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CN-121978346-A - Aptamer-mediated EXPAR-molecular beacon fluorescent biosensor and preparation method and application thereof

CN121978346ACN 121978346 ACN121978346 ACN 121978346ACN-121978346-A

Abstract

The invention discloses an aptamer-mediated EXPAR-molecular beacon fluorescence biosensor and a preparation method and application thereof. The construction and detection process of the sensor comprises three key steps of protein junction proper ligand, exponential isothermal amplification (EXPAR) and molecular beacon Fluorescence Resonance Energy Transfer (FRET). By ingenious design of the EXPAR system, the system realizes triple signal amplification, remarkably improves detection sensitivity, and can detect Abeta 42 and Abeta 40 amyloid with the level as low as 100 fM. In addition, based on the sequence specificity of the aptamer and the high specificity design of each nucleic acid sequence involved in the detection system, the method can detect the Abeta 42 amyloid and Abeta 40 amyloid or other substances simultaneously in the same system (such as a plasma sample), thereby improving the detection efficiency.

Inventors

  • CHEN TINGMEI
  • WU HAOYU
  • XIE GUOMING

Assignees

  • 重庆医科大学

Dates

Publication Date
20260505
Application Date
20260126

Claims (10)

  1. 1. A method for preparing an aptamer-mediated EXPAR-molecular beacon fluorescent biosensor, comprising the following steps: (1) The synthesis of the aptamer complex comprises the steps of mixing an Abeta 42 aptamer solution, an Abeta 42 primer solution, an Abeta 40 aptamer solution and an Abeta 40 primer solution, and reacting to obtain an Abeta 42/Abeta 40 aptamer complex mixed solution; (2) Magnetic bead coupling, namely after magnetic separation of the avidin-modified magnetic beads, adding the Abeta 42/Abeta 40 aptamer complex mixed solution prepared in the step (1) into the magnetic beads, and mixing and incubating the mixture at room temperature for coupling after full shaking and suspension; (3) The replacement and separation of primers, namely adding an Abeta 42 protein and Abeta 40 protein standard oligomer or a to-be-detected product containing Abeta 42 protein and Abeta 40 protein into the system obtained in the step (2), and incubating for 5-25 minutes at 25-45 ℃; (4) Preparing an EXPAR final product, mixing an Abeta 40 template X-X-Y, A beta 40 template Y-Y-Z, A beta 42 template X-X-Y, A beta 42 template Y-Y-Z, dNTPs with the supernatant obtained in the step (3) to prepare a reaction system, adding enzyme and buffer solution required by isothermal amplification, performing an EXPAR reaction at 55-65 ℃, and terminating the reaction to obtain the EXPAR final product; (5) Adding an Abeta 42 molecular beacon and an Abeta 40 molecular beacon into the EXPAR final product obtained in the step (4), and mixing and incubating at 49-60 ℃ to form the fluorescent biosensor; The nucleotide sequences of the Abeta 42 aptamer, abeta 42 primer, abeta 40 aptamer, abeta 40 primer, abeta 42 template X-X-Y, A beta 42 template Y-Y-Z, A beta 40 template X-X-Y, A beta 40 template Y-Y-Z, A beta 42 molecular beacon and Abeta 40 molecular beacon are sequentially shown as SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10.
  2. 2. The method of claim 1, wherein the reaction conditions in step (1) are 95℃denaturation for 5 minutes, followed by annealing to 25℃at a rate of 0.1℃per second to form an A.beta.42/A.beta.40 aptamer complex mixture.
  3. 3. The method of claim 1, wherein the incubation in step (3) is carried out at 25℃for 5 minutes.
  4. 4. The method according to claim 1, wherein the reaction system in the step (4) further comprises 10X NeBuffer r 3.1.1, vent (exo-) DNA polymerase, nt.BstNBI nicking endonuclease, 10X thermo pol reaction buffer, and the reaction is terminated by reacting at 55-60 ℃ for 16-19 minutes and then incubating at 80 ℃ for 15 minutes.
  5. 5. The preparation method of claim 1, wherein the mixed incubation is carried out at 49 ℃ for 8-15 minutes in the step (5).
  6. 6. An aptamer-mediated EXPAR-molecular beacon fluorescent biosensor, which is characterized by being prepared by the preparation method of any one of claims 1 to 5.
  7. 7. Use of an aptamer-mediated EXPAR-molecular beacon fluorescent biosensor according to claim 6 for detecting aβ42 and aβ40.
  8. 8. The use according to claim 7, characterized in that the use is: (1) Respectively reacting Abeta 42 and Abeta 40 protein standard substances with different concentrations in the fluorescent biosensor, respectively testing the fluorescence intensity of the fluorescent biosensor, and constructing a standard curve to obtain a linear equation; (2) And (3) reacting the sample to be detected in the fluorescent biosensor, measuring a fluorescent signal by adopting a fluorescent spectrometer, calculating the concentration of Abeta 42 and Abeta 40 proteins in the sample to be detected according to a standard curve or a linear equation, and judging whether the sample to be detected contains Abeta 42 and Abeta 40 proteins and the concentration.
  9. 9. The method according to claim 8, wherein the sample to be tested is plasma.
  10. 10. The method of claim 7, wherein the linear range of detection is Aβ40:1 pM-10 nM and Aβ42:0.5 pM-5 nM.

Description

Aptamer-mediated EXPAR-molecular beacon fluorescent biosensor and preparation method and application thereof Technical Field The invention belongs to the technical field of biomedicine, and relates to an aptamer-mediated EXPAR-molecular beacon fluorescence biosensor as well as a preparation method and application thereof. Background Alzheimer's Disease (AD) is a neurodegenerative disease, and is the most common cause of dementia, with hidden onset, and diagnosis, especially early diagnosis, is difficult. Alzheimer's disease has a tremendous impact on individuals, caregivers, and society in developed and developing countries. Aβ Amyloid (Amyloid-beta protein) is a 39-42 amino acid polypeptide, and its aggregation and deposition in brain tissue is a major cause of Alzheimer's disease. In particular Abeta 42 amyloid Aβ40 amyloid protein. From the pathological mechanism, the accumulation of soluble aβ42 and aβ40 oligomers in brain tissue is a key factor in the formation of amyloid plaques, a pathological change characteristic of alzheimer's disease, and aβ42 amyloid and aβ40 amyloid are biomarkers of the earliest occurrence of changes in levels in the biogenesis phase of alzheimer's disease, which begin to occur in cerebrospinal fluid and plasma (permeated from cerebrospinal fluid) in the early 17-23 years of the clinic. While aβ42 and aβ40 are also one of the most reliable diagnostic indicators of any stage, they can be used to aid in the differential diagnosis of AD or non-AD when patients enter MCI, dementia stages. Therefore, detecting the concentration of the polypeptide in cerebrospinal fluid or plasma is of great importance for screening and diagnosing Alzheimer's disease. The current detection techniques of Abeta 42 and Abeta 40 are mainly divided into conventional detection techniques and emerging detection techniques. Conventional detection technologies such as Elisa have low sensitivity and can only be used for qualitative detection, and chemiluminescence immune methods with higher sensitivity in the conventional detection technologies also face the problems of high cost, interference of amphotropic antibodies and the like, while emerging detection technologies such as single-molecule immune arrays, microfluidic chips and the like have high sensitivity and accuracy, but the problems of high cost, difficult implementation and the like are also not quite small. The detection of the Abeta 42 and Abeta 40 amyloid mainly depends on cerebrospinal fluid or a blood plasma sample, and at the present stage, the detection of the blood plasma Abeta 42 and Abeta 40 amyloid is gradually replaced by the detection of cerebrospinal fluid with larger traumatism because the detection of the blood plasma Abeta 42 and Abeta 40 amyloid has smaller traumatism, the prediction accuracy difference between a blood plasma model and a cerebrospinal fluid model has no statistical significance (p=0.44 and AUC=0.93), and the detection of a blood plasma biomarker index can also meet the clinical prediction requirement. Whereas the contents of aβ42 and aβ40 in the plasma of patients with alzheimer's disease are very small, conventional detection techniques are often inadequate. And the screening pressure is gradually increased due to the improvement of the prevalence rate of the Alzheimer's disease at present, and the adoption of an emerging method is impractical. In addition, in addition to absolute concentrations of aβ42 and aβ40, reduced cerebrospinal fluid and plasma aβ42/aβ40 concentration ratios are also associated with memory decline. In cerebrospinal fluid, this ratio correlates more significantly with decreased amyloid burden and cognitive function in the brain compared to single aβ42 levels. In plasma, several studies have found that there is a significant difference in the aβ42/aβ40 concentration ratio in patients and cognitive normal populations. The A.beta.42/A.beta.40 concentration ratio is also a very valuable indicator. However, the extremely low abundance of aβ42 and aβ40 in plasma highlights the urgent need for a detection method that is rapid, sensitive and capable of simultaneous detection of aβ42 and aβ40. Therefore, there is a need to develop a simple, highly sensitive and highly specific method for detecting aβ42 and aβ40. An Aptamer (Aptamer) is a single-stranded DNA or RNA molecule obtained by artificial screening, which can bind to a target molecule with high specificity and high affinity and undergo a special spatial structural change, and can play a role in signal conversion in detection of the target molecule. Since the screening of aβ42 and aβ40 ligands, they soon enter the public and are of great importance in the detection of aβ42 and aβ40. In recent years, isothermal amplification techniques have been increasingly used, which can be kept at a constant temperature during the amplification without requiring complicated thermal cycling steps, wherein EXPAR (exponential isothermal amplification) i