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CN-121994890-A - Tumor small extracellular vesicle electrochemical detection platform based on OR logic gate, and preparation method and application thereof

CN121994890ACN 121994890 ACN121994890 ACN 121994890ACN-121994890-A

Abstract

The invention discloses an or logic gate-based electrochemical detection platform for tumor small extracellular vesicles, a preparation method and application thereof, wherein the electrochemical detection platform comprises a DNA probe containing an aptamer, a substrate probe linker, a probe PAP and a gold electrode, and the DNA probe comprises three aptamers AP1, AP2 and AP3. The invention adopts the specificity of multiple aptamers to identify different proteins, realizes the high-sensitivity detection of the tumor small extracellular vesicles, solves the problems of insufficient signal intensity and difficult detection of low-abundance vesicles in a single aptamer identification mode, and on the other hand, compared with the traditional single protein detection scheme, the invention can synchronously capture the existence states of multiple target proteins on the surface of the tumor small extracellular vesicles, realizes the differential identification and characterization of the tumor small extracellular vesicles with different phenotypes, and provides more comprehensive molecular marker information for the accurate diagnosis of tumors.

Inventors

  • ZHANG HUI
  • Li Menlong

Assignees

  • 南京师范大学

Dates

Publication Date
20260508
Application Date
20260205

Claims (10)

  1. 1. The electrochemical detection platform for the tumor small extracellular vesicles based on the OR logic gate is characterized by comprising a DNA probe containing an aptamer, a substrate probe linker, a probe PAP and a gold electrode, wherein the DNA probe comprises three aptamers AP1, AP2 and AP3, the three aptamers AP1, AP2 and AP3 respectively specifically identify PTK7 protein, CD63 protein and EpCAM protein, the three aptamers AP1, AP2 and AP3 respectively contain a general L1 sequence, the sequences are complementarily matched with the substrate probe linker and construct a specific identification and cleavage site of Nb.BbvCI enzyme, a DNA1 fragment is generated after the Nb.BbvCI enzyme is acted, the PAP probe is modified on the surface of the gold electrode in advance and hybridized with the enzyme-cleaved product DNA 1to form a double-chain complex.
  2. 2. The or gate-based tumor small extracellular vesicle electrochemical detection platform according to claim 1, wherein the sequences of the aptamers AP1, AP2, AP3 are preferably respectively AP1： 5'-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GAA AAA AAA AAA AAT CCT CAG CAG TTA-3',AP2： 5'-CAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TAA CT AAA AAA AAT CCT CAG CAG TTA GCA TT-3',AP3： 5'-CAC TAC AGA GGT TGC GTC TGT CCC ACG TTG TCA TGG GGG GTT GGC CTG AAAAAAA AT CCT CAG CAG TTA ACC TCT GTA GTG-3'.
  3. 3. The or gate-based tumor small extracellular vesicle electrochemical detection platform of claim 1, wherein the sequence of the substrate probe linker is 5'-TCA GCA GGG AGG AAG ACA ATA TTA ACT GCT GAG GAT AAA CG-3'.
  4. 4. The or gate-based tumor small extracellular vesicle electrochemical detection platform according to claim 1, wherein the L1 sequence is 5'-AT CCT CAG CAG TTA-3', nb.BbvCI and the sequence of DNA1 formed after enzymatic cleavage is 5'-TCA GCA GGG AGG AAG ACA ATA TTA ACT GC-3'.
  5. 5. The or gate-based tumor small extracellular vesicle electrochemical detection platform according to claim 1, wherein the sequence of the probe PAP is 5'-CCT CCC TGC TGA A CAC ACA CAC AAA AAA AAA AAA AAA AAA AAA AAA CAC ACA CAC A GC AGT TAA TAT TG-3'.
  6. 6. A method of preparing the or gate-based tumor small extracellular vesicle electrochemical detection platform of claim 1, comprising the steps of: (1) Incubating tumor small extracellular vesicles with AP1, AP2 and AP3 to form sEVs-aptamer complex, exposing the same sequence L1, wherein enzyme cutting sites are embedded in the sequence; (2) Taking sEVs-aptamer complex prepared in the step (1), and carrying out incubation reaction with a substrate probe linker to construct a complete Nb.BbvCI enzyme cleavage site; (3) Incubating the probe PAP with the pretreated gold electrode, and immersing the probe PAP in 6-Mercaptoethanol (MCH) after incubation overnight to block non-specific sites, so as to obtain a modified electrode; (4) Incubating the solution obtained in the step (2) with the modified electrode obtained in the step (3), and using the detection electrode obtained after incubation as an electrochemical detection platform.
  7. 7. The method of claim 6, wherein the tumor small extracellular vesicles in step (1) are small extracellular vesicles secreted by CCRF-CEM cells, MCF-7 cells, heLa cells or Ramos cells.
  8. 8. The method for preparing the tumor small extracellular vesicles electrochemical detection platform based on OR logic gate according to claim 6, wherein the concentration of the tumor small extracellular vesicles is 2.75X10 2 -5.5×10 8 parts per mL, the concentration of a substrate probe linker is 1.5-3.5 mu M, the concentration of Nb.BvCI enzyme is 0.2-0.7U/mu L, and the concentration of probe PAP is 0.01-2 mu M.
  9. 9. Use of an or gate-based tumor small extracellular vesicles electrochemical detection platform according to claim 1 for detecting small extracellular vesicles.
  10. 10. The application according to claim 9, characterized in that it comprises the following steps: (1) The electrochemical detection platform is used as a working electrode, the platinum wire electrode is a counter electrode, the saturated calomel electrode is used as a reference electrode, and a three-electrode system is used for electrochemical analysis and detection; (2) The signal difference value obtained by detecting the tumor small extracellular vesicles with different concentrations is used for quantitatively analyzing and detecting the tumor small extracellular vesicles.

Description

Tumor small extracellular vesicle electrochemical detection platform based on OR logic gate, and preparation method and application thereof Technical Field The invention relates to the technical field of tumor small extracellular vesicles detection, in particular to an or logic gate-based electrochemical detection platform for tumor small extracellular vesicles, and a preparation method and application thereof. Background The small extracellular vesicles (Small Extracellular Vesicles, sEVs) are bilayer membranous vesicles of diameter 30-150 a nm secreted by the parent cell. The cell-to-cell hybrid protein contains substances such as lipid, protein, nucleic acid and the like, carries genetic materials of parent cells, is an important substance for information transmission among cells, and plays an important role in physiological and pathological processes. sEVs secreted by disease cells contains more biomarkers than sEVs of normal cells, which provides powerful support for early diagnosis and treatment of disease. The tumor sEVs surface carries not only transmembrane proteins such as CD9, CD63, CD81, etc., but also abundant characteristic proteins such as carcinoembryonic antigen (CEA), mucin-1 (MUC 1), epithelial cell adhesion molecule (EpCAM), protein tyrosine kinase 7 (PTK 7), etc. These characteristic proteins can specifically bind to antibodies, aptamers, etc. On the basis, the tumor sEVs is combined with a plurality of detection methods such as fluorescence, electrochemistry, electrochemiluminescence and the like, and a detection platform with high sensitivity and high specificity is provided for detecting the tumor sEVs. The concentration of the biomarker of the tumor sEVs in the early stage of the disease is very low, most of the existing detection methods of the tumor sEVs detect single protein, but the signal change obtained by the single protein is small, so that the low detection limit is not easy to obtain, and the low concentration of sEVs is difficult to detect. For this reason, a method for obtaining a low detection limit is urgently needed for early diagnosis of diseases. Disclosure of Invention The invention aims to solve the technical problems, and aims to provide an electrochemical detection platform for tumor small extracellular vesicles based on an OR logic gate, which constructs a multi-aptamer-polyprotein recognition system by designing a DNA probe containing various specific aptamers, takes the signal of the OR logic gate as an input signal, takes a RuHeX electrochemical signal as an output signal, and reflects the content of biomarkers on the surfaces of the tumor small extracellular vesicles by the change degree (delta I) of the signal. The invention adopts the specificity of multiple aptamers to identify different proteins, realizes the high-sensitivity detection of the tumor small extracellular vesicles, solves the problems of insufficient signal intensity and difficult detection of low-abundance vesicles in a single aptamer identification mode, and on the other hand, compared with the traditional single protein detection scheme, the invention can synchronously capture the existence states of multiple target proteins on the surface of the tumor small extracellular vesicles, realizes the differential identification and characterization of the tumor small extracellular vesicles with different phenotypes, and provides more comprehensive molecular marker information for the accurate diagnosis of tumors. The invention also provides a preparation method and application of the tumor small extracellular vesicle electrochemical detection platform based on the OR logic gate. According to the technical scheme, the tumor small extracellular vesicle electrochemical detection platform based on the OR logic gate is characterized by comprising a DNA probe containing an aptamer, a substrate probe linker, a probe PAP and a gold electrode, wherein the DNA probe comprises three aptamers AP1, AP2 and AP3, respectively and specifically recognizes PTK7 protein, CD63 protein and EpCAM protein, the three aptamers AP1, AP2 and AP3 all contain a universal sequence L1, the sequences can be complementarily matched with the substrate probe linker and construct specific recognition and cleavage sites of Nb.BbvCI enzyme, a DNA1 fragment is generated after the Nb.BbvCI enzyme acts, the PAP probe is modified on the surface of the gold electrode in advance and can be hybridized with the enzyme-cleaved product DNA1 to form a double-chain complex. Wherein the sequences of the aptamer AP1, the aptamer AP2 and the aptamer AP3 are respectively AP1： 5'-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GAA AAA AAA AAA AAT CCT CAG CAG TTA-3',AP2： 5'-CAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TAA CT AAA AAA AAT CCT CAG CAG TTA GCA TT-3',AP3： 5'-CAC TAC AGA GGT TGC GTC TGT CCC ACG TTG TCA TGG GGG GTT GGC CTG AAAAAAA AT CCT CAG CAG TTA ACC TCT GTA GTG-3'. Wherein the sequence of the substrate probe linker is 5'-TCA GCA GGG AG