Method for detecting intracellular cytochrome c based on near-infrared fluorescence lifetime imaging

A technology of fluorescence lifetime and detection method, which is applied in the field of chemistry and can solve problems such as reducing detection sensitivity

Pending Publication Date: 2022-03-08
SHENZHEN TECH UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] On the other hand, ultraviolet and visible light excitation can easily produce strong background fluorescence interference, which further reduces detection sensitivity.

Method used

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  • Method for detecting intracellular cytochrome c based on near-infrared fluorescence lifetime imaging
  • Method for detecting intracellular cytochrome c based on near-infrared fluorescence lifetime imaging
  • Method for detecting intracellular cytochrome c based on near-infrared fluorescence lifetime imaging

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] 1) Preparation of gold nanorod particles: 75 μL of HAuCl4 (0.01M) was added to 2.5 mL of CTAB (0.1M) to form a homogeneous mixture. Afterwards, 200 μL of NaBH4 (10 mM) was quickly injected into the above mixture under vigorous stirring, and the gold nanometer seed solution was obtained immediately. Afterwards, a gold nanorod growth solution containing CTAB (0.1M, 50mL), AgNO3 (0.01M, 0.5mL), HAuCl4 (0.01M, 2.5mL) and ascorbic acid (0.1M, 0.375mL) was prepared. Get gold seed solution 0.5mL and join in above-mentioned growth liquid, after reacting at room temperature for 2.5 hours, the solution is centrifuged (1000rpm, 15 minutes. It redisperses in deionized water.

[0029] 2) Preparation of gold nanorod@Cy5.5 complex: take gold nanorod particles (about 0.1nM, 2mL) in step 1), mix with 200μL (500nM) thiol nucleic acid aptamer overnight, centrifuge at 10000rpm for 15min, centrifuge twice Remove excess unconnected nucleic acid aptamer once, redisperse and dilute the precip...

Embodiment 2

[0032]Detection of carcinoembryonic antigen: the gold nanorod particles@Cy5.5 complex obtained in Example 1 (concentration in the cell culture medium is 0.025nM), incubated with the cells for 6 hours (same as in Example 1), and added After incubating 2 μM podidine in 2 mL DMEM at 37°C for two hours, under the excitation of 633nm near-infrared laser, the fluorescence lifetime imaging was tested, and the cell fluorescence imaging image is shown in Figure 5 As shown, the average lifetime is 820ps. Since etoposide can effectively induce cells to produce cytochrome c, the dose of etoposide is increased compared to Example 2, thus more cytochrome c can be induced, and the fluorescence lifetime of Cy5.5 molecules increases, The average lifespan continued to increase to 820 ps, ​​so it could be judged that intracellular cytochrome c was produced in large quantities.

Embodiment 3

[0034] The gold nanorod particles@Cy5.5 complex obtained in Example 1 (concentration in the cell culture medium is 0.025nM) was incubated with the cells for 6 hours (same step as in Example 1), and 5 μM of etoposide was added in 2 mL DMEM At 37°C, after incubation for two hours, under the excitation of 633nm near-infrared laser, test the fluorescence lifetime imaging, the cell fluorescence imaging picture is as follows Figure 6 As shown, the average lifetime is 980ps. Since etoposide can effectively induce cells to produce cytochrome c, the dose of etoposide is increased compared to Example 3, thereby inducing more cytochrome c, and the fluorescence lifetime of the Cy5.5 molecule increases, The average lifespan continued to increase to 980 ps, ​​so it could be judged that intracellular cytochrome c was produced in large quantities.

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Abstract

The embodiment of the invention discloses a method for detecting intracellular cytochrome c based on near-infrared fluorescence lifetime imaging, which comprises the following steps: mixing a gold nanorod particle solution marked with a nucleic acid aptamer and a near-infrared organic fluorescence molecular solution marked with a nucleic acid aptamer, centrifuging, dispersing the obtained precipitate in a buffer solution, and drying to obtain the intracellular cytochrome c based on near-infrared fluorescence lifetime imaging. The nucleic acid aptamers marked on the near-infrared organic fluorescent molecules are nucleotide sequences specifically combined with the cytochrome c, and the nucleic acid aptamers marked on the gold nanorod particles are nucleotide sequences capable of being combined with the nucleic acid aptamers marked on the near-infrared organic molecules; incubating and mixing the detection agent and the cells for at least 20 hours, exciting by adopting near-infrared light after the cells uptake the nano detection system, and testing the fluorescence lifetime in a near-infrared window; and obtaining the content of the cytochrome c in the cell according to a pre-detected relationship between the fluorescence lifetime and the content of the cytochrome c.

Description

technical field [0001] The embodiment of the present invention relates to the field of chemical technology, especially a method for detecting intracellular cytochrome c based on near-infrared fluorescence lifetime imaging. Background technique [0002] Cytochrome c is a common substance in biochemical reactions, especially an important biomarker in the early apoptosis process of cells. At present, cytochrome c detection methods mainly include ELISA method, Western blot analysis, mass spectrometry, optical / electrochemical detection methods, etc. Although these methods achieve highly sensitive detection of cytochrome c, they cannot directly detect intracellular cytochrome c. The development of a direct detection method for cytochrome c in cells has great significance in biological detection. [0003] In recent years, the development of nucleic acid aptamer technology and the development of nanotechnology have provided new ideas for the detection of intracellular cytochrome c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6428G01N2021/6432
Inventor 薛彬王丹周沧涛阮双琛
Owner SHENZHEN TECH UNIV
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