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Multifunctional magnetic DNA (deoxyribonucleic acid) nanosphere and preparation method and application thereof

A nanosphere and multifunctional technology, applied in the field of multifunctional magnetic DNA nanospheres and their preparation, can solve the problem of less DNA nanospheres, and achieve the effects of high selectivity, convenient separation and strong detection specificity

Active Publication Date: 2017-05-31
LINYI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few studies on DNA nanospheres at present, especially the multifunctional DNA nanospheres that can specifically recognize target cells as a carrier for targeted drug delivery and can detect intracellular glutathione have not yet been reported.

Method used

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  • Multifunctional magnetic DNA (deoxyribonucleic acid) nanosphere and preparation method and application thereof
  • Multifunctional magnetic DNA (deoxyribonucleic acid) nanosphere and preparation method and application thereof
  • Multifunctional magnetic DNA (deoxyribonucleic acid) nanosphere and preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0086] Embodiment 1: the synthesis of DNA-SP

[0087] Dissolve 0.6 μL of phosphorylated linear template strand (concentration: 100 μM) in 97.7 μL D-PBS solution (pH: 7.4), place the mixture in a PCR instrument, react at 95 °C for 2 min, and then cool down at 0.4 °C per minute. Slowly cool down to 55 degrees Celsius, then add 1.2 μL of poly (T) DNA primer single strand (concentration is 100 μM; sequence shown in SEQ ID NO.2), continue to cool down slowly to 25 degrees Celsius at a rate of 0.4 degrees Celsius per minute , to complete the annealing process, after which 0.5 μL T 4 DNA ligase, mix well and react at 25 degrees Celsius for 4 hours.

[0088] Next, the rolling circle amplification process was carried out, and 40 μL phi29 DNA polymerase (concentration: 10 U / μL), 40 μL dNTPs (concentration: 10 mM), and 10 μL D-PBS solution (pH: 7.4) were added to the above reaction solution, and mixed evenly Place it at 30°C for 10 hours, and finally place the reaction system at 65°C f...

Embodiment 2

[0091] Embodiment 2: the synthesis of MNP / DNA-SP

[0092] Dissolve 0.6 μL of phosphorylated linear template strand (100 μM concentration, sequence shown in SEQ ID NO.1) in 97.7 μL D-PBS solution (pH 7.4), place the mixture in a PCR instrument, and react at 95 degrees Celsius 2min, then slowly cool down to 55 degrees Celsius at a rate of 0.4 degrees Celsius per minute; then add 1.2 μL DNA primer single strand (concentration is 100 μM; the primer single strand refers to the DNA primer single strand modified biotin molecules, the sequence is as SEQ ID NO.3), continue to cool down slowly to 25 degrees Celsius at a rate of 0.4 degrees Celsius per minute, and complete the annealing process; then add 0.5 μL T 4 DNA ligase (2000 U / μL) and 10 μL streptavidin magnetic beads (10 mg / ml) were mixed evenly and reacted at 25 degrees Celsius for 4 hours.

[0093] Next, carry out the rolling circle amplification process, add 40 μL phi29 DNA polymerase (concentration: 10 U / μL), 40 μL dNTPs (co...

Embodiment 3

[0098] Embodiment 3: the preparation of MNP / DS-SP

[0099] Dissolve 0.6 μL of phosphorylated linear template strand (concentration: 100 μM) in 97.7 μL D-PBS solution (pH: 7.4), place the mixture in a PCR instrument, react at 95 °C for 2 min, and then cool down at 0.4 °C per minute. Slowly cool down to 55 degrees Celsius, then add 1.2 μL of poly (T) DNA primer single strand (concentration is 100 μM; sequence shown in SEQ ID NO.2), continue to cool down slowly to 25 degrees Celsius at a rate of 0.4 degrees Celsius per minute , to complete the annealing process, after which 0.5 μL T 4 DNA ligase, mix well and react at 25 degrees Celsius for 4 hours.

[0100] Next, the rolling circle amplification process was carried out, and 40 μL phi29 DNA polymerase (concentration: 10 U / μL), 40 μL dNTPs (concentration: 10 mM), and 10 μL D-PBS solution (pH: 7.4) were added to the above reaction solution, and mixed evenly Placed at 30°C for 10 hours, and finally heated the reaction system at 65...

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Abstract

The invention discloses a multifunctional magnetic DNA (deoxyribonucleic acid) nanosphere. The multifunctional magnetic DNA nanosphere comprises a DNA nanosphere combined with magnetic beads, and / or a DNA nanosphere combined with magnetic beads and containing double sulfur bonds. The multifunctional magnetic DNA nanosphere is characterized in that a phosphorylated linear DNA template single chain and a DNA primer single chain can complete the assembly of the nanosphere; in the nanosphere assembly process, the other DNA primer single chain is differently modified, and the phosphorylated linear DNA template single chain is subject to structural design, so as to assemble multiple types of functional groups in the nanosphere structure. The multifunctional magnetic DNA nanosphere has the advantages that the multifunctional magnetic DNA nanosphere can be used as a target transportation medicine carrier, so that the biological separation is easy; the DNA primer single chain of the multifunctional magnetic DNA nanosphere is modified by the double sulfur bonds, and the sulfhydryl compound in the cell can be detected.

Description

technical field [0001] The invention relates to the technical field of detection and targeted drug delivery of intracellular sulfhydryl compounds, in particular to a multifunctional magnetic DNA nanosphere and its preparation method and application. Background technique [0002] The complementarity of deoxyribonucleotides (DNA) is manifested in the complementarity of four nucleotides, cytosine (C), thymine (T), adenine (A) and guanine (G). In essence, the complementarity of DNA is the basis of DNA replication and transcription, creating a double helix structure of DNA double strands. Compared with the traditional idea that DNA is hereditary, more and more successful cases have been developed by combining its biocompatibility with nanotechnology, such as DNA nanocage, which is a structure that can effectively encapsulate enzymes, six Five out of 10 metabolizing enzymes tested exhibited 4 to 10 times higher turnover than normal metabolizing enzymes. DNA nanoflowers are also ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/11G01N21/64C12Q1/68A61K47/54A61K31/704A61K49/00A61P35/00
CPCA61K31/704A61K49/0017C12N15/11C12Q1/686G01N21/6428C12Q2531/125C12Q2521/501
Inventor 郭英姝王玉洁李双张书圣
Owner LINYI UNIVERSITY
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