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CN-121975520-A - Construction method of luminescence enhanced long afterglow nano probe and application of luminescence enhanced long afterglow nano probe in beta-hexa selective detection

CN121975520ACN 121975520 ACN121975520 ACN 121975520ACN-121975520-A

Abstract

The invention discloses a construction method of a luminescence enhanced long afterglow nano probe and application thereof in selective detection of beta-hexa, the construction method comprises the specific steps of S1 preparing long afterglow nano particles ZGOM through a hydrothermal synthesis method, and carrying out surface modification on the long afterglow nano particles to obtain ZGOM-NH 2 , S2 dissolving HaloTag ligand in 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, stirring at room temperature overnight, activating the ligand to form an active ester intermediate, and then adding ZGOM-NH 2 for reaction to obtain the long afterglow nano probe ZGOM@L. The ZGOM@L formed by surface modification of the traditional long afterglow material ZGOM has enhanced afterglow strength, and avoids interference of complex matrix background, so that detection sensitivity and selectivity in beta-hexa-selective detection application are improved.

Inventors

  • YAN XIUPING
  • XU QIN
  • ZHAO XU
  • CHEN LIJIAN

Assignees

  • 江南大学

Dates

Publication Date
20260505
Application Date
20251223

Claims (10)

  1. 1. The construction method of the luminescence enhanced long afterglow nano probe is characterized by comprising the following specific steps: s1, preparing long afterglow nano particles ZGOM by a hydrothermal synthesis method, and carrying out surface modification on the long afterglow nano particles to obtain ZGOM-NH 2 ; S2, dissolving a HaloTag ligand in 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, stirring overnight at room temperature, activating the ligand to form an active ester intermediate, and then adding ZGOM-NH 2 to react to prepare the long afterglow nano probe ZGOM@L.
  2. 2. The method for constructing the luminescence enhanced long persistence nanoprobe according to claim 1, wherein the specific steps of the step S1 are as follows: s11, dissolving Zn 2+ salt and Mn 2+ salt in pure water, and adding strong acid into the mixture while stirring to form a precursor solution; S12, dissolving GeO 2 in alkaline solution, adding the alkaline solution into the precursor solution in the step S11, regulating the pH to 8-10, stirring and mixing uniformly at room temperature, transferring the mixed solution into a high-pressure reaction kettle with a polytetrafluoroethylene lining, performing hydrothermal reaction, repeatedly washing the obtained product with ultrapure water for multiple times, and drying in vacuum to obtain long-afterglow nano particles Zn 2 GeO 4 :Mn which is marked as ZGOM; S13, dispersing long afterglow nano particles ZGOM in N, N-Dimethylformamide (DMF) by ultrasonic, dropwise adding APTES under stirring for reaction, and after the reaction is finished, centrifugally separating a product, washing with DMF and drying in vacuum to obtain ZGOM-NH 2 .
  3. 3. The method for constructing the luminescence enhanced long persistence nanoprobe according to claim 1, wherein the specific steps of the step S2 are as follows: S21, firstly, dissolving a HaloTag ligand in DMF, adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, and stirring overnight at room temperature to activate the ligand to form an active ester intermediate solution; s22, adding ZGOM-NH 2 and N, N-diisopropylethylamine into the active ester intermediate solution, carrying out ultrasonic treatment, and reacting for 12h under vigorous stirring; and S23, after the reaction is finished, centrifugally collecting a product, washing the product with ethanol and high-purity water for multiple times in sequence, and finally, freeze-drying the product to obtain a final product ZGOM@L.
  4. 4. The method for constructing the luminescence enhanced long persistence nanoprobe according to claim 2, wherein in the step S11, the molar ratio of Zn 2+ salt to Mn 2+ salt is 1:0.0025, both Zn 2+ salt and Mn 2+ salt are nitrates, and the strong acid is nitric acid, i.e. HNO 3 is added into Zn 2+ salt and Mn 2+ salt to form a precursor solution.
  5. 5. The method for constructing the luminescence enhanced long persistence nanoprobe according to claim 4, wherein in the step S12, geO 2 is dissolved in NaOH solution to prepare Na 2 GeO 3 solution of 0.5-1M, 1mmol of Na 2 GeO 3 solution is added into the precursor solution drop by drop, the pH is regulated to 9.0 by 28wt% of NH 3 ·H 2 O, the mixture is stirred for 1h at room temperature, the hydrothermal reaction is carried out at 220 ℃ for 6-8h, the temperature of vacuum freeze drying of the product is-40 ℃, the vacuum degree is 10Pa, and the time is 6-8h.
  6. 6. The method for constructing the luminescence enhanced long persistence nanoprobe according to claim 3, wherein, The ratio of HaloTag ligand to DMF in step S21 is 1mg to 1mL, and the mass ratio of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride to N-hydroxysuccinimide is 2 to 1; The ratio of ZGOM-NH 2 to N, N-Diisopropylethylamine (DIPEA) added in the step S22 is 1mg to 0.86 mu L; The product was collected by centrifugation at 13000 Xg for 10min in step S23, the temperature of the freeze-drying was-40℃and the time was 6-8h.
  7. 7. An application of a luminous enhanced long afterglow nano probe in selective detection of beta-hexa.
  8. 8. The use of the luminescence enhanced long persistence nanoprobe according to claim 7 in the selective detection of β -hexa, wherein the specific steps of the use are: S3, expressing and purifying LinB protein to obtain purified LinB enzyme solution; S4, modifying the HaloTag protein at the bottom of the pore plate to obtain a HaloTag protein detection plate; s5, detecting beta-hexa by adopting a HaloTag protein detection plate, wherein the detection method specifically comprises the following steps: S51, firstly, mixing ZGOM@L probe suspension and beta-HCH standard substances with different concentrations in HEPES buffer solution, wherein the pH value is 8.5, and preparing working solution; s52, adding the working solution into wells of a HaloTag protein detection plate, and adding LinB enzyme solution into each well, wherein the concentration of LinB enzyme solution is 2-6 mug.mu.L- 1 , dissolving the solution in 10mM HEPES buffer solution to make the final reaction volume of each well be 200 mu.L, wherein the final concentration of LinB enzyme solution is 1-3 mug.mu.L- 1 , and the final concentration of ZGOM@L is 0.25 mug.mu.L- 1 ; S53, incubating the reaction plate at room temperature for 2 hours, absorbing liquid in the hole after incubation, washing 2 times by using PBST, washing 2 times by using PBS to remove unreacted ZGOM@L probes, adding PBS into each hole, and detecting the long afterglow luminous intensity by using a multifunctional enzyme-labeled instrument.
  9. 9. The use of the luminescence enhanced long persistence nanoprobe according to claim 8 in the selective detection of β -hexa, wherein the specific steps of step S3 are as follows: S31, inoculating the transformed strain to a fresh LB culture medium, and adding IPTG at 37 ℃ to perform induction expression to obtain LinB protein with a hexahistidine tag; S32, after induction, centrifugally collecting thalli, re-suspending in a lysis buffer solution, adding a protease inhibitor, and carrying out ultrasonic disruption after suspending for 30 minutes; s33, loading the sample obtained by incubation on a gravity chromatographic column the next day, washing the non-specific adsorption protein with 12 times of column volume of washing buffer, and eluting the target protein with 20 times of column volume of high-concentration imidazole elution buffer; S34, collecting and eluting target protein, analyzing purity by SDS-PAGE, combining components containing purified LinB protein, washing with PBS buffer solution with pH value of 7.4, concentrating and split charging, and preserving at-80 ℃ for a long time to finally obtain purified LinB enzyme solution.
  10. 10. The use of the luminescence enhanced long persistence nanoprobe according to claim 8 in the selective detection of β -hexa, wherein the specific steps of step S4 are as follows: s41, adding a protein G solution into each well of a 96-well high-binding ELISA plate, and coating at 4 ℃ overnight; s42, after the coating is finished, washing 3 times by using PBST, and washing 2 times by using PBS; s43, adding PBS solution containing 3% (w/v) bovine serum albumin into each hole, sealing for 1 hour at room temperature, and washing again; S44, adding an anti-His antibody into each well, diluting with PBS at a ratio of 1:1000, incubating at 4 ℃ for overnight, adding 100 mu L of HaloTag protein solution into each well after washing, incubating for 2 hours at room temperature, and finally washing 2 times by using PBST, and washing 2 times by using PBS to prepare the HaloTag protein detection plate.

Description

Construction method of luminescence enhanced long afterglow nano probe and application of luminescence enhanced long afterglow nano probe in beta-hexa selective detection Technical Field The invention relates to the technical field of nano probes, in particular to a construction method of a luminescence enhanced long afterglow nano probe and application of the luminescence enhanced long afterglow nano probe in beta-hexa selective detection. Background Hexakis (Hexachlorocyclohexane, HCH), an organochlorine pesticide (OCP) that has been widely used, belongs to a Persistent Organic Pollutant (POP) that remains long-term after long-term agricultural application and continuously pollutes the global ecosystem. Due to the high stability, the hexakis-hexakis and isomers thereof are difficult to degrade in the environment, so that the hexakis-hexakis and isomers thereof can widely migrate and accumulate in water, soil, food chains and even human tissues and have biological amplification effects. The lipophilicity of hexakis makes it easy to biologically enrich in adipose tissue and biological fluids, and it has been demonstrated to have carcinogenic, teratogenic and endocrine disrupting properties, which pose a significant threat to human health. Although the implementation of the "Stockholm convention" has been disabled for six and six in 2009, the problems of environmental residue and illegal use still remain. Among the various isomers of hexakis, beta-hexakis (beta-HCH) exhibits the highest toxicity and environmental persistence. The outstanding stability of the isomer is derived from the fact that all chlorine atoms are in a flat bond configuration on a cyclohexane ring, so that the isomer is endowed with extremely strong degradation resistance. Furthermore, β -hexakis has a stronger tendency to accumulate in adipose tissue (10-30 times higher than other isoforms) and slower elimination kinetics (5 times slower elimination rate). More importantly, α -hexakis and γ -hexakis can be converted into the more thermodynamically stable β -isomer during the metabolic process, making it the ultimate metabolic destination of the hexakis isomer in the environment. Thus, β -hexakis is the major and often the only detectable isomer that is long-term in biological samples, and early studies have reported the detection of only β -hexakis in breast milk. Therefore, developing a high-sensitivity monitoring method specific to beta-hexa is important to realizing accurate risk assessment. In view of the significant risk of β -hexakis, it is critical to effectively monitor it. Currently, reliable detection methods rely mainly on laboratory techniques such as gas chromatography-mass spectrometry (GC-MS). Emerging sensing technologies such as electrochemical and colorimetric sensing technologies. However, the existing gas chromatography-mass spectrometry (GC-MS) has high detection sensitivity, but the sample pretreatment steps are complicated, special operation is needed, equipment is expensive, and quick and on-site analysis is difficult to realize. While emerging sensing technologies, such as electrochemical and colorimetric sensing technologies, are designed to address the above-mentioned deficiencies, in-situ detection with the premise of one-trillion detection sensitivity and high selectivity still presents significant challenges. Therefore, there is an urgent need to develop new detection strategies that combine high sensitivity, rapidity and portability characteristics. The long afterglow material is a material which absorbs energy such as visible light, ultraviolet light, X rays and the like and stores the energy, can still emit light after the energy is cut off, and can also be called as a light storage type luminescent material or a noctilucent material. Long persistence materials can store energy in traps, with continued luminescence after cessation of excitation of the material. Due to the remarkable characteristics, the long afterglow material starts to be applied in the fields of illumination, emergency indication, light energy storage, detection, traffic, military and the like. Therefore, the research and development of the novel molecular probe based on the near infrared luminescence long afterglow nanomaterial has wide application prospect in fluorescent environment detection analysis. Therefore, the invention provides a construction method of the luminescence enhanced long afterglow nano probe and application of the luminescence enhanced long afterglow nano probe in beta-hexa-selective detection, the anti-interference capability of the detection is improved, and the whole flow is rapid and simple, and has high sensitivity, high selectivity and rapidness and convenience. Disclosure of Invention The invention aims to provide a construction method of a luminescence enhanced long afterglow nano probe and application thereof in selective detection of beta-hexa, the purposes of improving the anti-interference capability of