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Method for detecting cytochrome c in living cell based on Raman-fluorescent dual-mode probe

A dual-mode probe and detection method technology, applied in the fields of nanomaterials and life sciences, can solve problems such as hindering applications and weak Raman scattering intensity, and achieve accurate and reliable results, significant Raman enhancement, and significant fluorescence quenching effects Effect

Active Publication Date: 2019-03-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most samples, especially biological samples, hinder the application of this technique in many fields due to their weak Raman scattering intensity.

Method used

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  • Method for detecting cytochrome c in living cell based on Raman-fluorescent dual-mode probe
  • Method for detecting cytochrome c in living cell based on Raman-fluorescent dual-mode probe
  • Method for detecting cytochrome c in living cell based on Raman-fluorescent dual-mode probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] The synthesis route of gold seeds is as follows:

[0061] 50 μL of 25 mM HAuCl 4 Add the solution to 4.7 mL of 0.1 M CTAC solution and inject 300 μL of freshly prepared pre-cooled 10 mM NaBH under vigorous stirring 4 solution, the solution changed from light yellow to orange and then stood at room temperature for 2h to consume excess NaBH 4 ,spare.

Embodiment 2

[0063] The synthetic route of AuNTs is as follows:

[0064] After the prepared seeds were diluted 10 times with 0.1M CTAC solution, two growth solutions were prepared: (A) 1.6mL of 0.1M CTAC solution was added to 8mL of ultrapure water, and then 40μL of 50mM HAuCl was added 4 and 15μL 10mM NaI; (B) 500μL 50mM HAuCl 4 Added to 40 mL of 0.05M CTAC, followed by 300 μL of 10 mM NaI. Growth solution A was used to grow CTAC-capped seeds into larger nanoparticles, while growth solution B was used for the growth of triangular sheets. Add 40 and 400 μL of 0.1M AA solutions to growth solutions A and B, respectively, and stir until the two solutions turn from light yellow to completely transparent, indicating that Au III Reverted to Au I . Then 100 μL of diluted seeds was added to growth solution A, and after stirring for no more than 5 s, 3.2 mL of this solution was immediately added to growth solution B and stirred for 5 s. Finally, the growth solution B was allowed to stand at ro...

Embodiment 3

[0066] Preparation of Raman-fluorescent dual-mode probes:

[0067] Add SH-PEG-COOH solution to the resuspended AuNTs and keep stirring for 1 h, and centrifuge at 3000 rpm for 10 min to remove excess unreacted reagents. Place the DNA solution in a water bath at a temperature higher than Tm for 5 min to activate the DNA. Then the Cyt c aptamer DNA1 solution was added to the AuNTs so that the final DNA1 concentrations were 100, 250, 500, 800, 1000 and 1200 nM ( image 3 ). It is well known that the intensity of absorbance decrease represents the affinity between NP and DNA to some extent. The DNA aqueous solution has a typical absorption band (about 260nm), and the decrease of this value can be used to prove that the nanoparticles are modified by DNA. As shown, ΔA260 increased significantly with increasing DNA1 concentration. When the concentration reaches 1000nM, ΔA260 is almost unchanged. Therefore, the optimal concentration of DNA1 final concentration is 1000 nM. The mix...

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Abstract

The invention discloses a method for detecting cytochrome c in a living cell based on a Raman-fluorescent dual-mode probe. The detecting method realizes dual-signal detection based on on-off conversion of Raman scattering and fluorescence resonance energy transfer through a nano-sensor which is constructed through assembling an adapter of the cytochrome c(Cyt c) on a gold triangle sheet (AuNTs). The method of the invention can simultaneously detect the Cyt c in the living cell through a Raman signal and a fluorescent signal. Furthermore the method has advantages of high detection singularity,high sensitivity and high accuracy. Furthermore compared with ELISA, the error is lower than 5%.

Description

technical field [0001] The invention relates to the fields of nanomaterials and life sciences, in particular to a method for quantitatively detecting cytochrome c (Cyt c) in living cells based on a Raman-fluorescent dual-mode probe. Background technique [0002] Apoptosis, the process of programmed cell death, is a complex and highly regulated process that has significant effects on cell growth and proliferation. As an important biomarker of early apoptosis, cytochrome c (Cyt c) is considered a key component of the intrinsic apoptotic pathway and often indicates a point of no return in apoptosis. Under normal conditions, Cyt c exists at the top of the mitochondrial membrane in cells, acting as an electron carrier in the mitochondrial membrane space between Cyt c reductase and Cyt c oxidase. When the cells are under pathological conditions, the mitochondrial membrane pores are opened, Cyt c is released from the mitochondria into the cytoplasm, activates the caspase apoptosis...

Claims

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Application Information

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IPC IPC(8): G01N21/65G01N21/64C12N15/115C09K11/06C09K11/58B82Y30/00B82Y40/00
CPCB82Y30/00B82Y40/00C09K11/06C09K11/58C12N15/115G01N21/6428G01N21/658G01N2021/6432
Inventor 马小媛张京娜王周平
Owner JIANGNAN UNIV
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