Search

CN-122012695-A - Signal amplification nucleic acid nano probe for detecting Abeta O and preparation method thereof

CN122012695ACN 122012695 ACN122012695 ACN 122012695ACN-122012695-A

Abstract

The invention provides a nucleic acid nano probe for detecting A beta O signal amplification, which comprises a signal output unit QDs, a metal organic framework MOFs, a CdSe/ZnS quantum dot, a DNA1 sequence, a DNA 2/DNA 1 sequence, a DNA 3/DNA 1 sequence, and a DNA1 sequence, wherein the signal output unit QDs is formed by synthesizing surface modified streptavidin modified CdSe/ZnS quantum dots through a thermosol method, the metal organic framework MOFs is formed by synthesizing porphyrin serving as an organic ligand through a hydrothermal method and serving as a carrier and simultaneously serving as a quencher of the CdSe/ZnS quantum dots by taking metal ion Zr as a center, the Aptamer sequence of the A beta O is designed according to the A beta O Aptamer sequence and is complementary with the A beta O Aptamer sequence, and the 5 'end of the DNA1 sequence is modified with a phosphorylating group, and the 3' end of the DNA1 sequence is modified with avidin. The invention also provides a preparation method of the nucleic acid nano probe for detecting the signal amplification of the Abeta O. The invention quantitatively detects Abeta O in peripheral blood and can provide a new thought and means for early diagnosis of Alzheimer's disease.

Inventors

  • CHENG XIAO
  • MA DANDAN

Assignees

  • 山西省人民医院

Dates

Publication Date
20260512
Application Date
20260210

Claims (5)

  1. 1. A signal amplified nucleic acid nanoprobe for detecting aβo, comprising: the signal output unit QDs is CdSe/ZnS quantum dots modified by surface modified streptavidin synthesized by a thermosol method; The metal organic frameworks MOFs are synthesized by a hydrothermal method by taking metal ions Zr as a center and porphyrin as an organic ligand, and are used as carriers and quenching agents of CdSe/ZnS quantum dots; Aptamer sequence of Abeta O; And a DNA1 sequence which is designed according to the Aptamer sequence of Abeta O and is complementary to the Aptamer sequence of Abeta O, wherein the 5 '-end of the DNA1 sequence is modified with a phosphorylating group, and the 3' -end of the DNA1 sequence is modified with avidin; Wherein; DNA1 sequence 5 '-GCCCCAACACAGGCTCTCTTCTCCGAGCCGGTCGA-Biotin-3' Apoligomer sequence of Abeta O5 '-PO4 3-AGAGAGCCTGTGTTGGGGCGGGTGCG-3'; the signal output units QDs are modified onto the DNA1 sequence by biotin-avidin reactions; the DNA1 sequence and the Aptamer sequence of Abeta O are combined together through base complementary pairing; The phosphorylating group is functionalized to the surface of metal organic frameworks MOFs nano-particles through DNA1 sequence.
  2. 2. A method for preparing a signal amplification nucleic acid nanoprobe for detecting aβo, comprising: Synthesizing surface-modified streptavidin-modified CdSe/ZnS quantum dots by a thermosol method, and taking the CdSe/ZnS quantum dots as a signal output unit QDs; Synthesizing metal organic frameworks MOFs taking metal ions Zr as a center and porphyrin as an organic ligand by a hydrothermal method, taking the MOFs as a carrier and simultaneously taking the MOFs as a quencher of CdSe/ZnS quantum dots; Designing a DNA1 sequence complementary to the Aptamer sequence of Abeta O according to the Aptamer sequence of Abeta O, and modifying a phosphorylating group at the 5 'end and modifying avidin at the 3' end; Modifying the signal output unit QDs onto the DNA1 sequence through biotin-avidin reaction, combining the DNA1 sequence and the Aptamer sequence of Abeta O through base complementary pairing, and functionalizing the DNA1 phosphorylating group onto the surface of MOFs nano-particle to construct the Abeta O responsive nucleic acid nano-probe.
  3. 3. The method for preparing a signal amplification nucleic acid nanoprobe for detecting Abeta O of claim 2, DNA1 sequence 5 '-GCCCCAACACAGGCTCTCTTCTCCGAGCCGGTCGA-Biotin-3' Apoligomer sequence of AβO 5'-PO4 3-AGAGAGCCTGTGTTGGGGCGGGTGCG-3'.
  4. 4. The method for preparing a signal amplification nucleic acid nano-probe for detecting aβ O according to claim 3, wherein the surface modified streptavidin modified CdSe/ZnS quantum dot is synthesized by a thermosol method, comprising the steps of: Adding Se powder into a NaOH solution, stirring for 10: 10 min under the protection of N 2 , adding Al powder, heating to 85 ℃, and performing ultrasonic treatment for 15: 15 min to generate a Na 2 Se precursor solution; adding Na 2 Se precursor solution into the cadmium perchlorate solution to prepare CdSe nuclear quantum dot solution; Adding ammonium methosulfate and zinc chloride solution into CdSe nuclear quantum dot solution, adding activated streptavidin after ultrasonic treatment at 85 ℃ for 25 min, stirring overnight, and purifying to obtain the surface modified streptavidin modified CdSe/ZnS quantum dot.
  5. 5. The method for preparing a signal amplification nucleic acid nanoprobe for detecting aβo according to claim 4, wherein a metal organic framework MOFs centering on a metal ion Zr and porphyrin as an organic ligand is synthesized by a hydrothermal method, comprising the steps of: 100 mg TCPP, 300 mg ZrOCl 2 and 2.8. 2.8 g phenylboronic acid reagent were added to 10 mL of N, N-dimethylformamide DMF to form a mixture solution; Transferring the mixture solution into a tetrafluoroethylene-lined hydrothermal reaction kettle, and reacting at 90 ℃ for 5 h; after the reaction, the mixture was centrifuged at 12000 rpm min and washed three times with DMF to obtain MOFs particles, which were kept at 4℃for further use.

Description

Signal amplification nucleic acid nano probe for detecting Abeta O and preparation method thereof Technical Field The invention belongs to the field of molecular biology, and particularly relates to a nucleic acid nano probe for detecting A beta O signal amplification and a preparation method thereof. Background Beta amyloid oligomers aβo are associated with cognitive dysfunction and disease severity in AD. A possible mechanism for aβo-induced toxicity might be their binding to cell membrane receptors, leading to enlargement of the pores on the membrane and acceleration of intracellular aggregation. Meanwhile, abeta O can also interact with neurons and glial cells, so that proinflammatory cascades such as mitochondrial dysfunction, oxidative stress increase and the like are activated, intracellular signal paths are damaged, calcium ion metabolism is interfered, and finally, neuronal apoptosis and cell death are induced. Thus, aβo is currently considered a promising potential candidate biomarker for early diagnosis of AD. In recent years, researchers have developed various methods for detecting the content of aβo in serum, such as enzyme-linked immunosorbent assay (ELISA), capillary electrophoresis, resonance Light Scattering (RLS), electrochemical detection, polymerase Chain Reaction (PCR). Although these methods successfully detect aβo, most require the use of expensive antibodies, which are susceptible to the surrounding environment and will generate excessive background signals that affect detection efficiency. Currently, the most commonly used method clinically is enzyme-linked immunosorbent assay (ELISA method) to detect the AβO content in serum. However, in ELISA, firstly, the antibody is expensive, secondly, ELISA signals are generated based on enzyme reaction dependent on time, the enzyme is sensitive to environmental factors (temperature, time and the like), is easily influenced by matrix components, the detection result is easily influenced, and the result distortion occurs, so that the method is restricted to be clinically used. Aptamer (Aptamer) is an oligonucleotide obtained by systematic evolution (SELEX) technology, can specifically identify ions, small molecules, proteins, cells, even tissues and the like, and has been widely used in the fields of biosensing, molecular detection, drug delivery and the like. Compared with the traditional antibody, the Aptamer has the outstanding advantages of high affinity, good stability, low cost, convenient automatic synthesis and the like. Tsukakoshi et al successfully isolated Abeta O-specific aptamers that have been successfully used in bioassays for AD-related studies in combination with gel shift and competitive screening. However, these methods are mainly output in a 1:1 signal conversion manner, which greatly limits the detection sensitivity of aβo and ultimately limits their application in early diagnosis of alzheimer's disease. Disclosure of Invention It is an object of the present invention to provide a nucleic acid nanoprobe for detecting signal amplification of aβ O and a method for preparing the same, and to provide at least advantages as will be described later. The invention also aims to provide a nucleic acid nano probe for detecting the signal amplification of the Abeta O, which has the advantages of good stability, low cost and high sensitivity, quantitatively detects the Abeta O in peripheral blood, and can improve the early diagnosis of AD and monitor the response of patients to treatment. The technical scheme of the invention is as follows: A signal amplified nucleic acid nanoprobe for detecting aβo, comprising: the signal output unit QDs is CdSe/ZnS quantum dots modified by surface modified streptavidin synthesized by a thermosol method; The metal organic frameworks MOFs are synthesized by a hydrothermal method by taking metal ions Zr as a center and porphyrin as an organic ligand, and are used as carriers and quenching agents of CdSe/ZnS quantum dots; Aptamer sequence of Abeta O; And a DNA1 sequence which is designed according to the Aptamer sequence of Abeta O and is complementary to the Aptamer sequence of Abeta O, wherein the 5 '-end of the DNA1 sequence is modified with a phosphorylating group, and the 3' -end of the DNA1 sequence is modified with avidin; Wherein; DNA1 sequence 5 '-GCCCCAACACAGGCTCTCTTCTCCGAGCCGGTCGA-Biotin-3' Apoligomer sequence of Abeta O5 '-PO4 3-AGAGAGCCTGTGTTGGGGCGGGTGCG-3'; the signal output units QDs are modified onto the DNA1 sequence by biotin-avidin reactions; the DNA1 sequence and the Aptamer sequence of Abeta O are combined together through base complementary pairing; The phosphorylating group is functionalized to the surface of metal organic frameworks MOFs nano-particles through DNA1 sequence. A method for preparing a signal amplified nucleic acid nanoprobe for detecting aβo, comprising the steps of: Synthesizing surface-modified streptavidin-modified CdSe/ZnS quantum dots by a thermosol method, and