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CN-120758596-B - Preparation method of double-ligand-protected electrochemiluminescence gold nanocluster and application of double-ligand-protected electrochemiluminescence gold nanocluster in detection of hOGG1

CN120758596BCN 120758596 BCN120758596 BCN 120758596BCN-120758596-B

Abstract

The invention relates to a preparation method of a double-ligand protected electrochemiluminescence gold nanocluster and application thereof in detecting hOGG1, wherein the electrochemiluminescence gold nanocluster is synthesized by taking methionine and beta-cyclodextrin as double ligands by a one-step method, and the obtained water-soluble electrochemiluminescence gold nanocluster has good biocompatibility, higher electrochemiluminescence efficiency and completely passivated nanomaterial performance. When the hOGG1 ECL sensor using the specific electrochemiluminescence gold nanocluster as an ECL luminophor and Triethylamine (TEA) as a coreactant is applied to detection of human 8-oxo guanine DNA glycosylase (hOGG 1), the sensor has higher sensitivity and good sensing potential. The sensor is simple to operate, good in repeatability and has important scientific significance and application value for clinical early diagnosis.

Inventors

  • YU LEI
  • ZHANG QIAO
  • SUN YUE
  • Heng Yaping
  • GUO YURONG
  • Xu Mengrui

Assignees

  • 齐鲁理工学院

Dates

Publication Date
20260508
Application Date
20250729

Claims (10)

  1. 1. The preparation method of the double-ligand-protected electrochemiluminescence gold nanocluster is characterized by comprising the following steps of adding a mixed solution of beta-cyclodextrin and sodium hydroxide into a mixed aqueous solution of HAuCl 4 ·3H 2 O and methionine under stirring, performing first incubation, and then adding sulfuric acid to obtain a gold nanocluster precipitate; The molar ratio of the HAuCl 4 ·3H 2 O, the methionine to the beta-cyclodextrin is 1:64:4.16; The molar ratio of the HAuCl 4 ·3H 2 O, the sodium hydroxide and the sulfuric acid is 1:44:56; the temperature of the first incubation is 37 ℃; The temperature of the second incubation is 80 ℃; the mass concentration of the ammonia water solution is 2%.
  2. 2. The process according to claim 1, wherein the aqueous mixture of HAuCl 4 ·3H 2 O and methionine is obtained by mixing an aqueous HAuCl 4 ·3H 2 O solution with an aqueous methionine solution, wherein the aqueous HAuCl 4 ·3H 2 O solution has a concentration of 12.5 mM and the aqueous methionine solution has a concentration of 30 mg/mL, and/or The beta-cyclodextrin and sodium hydroxide mixed solution is obtained by mixing beta-cyclodextrin aqueous solution and sodium hydroxide aqueous solution, wherein the concentration of the beta-cyclodextrin aqueous solution is 30 mg/mL, and the concentration of the sodium hydroxide aqueous solution is 22 mg/mL.
  3. 3. The method of claim 1, wherein the sulfuric acid has a concentration of 1M.
  4. 4. Use of the dual ligand protected electrochemiluminescence gold nanocluster prepared by the dual ligand protected electrochemiluminescence gold nanocluster preparation method of any one of claims 1-3 for detecting alogg 1.
  5. 5. A preparation method of the hOGG1 ECL sensor is characterized by comprising the following steps of mixing DNA1 and DNA2 in a hybridization buffer solution to obtain a DNA1/DNA2 hybridization product dsDNAs, adding dsDNAs into streptavidin-modified magnetic beads, and forming an MB/dsDNA probe through interaction reaction of biotin in dsDNAs and streptavidin, wherein the sequence of the DNA1 is 5'-CAG TCC GGA GGT G-biotin-3', and the sequence of the DNA2 is 5'-CAC CTC CGG ACT G-Fc-3'; Coating gold nanocluster solution on the surface of the GCE electrode subjected to surface pretreatment to obtain gold nanocluster/GCE, and then pre-oxidizing the gold nanocluster/GCE under the condition of continuous potential pulse; The gold nanocluster solution is a suspension formed by dispersing the double-ligand-protected electrochemiluminescence gold nanoclusters prepared by the preparation method of the double-ligand-protected electrochemiluminescence gold nanoclusters in ultrapure water according to any one of claims 1 to 3.
  6. 6. The method of claim 5, wherein the hybridization buffer has a composition of 10mM Tris, 1.0 mM EDTA, and 1.0M NaCl, a pH of 7.4, and/or The DNA hybridization temperature is 37 ℃ and the time is 30min, and/or The volume ratio of dsDNAs to streptavidin modified magnetic beads is 1:19, and/or The temperature of the interaction reaction of the biotin and the streptavidin is room temperature and the time is 30min.
  7. 7. The method of manufacturing a hOGG1 ECL sensor according to claim 5, wherein the surface pretreatment of the GCE electrode comprises polishing the GCE electrode with Al 2 O 3 slurry, followed by sequentially ultrasonic treatment with distilled water and ethanol, and/or The concentration of the gold nanocluster solution is 1mg/mL, and/or The pre-oxidation conditions were that the signaling probe gold nanoclusters in the gold nanoclusters/GCE were pulsed 60 s at a continuous potential of 1.0V in PBS containing 0.1M, 0.12M TEA, and ph=7.4.
  8. 8. Use of a shogg 1 ECL sensor prepared by the method of preparing a shogg 1 ECL sensor according to any one of claims 5-7 for monitoring cellular shogg 1 activity at a single cell level.
  9. 9. A method of using a shogg 1 ECL sensor prepared by a method of preparing a shogg 1 ECL sensor according to any one of claims 5-7 to monitor shogg 1 activity, comprising the steps of: After the MB/dsDNA probe is incubated with the reaction solution and the hOGG1 solution for one time, the first supernatant is removed through one-time magnetic separation, the MB/dsDNA probe washed by the buffer solution is incubated with the buffer solution containing HpaII restriction endonuclease for the second time, after the second magnetic separation, the second supernatant is added to the pre-oxidized gold nanocluster/GCE electrode for three times for incubation, and ECL measurement is carried out after pre-oxidation under the condition of continuous potential pulse.
  10. 10. The method of claim 9, wherein the ECL signal is tested under conditions of 5 s at an initial potential of 0V and 1 s at a final potential of 1.2V during ECL measurement, and/or ECL measurement was performed in 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 temperature of the primary incubation is 37 ℃ and the time is 2 h, and/or The concentration of HpaII restriction enzyme in the buffer solution containing HpaII restriction enzyme is 80U/mL, and/or The secondary incubation is carried out at a temperature of 37 ℃ for a time of 2 h, and/or The three incubations are at 37℃for a period of 50 min ℃and/or The volume ratio of the MB/dsDNA probe, the washing buffer solution and the buffer solution containing HpaII restriction enzyme is 5:1:1.

Description

Preparation method of double-ligand-protected electrochemiluminescence gold nanocluster and application of double-ligand-protected electrochemiluminescence gold nanocluster in detection of hOGG1 Technical Field The invention relates to the technical field of analytical chemistry and nano methods, in particular to a preparation method of an electrochemiluminescence gold nanocluster protected by methionine and beta-cyclodextrin double ligand and application of the electrochemiluminescence gold nanocluster in detection of hOGG 1. Background Electrochemical luminescence (ECL) technology combines the characteristics of electrochemistry and chemiluminescence, and has been attracting more and more attention in biosensing (immunoassay and genetic analysis strategies) application by virtue of its inherent advantages of high sensitivity, zero background signal, simple setup, good controllability, short time consumption, etc. ECL luminophores are known to play a vital role in the development and application of ECL. In addition, various types of ECL luminophores (e.g. organic small molecules, polymers and inorganic materials) have also been widely developed. Gold nanoclusters are currently widely studied ECL emission materials because of their near infrared ECL emission, easy surface modification, good biocompatibility and environmental friendliness. However, gold nanoclusters generally suffer from poor ECL efficiency due to slow charge transfer during the electro-generation process, which remains a major challenge, limiting widespread use in ECL bioassays. Therefore, researchers explore various surface modification capping agents to prepare luminescent gold nanotubes, so that ECL emission wavelength of the gold nanotubes is expanded from a visible light range to a near infrared region, and the near infrared ECL is utilized to have the advantages of large tissue penetration depth, small photochemical harm, low background interference and the like, thereby improving the detection sensitivity of targets. But still further improvements in achieving more sensitivity and the like are needed. Disclosure of Invention Aiming at the problems existing in the prior art, the invention provides a preparation method of a double-ligand-protected electrochemiluminescence gold nanocluster and application thereof in detecting hOGG1, and the water-soluble electrochemiluminescence gold nanocluster is synthesized by taking methionine and beta-cyclodextrin as double ligands by a one-step method, so that the water-soluble electrochemiluminescence gold nanocluster has good biocompatibility, higher electrochemiluminescence efficiency and completely passivated nanomaterial performance, and can be used for sensitive detection of hOGG 1. In order to achieve the above purpose, the present invention adopts the following technical scheme: A preparation method of an electrochemiluminescence gold nanocluster protected by double ligands comprises the following steps: Under the stirring condition, adding a mixed solution of beta-cyclodextrin and sodium hydroxide into a mixed aqueous solution of HAuCl 4·3H2 O and methionine, performing first incubation, and then adding sulfuric acid to obtain gold nanocluster precipitate; And dissolving the gold nanocluster precipitate in an ammonia water solution, performing second incubation, and separating to obtain the double-ligand-protected electrochemiluminescence gold nanoclusters (Met/beta-CD-Au NCs). Further, the molar ratio of HAuCl 4·3H2 O, methionine to beta-cyclodextrin is 1:64:4.16. Further, the mixed aqueous solution of HAuCl 4·3H2 O and methionine is obtained by mixing an aqueous solution of HAuCl 4·3H2 O and an aqueous solution of methionine, preferably, the concentration of the aqueous solution of HAuCl 4·3H2 O is 12.5 mM, and the concentration of the aqueous solution of methionine is 30 mg mL −1. Further, the mixed solution of the beta-cyclodextrin and the sodium hydroxide is obtained by mixing an aqueous solution of the beta-cyclodextrin with an aqueous solution of the sodium hydroxide, and preferably, the concentration of the aqueous solution of the beta-cyclodextrin is 30 mg mL −1, and the concentration of the aqueous solution of the sodium hydroxide is 22 mg mL −1. Further, the molar ratio of HAuCl 4·3H2 O, sodium hydroxide to sulfuric acid was 1:44:56. Further, the concentration of sulfuric acid is 1M, and the mass concentration of the ammonia water solution is 2%. Further, the temperature of the first incubation was 37 ℃ for a time of 10h. Further, the second incubation is at a temperature of 80 ℃ for a time of 20 min. Further, the separation is centrifugation, the rotation speed of the centrifugation is 10000 rpm, and the time is 5 min. The invention also provides the double-ligand-protected electrochemiluminescence gold nanocluster prepared by the preparation method of the double-ligand-protected electrochemiluminescence gold nanocluster. The invention also provides an application of the doubl