Preparation method of dual-ligand protected electrochemiluminescence gold nanoclusters and application thereof in detection of hogg1

Abstract

The application relates to a preparation method of a dual-ligand protected electrochemiluminescence gold nanocluster and application of the electrochemiluminescence gold nanocluster in detection of hOGG1. The electrochemiluminescence gold nanocluster is synthesized in a one-step method with methionine and beta-cyclodextrin as dual ligands. The obtained water-soluble electrochemiluminescence gold nanocluster has good biocompatibility, high electrochemiluminescence efficiency and complete nanomaterial performance. When the specific electrochemiluminescence gold nanocluster is used as an ECL luminophore, and an hOGG1 ECL sensor with triethylamine (TEA) as a co-reactant is applied to detection of human 8-oxoguanine DNA glycosylase (hOGG1), the sensor has higher sensitivity and shows good sensing potential. The sensor is simple to operate, has good repeatability, and has important scientific significance and application value for early clinical diagnosis.

Description

Technical Field

[0001] This invention relates to the fields of analytical chemistry and nanotechnology, and in particular to a method for preparing electrochemiluminescent gold nanoclusters protected by methionine and β-cyclodextrin dual ligands and its application in detecting hOGG1. Background Technology

[0002] Electrochemiluminescence (ECL) technology combines the characteristics of electrochemistry and chemiluminescence. With its inherent advantages such as high sensitivity, zero background signal, simple setup, good controllability, and short processing time, it has attracted increasing attention in biosensing applications (immunoassay and gene analysis strategies). As is well known, ECL emitters play a crucial role in the development and application of ECL. Furthermore, various types of ECL emitters (such as small organic molecules, polymers, and inorganic materials) have been extensively developed.

[0003] Gold nanoclusters have become widely studied ECL emitting materials due to their near-infrared ECL emission, ease of surface modification, good biocompatibility, and environmental friendliness. However, the slow charge transfer during electrogeneration leads to generally poor ECL efficiency in gold nanoclusters, which remains a major challenge and limits their widespread application in ECL bioassays. To address this, researchers have explored various surface-modifying capping agents to prepare luminescent gold nanotubes, extending the ECL emission wavelength of gold nanotubes from the visible light range to the near-infrared region. Utilizing the advantages of near-infrared ECL, such as large tissue penetration depth, low photochemical hazard, and low background interference, the detection sensitivity of targets has been improved. However, further improvements are needed to achieve even greater sensitivity. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a method for preparing dual-ligand protected electrochemiluminescent gold nanoclusters and its application in detecting hOGG1. The water-soluble electrochemiluminescent gold nanoclusters are synthesized in one step using methionine and β-cyclodextrin as dual ligands. They not only have good biocompatibility but also high electrochemiluminescence efficiency and fully passivated nanomaterial properties, which can be used for the sensitive detection of hOGG1.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for preparing electrochemiluminescent gold nanoclusters protected by dual ligands includes the following steps:

[0007] Under stirring conditions, a mixture of β-cyclodextrin and sodium hydroxide was added to a mixed aqueous solution of HAuCl4·3H2O and methionine, followed by a first incubation, and then sulfuric acid was added to obtain gold nanoclusters precipitate.

[0008] The gold nanoclusters were precipitated and dissolved in an ammonia solution, and then subjected to a second incubation. After separation, dual-ligand protected electrochemiluminescent gold nanoclusters (Met / β-CD-Au NCs) were obtained.

[0009] Furthermore, the molar ratio of HAuCl4·3H2O, methionine, and β-cyclodextrin is 1:64:4.16.

[0010] Further, the mixed aqueous solution of HAuCl4·3H2O and methionine is obtained by mixing an aqueous solution of HAuCl4·3H2O and an aqueous solution of methionine; preferably, the concentration of the aqueous solution of HAuCl4·3H2O is 12.5 mM, and the concentration of the aqueous solution of methionine is 30 mg / mL. −1 .

[0011] Further, the β-cyclodextrin and sodium hydroxide mixed solution is obtained by mixing an aqueous solution of β-cyclodextrin and an aqueous solution of sodium hydroxide; preferably, the concentration of the aqueous solution of β-cyclodextrin is 30 mg / mL. −1 The concentration of the sodium hydroxide aqueous solution is 22 mg / mL. −1 .

[0012] Furthermore, the molar ratio of HAuCl4·3H2O, sodium hydroxide, and sulfuric acid is 1:44:56.

[0013] Furthermore, the concentration of the sulfuric acid is 1 M, and the mass concentration of the ammonia solution is 2%.

[0014] Furthermore, the first incubation temperature is 37°C, and the time is 10 hours.

[0015] Furthermore, the second incubation temperature is 80°C and the time is 20 min.

[0016] Furthermore, the separation is performed by centrifugation at a speed of 10,000 rpm for 5 minutes.

[0017] The present invention also provides a dual-ligand protected electrochemiluminescent gold nanoclusters prepared by the above-mentioned dual-ligand protected electrochemiluminescent gold nanoclusters preparation method.

[0018] The present invention also provides the application of the dual-ligand protected electrochemiluminescent gold nanoclusters prepared by the above-described method in the detection of hOGG1.

[0019] This invention also provides a method for fabricating an hOGG1 ECL sensor, comprising the following steps:

[0020] Probe: DNA1 and DNA2 are mixed in hybridization buffer to perform DNA hybridization, resulting in DNA1 / DNA2 hybridization product dsDNAs probe; the dsDNAs probe is added to streptavidin-modified magnetic beads, and the interaction between the biotin in the DNA1 / DNA2 hybridization product dsDNAs probe (i.e., biotin in DNA1) and streptavidin forms an MB / dsDNA probe.

[0021] Electrode and its pre-oxidation: A gold nanocluster solution was coated on the surface of the pretreated GCE electrode to obtain gold nanocluster / GCE; then the gold nanocluster / GCE was pre-oxidized under continuous potential pulse conditions.

[0022] The gold nanocluster solution is a suspension formed by dispersing the dual-ligand protected electrochemiluminescent gold nanoclusters obtained by the above-mentioned method in ultrapure water.

[0023] Further, the sequence of DNA1 is shown in SEQ ID No. 1, and the sequence of its complementary DNA2 is shown in SEQ ID No. 2. Cleavage sites are underlined.

[0024] Furthermore, the hybridization buffer consists of 10 mM Tris, 1.0 mM EDTA and 1.0 M NaCl, with a pH of 7.4.

[0025] Furthermore, the DNA hybridization was performed at a temperature of 37°C for 30 minutes.

[0026] Furthermore, the concentration of the dsDNAs is 10 μM.

[0027] Furthermore, the volume ratio of the dsDNAs to the streptavidin-modified magnetic beads is 1:19.

[0028] Furthermore, the interaction reaction between the biotin and the streptavidin is carried out at room temperature for 30 minutes.

[0029] Furthermore, the surface pretreatment of the GCE electrode includes: polishing the GCE electrode with Al2O3 slurry, and then ultrasonically treating it sequentially with distilled water and ethanol; preferably, the ultrasonic treatment with distilled water takes 3 minutes and the ultrasonic treatment with ethanol takes 3 minutes.

[0030] Furthermore, the concentration of the gold nanocluster solution is 1 mg / mL. −1 The volume of the gold nanocluster solution coated on the surface of the pretreated GCE electrode was 10 μL.

[0031] Furthermore, the pre-oxidation conditions are as follows: the signal probe gold nanoclusters in the gold nanoclusters / GCE are subjected to a continuous potential pulse of 1.0 V for 60 s in 0.1 M PBS containing 0.12 M TEA and pH=7.4.

[0032] The present invention also provides the application of the hOGG1 ECL sensor prepared by the above-mentioned method in the screening of inhibitors or in monitoring the hOGG1 activity of cells at the single-cell level.

[0033] The present invention also provides a method for using the hOGG1 ECL sensor prepared by the above-described method to monitor hOGG1 activity, comprising the following steps:

[0034] After the MB / dsDNA probe was incubated once with the reaction solution and hOGG1 solution, the first supernatant was removed by magnetic separation. The MB / dsDNA probe, washed with buffer, was then incubated a second time with buffer containing HpaII restriction endonuclease. After a second magnetic separation, the second supernatant was added to the pre-oxidized gold nanoclusters / GCE electrode for a third incubation. ECL measurement was performed after pre-oxidation under continuous potential pulse conditions. The ECL signal was recorded by an ECL analyzer using a step-pulse method.

[0035] Furthermore, the pre-oxidation conditions after three incubations are the same as those for the gold nanoclusters / GCE pre-oxidation.

[0036] Furthermore, the test conditions for the step pulse method are: 5 seconds at an initial potential of 0 V and 1 second at a final potential of 1.2 V.

[0037] Furthermore, the ECL measurement was performed in a 100 mM TEA + PBS solution at pH 7.4.

[0038] Furthermore, the reaction solution comprises 160 μM SAM, 2.0 μL 1×NEBuffer 2, 50 mM NaCl, 10 mM Tris−HCl, 10 mM MgCl2 and 1 mM DTT.

[0039] Furthermore, the volume ratio of the MB / dsDNA probe to the reaction solution is 1:2.

[0040] Furthermore, the incubation temperature is 37°C and the time is 2 hours.

[0041] Furthermore, the buffer solution is 1× CutSmart.

[0042] Furthermore, the concentration of HpaII restriction endonuclease in the buffer solution containing HpaII is 80 U / mL. −1 .

[0043] Furthermore, the secondary incubation temperature is 37°C, and the time is 2 hours.

[0044] Furthermore, the three incubations were performed at a temperature of 37°C for 50 minutes.

[0045] Furthermore, the volume ratio of the MB / dsDNA probe, the buffer solution, and the buffer solution containing HpaII restriction endonuclease is 5:1:1.

[0046] The hOGG1 ECL sensor provided by this invention employs dual-ligand protected electrochemiluminescent gold nanoclusters (Met / β-CD-Au NCs) as the ECL luminescent agent and TEA (triethylamine) as a co-reactant. The Met / β-CD-Au NCs can generate a significant ECL emission signal. In the presence of hOGG1, the MB / dsDNA probe is digested by the restriction endonuclease HpaII, leading to the release of Fc from the magnetic beads. β-CD (i.e., β-cyclodextrin) specifically recognizes the released Fc through guest-host interactions, thereby quenching the ECL signal. The constructed sensing platform can sensitively and selectively detect hOGG1 by monitoring changes in ECL intensity (sensitive detection of hOGG1 is crucial for clinical diagnosis). The hOGG1 ECL sensor provided by this invention uses dual-ligand protected electrochemiluminescent gold nanoclusters (Met / β-CD-Au NCs) as the ECL luminescent agent, which can sensitively and selectively detect hOGG1 in human serum.

[0047] The present invention has the following advantages:

[0048] 1. The present invention provides a one-step method for synthesizing water-soluble, dual-ligand-protected electrochemiluminescent gold nanoclusters using methionine and β-cyclodextrin as dual ligands. This method is environmentally friendly, simple and quick to operate, and has good reproducibility. The water-soluble, dual-ligand-protected electrochemiluminescent gold nanoclusters prepared by this method have good biocompatibility and exhibit high electrochemiluminescence efficiency and fully passivated nanomaterial properties. In particular, when used for the detection of hOGG1, it has higher sensitivity compared with existing technologies.

[0049] 2. The preparation method of the hOGG1 ECL sensor provided by this invention, on the one hand, achieves excellent near-infrared ECL emission performance by using specific gold nanoclusters as ECL emitting agents and triethylamine (TEA) as a co-reactant; on the other hand, it further improves the ECL efficiency of gold nanoclusters by utilizing a pre-oxidation strategy. When the hOGG1 ECL sensor using metal nanoclusters is applied to the detection of human 8-oxoguanine DNA glycosylase (hOGG1), it exhibits good sensing potential: it can detect molecules in the range of 0.0005~5 U / mL. −1 Sensitive detection of hOGG1 within the specified range, with a detection limit of 1.8 × 10⁻⁶. −4 U mL −1 In modern bioanalysis, this method offers a new perspective for improving ECL response by constructing a Met / β-CD-Au NCs / TEA system. Furthermore, this sensor is simple to operate, exhibits good repeatability, and has significant scientific and practical value for early clinical diagnosis. Attached Figure Description

[0050] Figure 1 This is a high-magnification transmission electron microscope image of Met / β-CD-Au NCs prepared in the embodiments of the present invention.

[0051] Figure 2 This is a histogram showing the size distribution of Met / β-CD-Au NCs prepared in this invention.

[0052] Figure 3 The UV absorption and fluorescence spectra of Met / β-CD-Au NCs prepared in this invention are shown.

[0053] Figure 4 The image shows the fluorescence lifetime curves of the Met / β-CD-Au NCs prepared in this invention.

[0054] Figure 5 The image shows the electrochemiluminescence spectrum of the Met / β-CD-Au NCs prepared in this invention.

[0055] Figure 6 The image shows the FT-IR spectrum of the Met / β-CD-Au NCs prepared in this invention.

[0056] Figure 7 The image shows the XPS spectrum of the Met / β-CD-Au NCs prepared in this invention.

[0057] Figure 8 The images show the electrochemiluminescence intensity-potential diagrams of the gold nanoclusters / GCE prepared in this invention and the pre-oxidized gold nanoclusters / GCE.

[0058] Figure 9 This is the electrochemiluminescence response curve of the hOGG1 ECL sensor assembly process in this invention.

[0059] Figure 10 This is the electrochemical impedance spectroscopy curve of the hOGG1 ECL sensor assembly process in this embodiment of the invention.

[0060] Figure 11 The hOGG1 ECL sensor prepared in this invention is suitable for applications ranging from 0.0005 to 5 U / mL. −1 Electrochemiluminescence response curve of hOGG1.

[0061] Figure 12 The hOGG1 ECL sensor prepared in this invention is suitable for applications ranging from 0.0005 to 5 U / mL. −1 The linear calibration curve of hOGG1.

[0062] Figure 13 This is a selectivity test diagram of the hOGG1 ECL sensor prepared in this embodiment of the invention.

[0063] Figure 14 This is a stability test diagram of the hOGG1 ECL sensor prepared in this embodiment of the invention.

[0064] Figure 15 This is a repeatability test diagram of the hOGG1 ECL sensor prepared in this embodiment of the invention.

[0065] Figure 16 A process flow diagram of the fabrication process of the hOGG1 ECL sensor provided for the implementation of this invention. Detailed Implementation

[0066] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Those skilled in the art can make non-essential improvements and adjustments to this invention based on the above description.

[0067] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0068] S-adenosyl-L-methionine (SAM) and streptavidin-modified magnetic beads were purchased from New England Biolabs. Oligonucleotides (DNA1 and DNA2) were synthesized using bioengineering techniques. The DNA1 sequence, as analyzed by [Company Name] (Shanghai, China), is shown in SEQ ID No. 1, and the complementary sequence of DNA2 is shown in SEQ ID No. 2. Cleavage sites are underlined.

[0069] Example

[0070] This embodiment provides an hOGG1 ECL sensor, and its fabrication process flow diagram is shown below. Figure 16 As shown, the specific steps include the following:

[0071] Dual-ligand protected electrochemiluminescent gold nanoclusters

[0072] Under stirring conditions, 0.5 mL of 12.5 mM HAuCl4·3H2O was mixed with 2 mL of 30 mg / mL HAuCl4·3H2O. −1 Mix with methionine, then add 1 mL of a 30 mg / mL solution. −1 β-cyclodextrin, and 0.5 mL of 22 mg / mL solution. −1 The sodium hydroxide solution was added, and then the mixture was incubated in an oven at 37°C for 10 h. Then, 0.35 mL of 1 M sulfuric acid was added to obtain gold nanoclusters precipitate.

[0073] The above-mentioned gold nanoclusters were dissolved in 3 mL of 2% ammonia solution, and then incubated for 20 min in an oven at 80 °C. After centrifugation at 10,000 rpm for 5 min, dual-ligand protected electrochemiluminescent gold nanoclusters (Met / β-CD-Au NCs) were obtained.

[0074] The electrochemiluminescent gold nanoclusters protected by the above-prepared dual ligands were subjected to the following tests:

[0075] High-magnification transmission electron microscope (TEM) images obtained using an HT 7700 transmission electron microscope are shown below. Figure 1 As shown, by Figure 1 It can be seen that the particle size of Met / β-CD-Au NCs is uniform;

[0076] exist Figure 1 Based on the obtained data, a histogram of the size distribution of gold nanoclusters was constructed, with size as the x-axis and the percentage of each size as the y-axis, as shown below. Figure 2 As shown. By Figure 2It can be seen that the Met / β-CD-Au NCs have a uniform particle size of 2.7±0.3 nm and exhibit a monodisperse distribution.

[0077] Figure 3 The UV absorption and fluorescence spectra of Met / β-CD-Au NCs are shown below. Figure 3 It can be seen that in the ultraviolet range, the absorption spectrum of Met / β-CD-Au NCs decreases significantly with increasing wavelength. The fluorescence emission spectrum of Met / β-CD-Au NCs shows a strong peak at 611 nm and a weak peak at 825 nm.

[0078] Figure 4 These are the fluorescence lifetime curves of Met / β-CD-Au NCs fitted using an exponential model. Figure 4 It can be seen that the fluorescence lifetime of Met / β-CD-Au NCs is 148.44 ns at the emission wavelength of 611 nm.

[0079] Figure 5 The electrochemiluminescence spectrum of Met / β-CD-Au NCs is shown. The electrochemiluminescence spectrum at 816 nm is similar to that of NCs. Figure 3 Compared to the fluorescence spectrum of the original, Met / β-CD-Au NCs exhibit a redshift of 205 nm.

[0080] To further demonstrate the formation of Met / β-CD-Au NCs, FT-IR spectra were obtained. The FT-IR spectra of Met / β-CD-Au NCs are as follows: Figure 6 As shown, by Figure 6 It can be seen that -SH is at 2062 cm −1 The disappearance of the weak stretching vibration at the point confirms the formation of the Au−S bond.

[0081] Figure 7 XPS spectra of Met / β-CD-Au NCs, from Figure 7 It can be seen that Met / β-CD-Au NCs are mainly composed of Au, S, C, N and O. The high-resolution XPS of Au is shown in Au 4f. 7 / 2 (84.1 eV) and Au 4f 5 / 2 (87.8 eV)( Figure 7 The illustration shows two strong peaks, indicating that Au(I) and Au(0) metallic states coexist within Au NCs.

[0082] The above Figures 1-7 The results show that this embodiment successfully prepared Met / β-CD-Au NCs protected by methionine and β-cyclodextrin dual ligands.

[0083] hOGG1 ECL sensor

[0084] Probe: The sequence is 5′-CAG T CC GG A GGT G-biotin-3′ DNA1, whose complementary sequence is 5′-CAC CT. C CGG DNA2 of ACT G-Fc-3′ (cutting sites are underlined) was mixed in hybridization buffer (containing 10 mM Tris, 1.0 mM EDTA and 1.0 M NaCl, pH 7.4) and hybridized at 37 °C for 30 min to obtain DNA1 / DNA2 hybridization products dsDNAs; 10 μL of the above dsDNAs at a concentration of 10 μM was added to 190 μL of streptavidin-modified magnetic beads, and the biotin in the DNA1 / DNA2 hybridization product reacted with streptavidin at room temperature for 30 min to form MB / dsDNA probes;

[0085] Electrode Coating: The GCE electrode was polished with Al2O3 slurry, and then ultrasonically treated with distilled water and ethanol for 3 min each, respectively. A 1 mg / mL solution was then coated onto the surface of the GCE electrode after the above surface pretreatment. −1 A solution of gold nanoclusters (obtained by dissolving the above-mentioned dual-ligand protected electrochemiluminescent gold nanoclusters in ultrapure water) was prepared to obtain gold nanoclusters / GCE.

[0086] Pre-oxidation: The above gold nanoclusters / GCE were pre-oxidized in 0.1 M pH 7.4 PBS (containing 0.12 M TEA) under a continuous potential pulse of 1.0 V for 60 s.

[0087] The hOGG1 ECL sensor described above is used to detect hOGG1 according to the following method:

[0088] 10 μL of the above-mentioned MB / dsDNA probe was incubated with 20 μL of reaction solution (containing 160 μM SAM, 2.0 μL 1×NEBuffer 2 (50 mM NaCl, 10 mM Tris−HCl, 10 mM MgCl2, 1 mM DTT) and different concentrations of hOGG1 solution at 37°C for 2 h. After incubation, the first supernatant was removed by magnetic separation. The MB / dsDNA probe, washed with 2 μL of 1×CutSmart buffer, was then incubated with 2 μL of 80 U / mL solution. -1The buffer containing HpaII restriction endonuclease was incubated twice at 37°C for 2 h. After two magnetic separations, the second supernatant was added to the pre-oxidized gold nanoclusters / GCE electrode and incubated three times for 50 min. Pre-oxidation was then performed under continuous potential pulse conditions (in 0.1 M pH 7.4 PBS (containing 0.12 M TEA) with a continuous potential pulse of 1.0 V for 60 s) before ECL measurement. The ECL measurement was performed in 100 mM TEA + PBS solution (pH=7.4). The ECL signal was measured at an initial potential of 0 V for 5 s and a final potential of 1.2 V for 1 s.

[0089] DNA1 / DNA2 consists of two complementary dsDNA strands. DNA1 assembles onto streptavidin-coated magnetic beads (MBs) via a specific streptavidin-biotin interaction, subsequently hybridizing with DNA2 to form a dsDNA probe containing a specific hOGG1 recognition sequence. In the presence of the target, the CpG dinucleotide site at 5′-CCGG-3′ cannot be methylated, leading to the digestion of the dsDNA probe by hOGG1. After magnetic separation, β-CD recognizes ferrocene (Fc) released from the MB via guest-host interactions, quenching ECL emission.

[0090] Figure 8 The graphs show the electrochemiluminescence intensity-potential of gold nanoclusters / GCE and pre-oxidized gold nanoclusters / GCE. Figure 8 It can be seen that the strength of the pre-oxidized gold nanoclusters / GCE (red) ECL is significantly improved.

[0091] Figure 9 The electrochemiluminescence response curves during the assembly process of the hOGG1 ECL sensor are shown below. Figure 9 As shown, bare GCE did not exhibit a significant electrochemiluminescence signal (curve a). The ECL release of Ox-Met / β-CD-Au NCs-TEA / GCE reached its maximum (curve b). Pre-oxidation (Ox-Fc / Met / β-CD-Au NCs-TEA / GCE) after incubation of the supernatant from the reaction with hOGG1 with Met / β-CD-Au NCs / GCE (curve c) resulted in a significantly lower ECL signal compared to Ox-Met / β-CD-Au NCs-TEA / GCE. These results demonstrate the successful fabrication of the biosensor.

[0092] Figure 10 The electrochemical impedance spectroscopy curves for the hOGG1 ECL sensor assembly process are shown below. Figure 10 As shown, the charge transfer resistance (R) of Met / β-CD-Au NCs / GCEct The R0 of Met / β-CD-Au NCs is close to that of bare GCE (121 Ω), indicating that Met / β-CD-Au NCs have good conductivity and a large specific surface area. Meanwhile, the R0 of Ox−Fc / Met / β−CD−Au NCs-TEA / GCE is much smaller. ct The value increased to 9.08 kΩ (curve c), further confirming that Fc was added to the electrode. The change in electrode impedance corresponds to the assembly process of the biosensor, indicating that the electrode assembly was successful.

[0093] Figure 11 The hOGG1 ECL sensor is suitable for applications ranging from 0.0005 to 5 U / mL. −1 The electrochemiluminescence response curve of hOGG1 is shown below. Figure 11 As shown, from 0.0005 U / mL −1 Up to 5 U mL −1 As the concentration of hOGG1 increases, the response of ECL gradually decreases. Figure 12 The hOGG1 ECL sensor is suitable for applications ranging from 0.0005 to 5 U / mL. −1 The linear calibration curve of hOGG1, such as Figure 12 As shown, the ECL signal exhibits a very strong linear correlation with the logarithm of hOGG1 concentration, and the corresponding linear regression equation is I. ECL =0.2467 – 0.2179lg C(R 2 = 0.9955), the resulting LOD is 1.8 × 10 −4 U mL −1 The obtained biosensor enables sensitive detection of hOGG1.

[0094] Figure 13 The selective test diagram for the hOGG1 ECL sensor is shown below. Figure 13 As shown, the ECL intensity reduction was minimal in both interfering and blank samples, while the target hOGG1 caused a significant reduction in ECL intensity. Furthermore, the ECL response to hOGG1 was comparable to that observed in a mixture of hOGG1 and potential interfering substances, further demonstrating the high selectivity of this biosensor.

[0095] Figure 14 The stability test results for the hOGG1 ECL sensor are shown below. Figure 14 It can be seen that this biosensor can detect 0.01 U / mL by continuously stepping at a potential from 0 to 1 V for 12 cycles. −1 The RSD of hOGG1 is 2.12%, indicating that it has good operational stability.

[0096] Figure 15This is a repeatability test graph for the hOGG1 ECL sensor, specifically obtained by measuring 1 U mL. −1 hOGG1 is used to assess reproducibility. For example... Figure 15 It was found that there were no significant differences among the seven electrodes, with an RSD as low as 2.35% (1 U mL). −1 This indicates that the designed biosensor has good reproducibility.

[0097] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing electrochemiluminescent gold nanoclusters protected by dual ligands, characterized in that, The process includes the following steps: Under stirring conditions, a mixed solution of β-cyclodextrin and sodium hydroxide is added to a mixed aqueous solution of HAuCl4·3H2O and methionine, followed by a first incubation, and then sulfuric acid is added to obtain gold nanoclusters precipitate. The gold nanoclusters were precipitated and dissolved in an ammonia solution, and then subjected to a second incubation. After separation, dual-ligand protected electrochemiluminescent gold nanoclusters were obtained. The molar ratio of HAuCl4·3H2O, methionine, and β-cyclodextrin is 1:64:4.

16. The molar ratio of HAuCl4·3H2O, sodium hydroxide, and sulfuric acid is 1:44:56; The first incubation temperature is 37°C; The second incubation temperature is 80℃; The mass concentration of the ammonia solution is 2%.

2. The preparation method according to claim 1, characterized in that, The mixed aqueous solution of HAuCl4·3H2O and methionine was obtained by mixing an aqueous solution of HAuCl4·3H2O with an aqueous solution of methionine; the concentration of the aqueous solution of HAuCl4·3H2O was 12.5 mM, and the concentration of the aqueous solution of methionine was 30 mg / mL; and / or The β-cyclodextrin and sodium hydroxide mixed solution is obtained by mixing an aqueous solution of β-cyclodextrin and an aqueous solution of sodium hydroxide; the concentration of the aqueous solution of β-cyclodextrin is 30 mg / mL and the concentration of the aqueous solution of sodium hydroxide is 22 mg / mL.

3. The preparation method according to claim 1, characterized in that, The concentration of the sulfuric acid is 1 M.

4. The application of the dual-ligand protected electrochemiluminescent gold nanoclusters prepared by the method of any one of claims 1-3 in the detection of hOGG1.

5. A method for fabricating an hOGG1 ECL sensor, characterized in that, The process includes the following steps: Probe: DNA1 and DNA2 are mixed in hybridization buffer to perform DNA hybridization, yielding DNA1 / DNA2 hybridization products dsDNAs; the dsDNAs are added to streptavidin-modified magnetic beads, and an MB / dsDNA probe is formed through the interaction between biotin in the dsDNAs and streptavidin; the DNA1 sequence is 5′-CAG TCC GGA GGT G-biotin-3′, and the DNA2 sequence is 5′-CAC CTC CGG ACT G-Fc-3′; Electrode and its pre-oxidation: A gold nanocluster solution was coated on the surface of the pretreated GCE electrode to obtain gold nanocluster / GCE; then the gold nanocluster / GCE was pre-oxidized under continuous potential pulse conditions. The gold nanocluster solution is a suspension formed by dispersing the dual-ligand protected electrochemiluminescent gold nanoclusters obtained by the preparation method of dual-ligand protected electrochemiluminescent gold nanoclusters according to any one of claims 1-3 in ultrapure water.

6. The method for fabricating the hOGG1 ECL sensor as described in claim 5, characterized in that, The hybridization buffer consists of 10 mM Tris, 1.0 mM EDTA, and 1.0 M NaCl, with a pH of 7.4; and / or The DNA hybridization was performed at a temperature of 37°C for 30 minutes; and / or The volume ratio of the dsDNAs to the streptavidin-modified magnetic beads is 1:19; and / or The interaction reaction between biotin and streptavidin was carried out at room temperature for 30 minutes.

7. The method for fabricating the hOGG1 ECL sensor as described in claim 5, characterized in that, The surface pretreatment of the GCE electrode includes: polishing the GCE electrode with Al2O3 slurry, followed by ultrasonic treatment with distilled water and ethanol in sequence; and / or The concentration of the gold nanocluster solution is 1 mg / mL; and / or The pre-oxidation conditions are as follows: the signal probe gold nanoclusters in the gold nanoclusters / GCE are pulsed at a continuous potential of 1.0 V for 60 s in 0.1 M PBS containing 0.12 M TEA and pH=7.

4.

8. The application of the hOGG1 ECL sensor prepared by the method of any one of claims 5-7 in monitoring cellular hOGG1 activity at the single-cell level.

9. A method for using the hOGG1 ECL sensor prepared by the method of any one of claims 5-7 to monitor hOGG1 activity, characterized in that, Includes the following steps: After the MB / dsDNA probe was incubated once with the reaction solution and hOGG1 solution, the first supernatant was removed by magnetic separation. The MB / dsDNA probe, washed with buffer, was incubated a second time with buffer containing HpaII restriction endonuclease. After two magnetic separations, the second supernatant was added to the pre-oxidized gold nanoclusters / GCE electrode for a third incubation. ECL measurement was performed after pre-oxidation under continuous potential pulse conditions.

10. The method of use as described in claim 9, characterized in that, The test conditions for the ECL signal during ECL measurement are: 5 s at an initial potential of 0 V, 1 s at a final potential of 1.2 V; and / or ECL measurements were performed in a 100 mM TEA + PBS solution at pH 7.4; and / or The volume ratio of the MB / dsDNA probe to the reaction solution is 1:2; and / or The incubation temperature was 37°C, and the time was 2 hours; and / or The concentration of HpaII restriction endonuclease in the buffer solution is 80 U / mL; and / or The secondary incubation was carried out at a temperature of 37°C for 2 hours; and / or The three incubations were performed at a temperature of 37°C for 50 minutes; and / or The volume ratio of the MB / dsDNA probe, washing buffer, and buffer containing HpaII restriction endonuclease is 5:1:1.

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