SERS-fluorescent dual mode probe based on DNA chain replacement, and preparation method and application of SERS-fluorescent dual mode probe

A DNA chain and fluorescence technology, applied in the field of bioanalytical chemistry, can solve the problems of unstable signal, inability to accurately observe the position and change of active substances in cells, complicated operation, etc., and achieve the effect of stable nature.

Active Publication Date: 2019-10-29
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although many fluorescence detection methods have appeared, the operation is cumbersome, the signal is unstable, and the position and change of active substances in cells cannot be accurately observed

Method used

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  • SERS-fluorescent dual mode probe based on DNA chain replacement, and preparation method and application of SERS-fluorescent dual mode probe
  • SERS-fluorescent dual mode probe based on DNA chain replacement, and preparation method and application of SERS-fluorescent dual mode probe
  • SERS-fluorescent dual mode probe based on DNA chain replacement, and preparation method and application of SERS-fluorescent dual mode probe

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Embodiment 1

[0035] A method for preparing a SERS-fluorescent dual-mode probe based on DNA strand displacement, such as figure 1 As shown, it specifically includes the following steps:

[0036] 1) Fe 3 o 4 Preparation: Take 1~5g FeCl 2 4H 2 O and 1~5g FeCl 3 ·6H 2 Dissolve O in 20mL ultrapure water, filter through filter paper and dilute 10-30mL, and after a few minutes of nitrogen gas, add 10-30mL ammonia water to the above solution drop by drop, in N 2 React under protection for 30 minutes and age for 1 to 3 hours. After the reaction, the heat source was removed and cooled to room temperature, separated by a magnet, washed repeatedly with water and ethanol several times, dried in an oven overnight and ground into powder for later use.

[0037] 2) Fe 3 o 4 @Au's preparation:

[0038] 2a) Fe 3 o 4 - Preparation of DA: weigh 10-30mg Fe 3 o 4 Dissolve in THF, add 2mL dopamine hydrochloride aqueous solution, sonicate evenly, stir overnight at room temperature, centrifuge to remo...

Embodiment 2

[0050] SERS-fluorescent dual-mode probes for SERS quantitative detection of VEGF in vitro, such as Figure 4 shown.

[0051] The Raman detection in the constructed detection method includes the following steps: In this embodiment, human melanoma cell A375 is taken as an example. After the A375 cells grow to the logarithmic phase, the cells are divided into 6 Inoculate 100 μL onto a sterile quartz plate at a density of 1 / mL, incubate for 24 hours until the wall is completely adhered, suck out the old medium, add DNA containing DNA and mix well 1-2 Modified Fe 3 o 4 @Au probe medium 1mL, co-culture with cells for 24h, suck out the old medium and wash it twice with PBS, then add DNA mixed with the same concentration 3 The basal medium was co-cultured for 0, 0.5, 1, 1.5, 2, 2.5 and 3 hours respectively, then the old medium was aspirated, washed twice with PBS, and SERS imaging was performed immediately.

[0052] in, Figure 4 -A is the SERS signal diagram of the SERS-fluoresc...

Embodiment 3

[0054] Qualitative fluorescence imaging analysis of SERS-fluorescent dual-mode probes, such as Figure 5 shown.

[0055] Fluorescence detection in the constructed detection method includes the following steps: In this embodiment, human melanoma cell A375 is taken as an example. After the cells grow to the logarithmic growth phase, the cells are 6 Inoculate 100 μL onto a fluorescent confocal dish at a density of cells / mL, incubate for 24 hours and wait for complete attachment, suck out the old medium, add DNA containing DNA and mix well 1-2 Modified Fe 3 o 4 @Au medium 1mL, co-culture with cells for 24h, suck out the old medium and wash it twice with PBS, then add DNA mixed with the same concentration 3 The basal medium was co-cultured for 0, 1, 10, 20, and 30 min respectively; then the old medium was aspirated, washed twice with PBS, and immediately performed fluorescence imaging.

[0056] The results showed that due to DNA 3 Displacement of DNA by strand displacement and...

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Abstract

The invention discloses an SERS-fluorescent dual mode probe based on a DNA chain replacement principle. The SERS-fluorescent dual mode probe is prepared from a DNA1-2 double strand chain modified by aferroferric oxide magnetic composite material with a certain particle size and another DNA3 single strand chain; and the DNA1 and the DNA3 contain the same aptamer, the tail end of the DNA2 is modified with a fluorogen FAM6, and the tail end of the DNA3 is modified with a fluorogen Cy5. The invention further discloses a preparation method and application of the SERS-fluorescent dual mode probe based on the DNA chain replacement principle. According to the SERS-fluorescent dual mode probe based on the DNA chain replacement principle, two types of signals of raman and fluorescence can be used for simultaneously detecting biomarkers in superficial living cells, detecting is fast, convenient and efficient, the specificity is high, the sensitivity is high, and the accuracy is high.

Description

technical field [0001] The invention relates to the technical field of bioanalytical chemistry, in particular to a DNA strand replacement-based SERS-fluorescence dual-mode probe, a preparation method and application thereof. Background technique [0002] Due to the high incidence of cancer, people's quality of life has declined significantly. Even though the incidence rate of some tumors is low, the degree of malignancy is high, and the metastasis occurs early, resulting in high mortality. Therefore, early detection, diagnosis and treatment of cancer are more important. The current common cancer prediction method is to detect some abnormal biological factors in the lesion area, including enzymes, proteins, DNA, RNA, etc., and use them as important biomarkers for the deterioration of early cancer cells. Although many fluorescent detection methods have been developed, their operations are cumbersome, the signal is unstable, and the position and changes of active substances in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/682C12N15/11
CPCC12Q1/682C12Q2537/1373C12Q2563/107C12Q2563/143C12Q2565/632Y02A50/30
Inventor 张卓旻黄路李攻科
Owner SUN YAT SEN UNIV
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