = 1:1) to the ELISA plate and shake it for 20 minutes to activate the carboxyl groups. Then, rinse the ELISA plate three times with distilled water. Immediately add the T-rich DNA chain synthesized in step (1) above and let it react at room temperature for 30 minutes to allow the sulfhydryl and carboxyl groups to react.

[0066] d. Add 200 μL of 0.05% Tris-HCl buffer to the microplate wells and wash three times to remove unbound DNA. Then, invert the microplate onto filter paper to completely absorb the moisture.

[0067] e. Add 100 μL of 0.1 mg / mL Fe3O4-NH2 dispersion to the enzyme-labeled wells and incubate at room temperature for 30 minutes;

[0068] f. Add 200 μL of 0.05% Tris-HCl buffer to the ELISA wells and wash three times to remove unbound DNA. Then, invert the ELISA plate onto filter paper to completely absorb the moisture.

[0069] g. Add 90 μL of Na2CO3-NaHCO3 (0.1 M pH 11.0) buffer, 5 μL of Luminol (500 μM), and 5 μL of H2O2 to the ELISA wells. Measure the chemiluminescence intensity using a microplate reader, setting the test interval to 1 s.

[0070] Detection and analysis of H2O2:

[0071] Based on the above steps (1) and (2), the concentration of H2O2 in step (2)g was changed (final concentration: 0, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 3000, 5000, 8000 μM), and the other steps remained unchanged. The luminescence intensity of the measurement system changed with the increase of the concentration of H2O2, based on which the detection and analysis of H2O2 was achieved.

[0072] Detection and analysis of TdT:

[0073] Based on the above steps (1) and (2), the concentration of TdT in step (1) was changed (final concentration: 0, 0.2, 0.4, 0.6, 1, 2, 4, 6, 10, 20, 60, 100, 200, 400, 1000, 2000, 4000, 6000 U / L), and the other steps remained unchanged. The luminescence intensity of the measurement system changed with the increase of TdT concentration, and the detection and analysis of TdT was achieved based on this.

[0074] Screening of inhibitor PP:

[0075] Based on the above experimental steps, different concentrations of PP (final concentrations: 0, 0.01, 0.03, 0.05, 0.1, 0.3, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 mM) were added to the step (1) of extending the T-rich DNA chain. The other steps remained unchanged. The changes in the luminescence intensity of the measurement system with the increase in the concentration of PP were measured, based on which the inhibitor PP was screened.

[0076] 2. Preparation of Electrochemical Sensors

[0077] (1) A gold electrode (Au, 2 mm in diameter) was polished on suede with 1.0, 0.3, and 0.05 μm alumina powder for 20 s, respectively. The electrode was then ultrasonically cleaned with water for 1 min twice and ultrasonically cleaned with ethanol for 20 s. The electrode was then dried with nitrogen and set aside.

[0078] (2) 1500 nM 2 μL ssDNA (300 nM) solution was dropped onto the surface of the gold electrode and then incubated in a 4 °C refrigerator overnight. Afterwards, the electrode was immersed in a 1.0 mM 6-mercapto-1-hexanol (MCH) solution for 30 min to remove unbound DNA. The electrode was then slowly rinsed with PBS (0.1 M pH 7.0).

[0079] (3) 4000U / L 2μL TdT (800U / L), 1mM 1μL dTTP (0.1mM), 2μL 5×TdT reaction buffer, and 5μL distilled water were sequentially drop-coated on the surface of electrode (2), and then placed in a biological incubator at 37°C for 200min, and then the electrode was slowly rinsed with PBS (0.1M pH 7.0);

[0080] (4) 4 μL of Fe3O4-NH2 prepared in step 1 above was dripped onto the electrode in step (3) above, kept in the dark at room temperature for 30 min, and then slowly rinsed with distilled water before conducting a series of electrochemical experiments;

[0081] (5) The electrode prepared above was placed in a total volume of 1 mL of PSB buffer (0.1 M pH 7.0), which contained 400 mM 5 μL H2O2 (2 mM).

[0082] Detection and analysis of H2O2:

[0083] Based on the above step 2, the concentration of H2O2 in step (5) was changed (final concentration: 0, 0.00001, 0.00002, 0.00005, 0.0001, 0.0002, 0.0005, 0.001, 0.002, 0.005, 0.02, 0.05, 0.2, 0.5, 1, 2, 5, 10 mM), and the other steps remained unchanged. The electrochemical signal of the measurement system changed with the increase of the concentration of H2O2, based on which the detection and analysis of H2O2 was achieved.

[0084] Detection and analysis of TdT:

[0085] Based on the above step 2, the concentration of TdT in step (3) was changed (final concentration: 0, 1, 1.5, 3, 5, 7, 10, 15, 30, 50, 70, 100, 150, 300, 500, 800, 1500, 2000 U / L), and the other steps remained unchanged. The electrochemical signal of the measurement system changed with the increase of TdT concentration, based on which the detection and analysis of TdT was achieved.

[0086] Screening of inhibitor PP:

[0087] Based on the above experimental steps, different concentrations of PP were added in step (3) (final concentrations: 0, 0.05, 0.1, 0.15, 0.2, 0.4, 0.6, 0.8, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5 mM), and the other steps remained unchanged. The changes in the electrochemical signal of the measurement system with the increase in the concentration of PP were measured, based on which the inhibitor PP was screened.

[0088] Example 3 Feasibility Analysis of Chemiluminescence and Electrochemical Sensors

[0089] The chemiluminescence intensity of the sensor prepared above was detected by an enzyme marker. Figure 1 As shown in A. Only when TdT product DNA@Fe3O4-NH2 exists in the system, the chemiluminescence intensity is very significant. Under other conditions, there is either only background signal or weak chemiluminescence intensity. This shows that the introduction of TdT product DNA@Fe3O4-NH2 promotes the catalytic reduction of H2O2 and enhances the chemiluminescence intensity of the system. This is because the presence of ultra-long DNA can increase the local concentration of magnetic nanoparticles, forming a large magnetic bioaggregate and exerting super catalytic ability. In addition, the prepared electrochemical sensor has an obvious CV signal near -0.75V, which is Figure 1 As shown in Figure B, TdT product DNA can absorb a large amount of Fe₃O₄-NH₂, effectively catalyzing the reduction of H₂O₂, resulting in a significant electrochemical signal. These experimental observations demonstrate the successful construction of this dual-mode sensor and that the aggregate exhibits strong catalytic activity.

[0090] Example 4 Analysis and Detection of H2O2, TdT and Its Inhibitor PP Using Chemiluminescence and Electrochemical Sensors

[0091] Based on the above examples 1 and 2, dual signal detection and analysis of H2O2 is realized, and the results are as follows: Figure 2 As shown in Figure 2, under the condition of fixed TdT concentration, the chemiluminescence intensity and electrochemical signal increased with the increase of H2O2 concentration. Figure 2 As shown in Figure A, the chemiluminescence intensity of the sensor and the logarithmic concentration of H2O2 show a good linear relationship. The linear correlation equation of the chemiluminescence intensity difference to the logarithmic concentration of H2O2 is: y = 5.06x + 4.73, R 2 =0.9970, the detection range is 0.1~3000μM, and the detection limit is 0.028μM; Figure 2 As shown in Figure B, the electrochemical signal of the sensor and the logarithmic concentration of H2O2 show a good linear relationship. The linear correlation equation of the current difference to the logarithmic concentration of H2O2 is: y = -24.03x -114.18, R 2 =0.9940, the detection range is 0.00001~10mM, and the detection limit is 3.24nM.

[0092] Based on the above embodiments 1 and 2, dual signal detection and analysis of TdT is realized, and the results are as follows: Figure 3As shown in the figure, under the condition of fixed H2O2 concentration, the chemiluminescence intensity and electrochemical signal increase with the increase of TdT concentration. When synthesizing DNA, as the amount of TdT added increases, the length and number of synthesized DNA chains increase, the amount of Fe3O4-NH2 adsorbed increases, the redox ability of H2O2 increases, and the dual signal also increases accordingly. Figure 3 As shown in Figure A, the chemiluminescence intensity of the sensor and the logarithmic concentration of TdT show a good linear relationship. The linear correlation equation of the chemiluminescence intensity difference to the logarithmic concentration of TdT is: y = 5.53x + 4.35, R 2 =0.9942, the detection range is 0.2~2000U / L, and the detection limit is 0.067U / L; Figure 3 As shown in Figure B, the electrochemical signal of the sensor and the logarithmic concentration of TdT show a good linear relationship. The linear correlation equation of the current difference to the logarithmic concentration of TdT is: y = -45.44x + 6.09, R 2 =0.9928, the detection range is 1-800U / L, and the detection limit is 0.35U / L.

[0093] Based on the above examples 1 and 2, dual signal detection analysis of TdT inhibitor PP was achieved, and the results are as follows: Figure 4 As shown in the figure, under the condition of fixed H2O2 concentration, the chemiluminescence intensity and electrochemical signal weakened with the increase of PP concentration. The addition of inhibitor PP inhibited the activity of TdT, and its DNA chain extension effect weakened with the increase of inhibitor PP concentration. Figure 4 A and Figure 4 As shown in B, the IC of PP 50 They are 1.16mM and 0.87mM respectively.

[0094] Example 5 Analysis of specificity and anti-interference ability

[0095] To verify the specificity and anti-interference ability of the dual signal detection of the sensor prepared above for H2O2 and TdT, the results of H2O2 are as follows Figure 5 As shown, the TdT results are as follows Figure 6As shown in the figure, A is the specificity analysis, and B is the anti-interference ability analysis. When detecting H2O2, the addition of other types of control substances such as ascorbic acid (AA), uric acid (UA), lysine (Lys), cysteine ​​(Cys), citric acid (CA), and dopamine (DA) did not interfere with the detection of H2O2. When detecting TdT, the addition of other types of control substances such as alkaline phosphatase (ALP), lysozyme (LZM), acetylcholinesterase (AChE), choline oxidase (ChOx), glucose oxidase (GOx), and thrombin (TB) did not interfere with the detection of TdT. This shows that the prepared sensor has good specificity for the target.

[0096] Example 6 Detection of TdT in Jurkat cells

[0097] The cells selected in this patent are Jurkat cells of the T cell leukocyte lineage. Extracting TdT from Jurkat cells involves several key steps, including cell culture, collection, lysis, and enzyme purification. Jurkat cells are a human T lymphocyte leukemia cell line, commonly used to study T cell function and signal transduction. The following are the basic steps for extracting TdT enzyme: ① Cell culture: Cell growth conditions, add 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin to a suitable culture medium (such as RPMI 1640), and culture at 37°C and 5% CO2; Cell expansion, wait until the cells grow to the logarithmic growth phase, usually the cell density reaches 1×10 6 When the cell count is 200 cells / mL, proceed to the next step. ②Cell collection and lysis: Collect cells and collect the cell pellet by centrifugation (300g, 5min, 4℃); Lyse cells by resuspending the cell pellet in lysis buffer (such as PBS buffer containing non-ionic surfactant NP-40), gently shaking to fully lyse the cells. Protease inhibitors can be added to prevent protein degradation; Centrifuge to remove cell debris. Centrifuge at high speed (such as 10000g, 10min) at 4℃ to remove cell debris and collect the supernatant. The supernatant is the lysis solution containing TdT enzyme. ③ Purification of TdT enzyme: Affinity chromatography: Use a specific antibody (such as an anti-TdT antibody) to immobilize it on an affinity chromatography column. Pass the lysate through the column, and the TdT enzyme is captured by the specific antibody. Elution: Use a high-salt buffer or a buffer containing a competitive ligand to elute the column, and collect the eluate, which contains the purified TdT enzyme. Concentration and desalting: Remove the high salt content from the eluate through dialysis or using a concentration column, while concentrating the TdT enzyme. Appropriate buffer: Store the purified TdT enzyme at -80°C in a buffer containing a stabilizer (such as glycerol) to avoid repeated freezing and thawing. The same method is used to extract TdT from HeLa cells.

[0098] The TdT enzyme obtained above was used to replace the TdT in Example 2 to complete the relevant experiments, and the results are as follows: Figure 7 As shown. Figure 7 As shown in A, as the number of Jurkat cells added to the system increases, the chemiluminescence intensity and electrochemical signal increase, indicating that the changes in the relevant signals are caused by TdT. To further prove that the changes in the chemiluminescence and electrochemical signals are caused by the presence of TdT in the system, different concentrations of inhibitors were introduced under the condition of fixing the number of Jurkat cells at 20,000 cells / mL. The results are shown in Figure 7 As shown in Figure B, as the concentration of the inhibitor PP increases, the chemiluminescence intensity and electrochemical signal decrease, indicating that Jurkat cells contain TdT and PP inhibits the related activity of TdT. The detection analysis of TdT can be linked to the analysis of TdT content in Jurkat cells. Subsequently, the laboratory cultured Hela cells and Jurkat cells were compared and analyzed, and the results are shown in Figure 2. Figure 7 As shown in C, the chemiluminescence intensity and electrochemical signal of the system with Hela cells are both lower than those of Jurkat cells, indicating that the TdT content in Hela cells is lower than that in Jurkat cells.

[0099] It should also be noted that the above embodiments do not limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art may make changes, modifications, additions or substitutions within the spirit of the present invention, which shall also fall within the scope of protection of the claims of the present invention.

Claims

1. Preparation method of chemiluminescence-electrochemical dual-mode sensor based on magnetic bioaggregates and its biological application, the mechanism of which is as follows: This patent demonstrates an innovative biosensing technology, which innovatively uses ultra-long DNA to guide magnetic nanomaterials to attach to the substrate surface, thereby forming magnetic bioaggregates. Using hydrogen peroxide (H2O2) as a signal transmission medium, this technology successfully implemented chemiluminescence and electrochemical analysis of terminal deoxyribonucleotidyl transferase (TdT) activity. This is the first report of the use of a chemiluminescence sensor for TdT activity, highlighting the innovation of this technology; this technology integrates chemiluminescence and electrochemical analysis into a dual-mode analysis method, effectively avoiding false positive or false negative signal interference; for the first time, TdT activity monitoring is achieved through material aggregation-induced signal enhancement, laying a solid foundation for accurate analysis of TdT activity in Jurkat cells.

2. The method for preparing a chemiluminescence-electrochemical dual-mode sensor based on magnetic bioaggregates and its biological application according to claim 1, characterized in that: This patent uses a dual-signal method of chemiluminescence and electrochemical CV to detect and analyze different targets H2O2, TdT and its inhibitor PP. In the chemiluminescence detection mode, the sensor can detect the lowest concentration of H2O2 of 0.028μM, the activity detection limit of TdT is 0.067U / L, and for its inhibitor PP, IC 50 In the electrochemical detection mode, the sensor has a detection limit of 3.24 μM for H2O2 and a detection limit of 0.35 U / L for TdT activity. The IC 50 is 0.87mM.

3. The method for preparing a chemiluminescence-electrochemical dual-mode sensor based on magnetic bioaggregates and its biological application according to claims 1-2, characterized in that: The dual-mode analysis technology defined in this patent was used to validate TdT expression in Jurkat cells. The results showed that Jurkat cells do contain a certain amount of TdT, while Hela cells, in contrast, have significantly lower levels of TdT. This finding not only confirms the effectiveness of the dual-mode sensor for biomarker detection but also provides an experimental basis for further investigation of TdT's potential as a leukemia marker.

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