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Multivalent aptamer functionalized DNA nano-structure probe as well as preparation method and application thereof

A nanostructure and functionalization technology, applied in the field of biomedicine, achieves the effects of stable structure, improved binding efficiency, and simple preparation method

Pending Publication Date: 2022-06-24
NANJING UNIV OF POSTS & TELECOMM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the multivalent aptamer-functionalized DNA nanostructured probes suitable for the efficient enrichment of CTCs have not been reported yet.

Method used

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  • Multivalent aptamer functionalized DNA nano-structure probe as well as preparation method and application thereof
  • Multivalent aptamer functionalized DNA nano-structure probe as well as preparation method and application thereof
  • Multivalent aptamer functionalized DNA nano-structure probe as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Multivalent aptamer functionalized DNA nanostructure probe and its preparation and structure characterization experiment

[0043] 1. Multivalent aptamer-functionalized DNA nanostructure probes

[0044] like figure 1As shown in the figure, one side of the DNA nanostructure of the probe has multiple DNA nucleic acid aptamers, which can specifically bind to protein receptors on the membrane surface of circulating tumor cells; the other side has multiple biotin sites, which can bind to streptavidin Avidin-modified magnetic particles; the probe is synthesized from a total of six DNA single strands of S1, S2, N1, N2, N3, and SYL3C, as follows:

[0045] Described S1 is shown as SEQ ID NO.1, specifically:

[0046] 5'-CAGGGCTGGGCATAGAAGTCAGGGCAGAGACGAGTTGAGAATACGAGT-TEG-Biotin-3';

[0047] The S1 is a sequence of modified biotin molecules, which can be combined with streptavidin-modified magnetic particles.

[0048] Described S2 is as shown in SEQ ID NO.2, specific...

Embodiment 2

[0066] Using the multivalent aptamer-functionalized DNA nanostructure probe prepared in Example 1 to realize the capture of human breast cancer cells (MCF-7) and the cell number gradient experiment, the details are as follows:

[0067] (1) MCF-7 cell staining: The passaged MCF-7 cells were placed at 37°C in 5% CO containing 2 After 48h, use 1×PBS (containing NaCl, KCl, Na 2 HPO 4 , KH 2 PO 4 , pH=7.4) to wash, remove the washing liquid, add DMEM without fetal bovine serum (FBS), and 5 μL of cell membrane dye DIO (1 mM), mix well and place in a cell incubator for staining for 20 min.

[0068] (2) MCF-7 cell digestion: After staining, remove the stain from the culture flask and wash with 1×PBS at least three times to avoid excess stain affecting cell viability, add trypsin (containing ethylenediaminetetraacetic acid (EDTA) EDTA) 0.02%), place the culture flask in the cell culture incubator again for about 2 min, then supplement with FBS-containing high-glucose medium DMEM, s...

Embodiment 3

[0080] The multivalent aptamer functionalized DNA nanostructure probe prepared in Example 1 was used to realize the capture of human pancreatic cancer cells (BxPC-3) and the cell number gradient experiment, as follows:

[0081] Referring to the operation of capturing MCF-7 with the multivalent aptamer-functionalized DNA nanostructure probe in Example 2, the difference in this example is that the cultured cells are BxPC-3, and the rest of the steps are the same as those in Example 2.

[0082] Experimental results: In this example, the capture efficiency of the multivalent aptamer-functionalized DNA nanostructure probe to capture BxPC-3 is as follows: Figure 5 As shown, the average capture efficiency of BxPC-3 was 52.9%.

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Abstract

The invention discloses a multivalent aptamer functionalized DNA nanostructure probe as well as a preparation method and application thereof, one side of the DNA nanostructure of the probe is provided with a plurality of DNA aptamers, and the DNA aptamers can be specifically combined with a circulating tumor cell membrane surface protein receptor; a plurality of biotin sites are arranged on the other side of the substrate and can be combined with streptavidin modified magnetic particles; the probe is synthesized by six DNA single chains, namely S1, S2, N1, N2, N3 and SYL3C, and DNA sequences of the S1, the S2, the N1, the N2, the N3 and the SYL3C are respectively shown as SEQ ID NO.1-6. The probe disclosed by the invention can realize target cell recognition based on different expression levels of biomarkers on the surface of a cell membrane, the probe disclosed by the invention is simple to synthesize, high in CTCs capturing efficiency and easy to operate, and meanwhile, the enriched CTCs can be released through DNA hydrolase, so that the probe has potential significance on pathological diagnosis and other subsequent researches of the CTCs.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a multivalent aptamer functionalized DNA nanostructure probe and a preparation method and application thereof. Background technique [0002] In 1869, Australian physician Ashworth first discovered and proposed the concept of circulating tumor cells (Circulating Tumor Cells, CTCs) in the blood of patients who died of cancer (Aust Med J, 1869, 14, 146-149). CTCs are derived from primary tumors or from Cancer cells that shed, invade, and enter the circulatory system at metastatic sites. Studies have shown that the number of CTCs in blood is closely related to the development of cancer patients, so real-time monitoring of changes in the number of CTCs in blood is very important for evaluating cancer metastasis and curative effect. Although the detection of CTCs has important clinical research and application value for the early diagnosis of cancer, the content of CTC...

Claims

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Application Information

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IPC IPC(8): C12N15/115G01N33/569C12N5/09B82Y5/00B82Y40/00
CPCC12N15/115G01N33/56966C12N5/0693B82Y5/00B82Y40/00C12N2310/16C12N2509/00Y02A50/30
Inventor 朱丹昂磊汪联辉晁洁魏青云
Owner NANJING UNIV OF POSTS & TELECOMM
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