Method for Raman-fluorescence dual-mode detection of calcium ions based on dual-mode nanoprobes

A nanoprobe and dual-mode technology, applied in the fields of nanomaterials and life sciences, can solve problems such as easy hydrolysis, achieve stable properties, low synthesis and detection costs, and easy loading into cells

Active Publication Date: 2022-04-05
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, acetoxymethyl ester (AM) probes are prone to hydrolysis and need to be kept dry during use

Method used

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  • Method for Raman-fluorescence dual-mode detection of calcium ions based on dual-mode nanoprobes
  • Method for Raman-fluorescence dual-mode detection of calcium ions based on dual-mode nanoprobes
  • Method for Raman-fluorescence dual-mode detection of calcium ions based on dual-mode nanoprobes

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] (1) Boil 100mL of 1mM chloroauric acid solution, add 15mL of 1% sodium citrate solution and stir vigorously. After reacting for 10min, the color of the solution turns wine red. After terminating the reaction, cool to room temperature to obtain the gold seed solution. Gold seeds were stored at 4°C until use. The gold species were characterized by TEM electron microscope and ultraviolet-visible absorption spectrum, the results are as follows figure 1 , where A is a TEM electron microscope image, and B is an ultraviolet-visible absorption spectrum image.

[0071] (2) Mix 200 μL of 3 mM silver nitrate solution and 100 μL of 0.1 M ascorbic acid solution evenly, and quickly add to 20 mL of 0.25 mM chloroauric acid solution containing 200 μL of gold species. The reaction solution was centrifuged at 4000 rpm for 15 min to obtain AuNSs, which were resuspended in 2 mL of 0.05% Tween-20. The nano-venus was characterized by TEM electron microscope and ultraviolet-visible absorpti...

Embodiment 2

[0078] Embodiment 2 Hybridization experiment of substrate chain (EtNa-Sub) and EtNa-Enz-AuNSs

[0079] A certain concentration of EtNa-Sub was added to the 1 nM EtNa-Enz-AuNSs solution. The mixture was incubated at 37°C for 3h, then the mixture was centrifuged at high speed (5000rpm, 15min) to remove excess substrate chains, and resuspended in 10mM Tris-HCl buffer (80mM LiCl, 0.01%Tween-20, pH 8.0) to obtain dual-mode nanoprobes. In order to obtain the maximum loading capacity, the concentration of EtNa-Sub solution was changed to 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 35 μM and 50 μM for optimization, and the SERS enhancement of Cy5 in AuNSs was used to prove the relationship between EtNa-Sub and EtNa-Enz-AuNSs hybridize. The result is as Figure 5 shown.

[0080] Such as Figure 5 , with the increase of EtNa-Sub concentration, Cy5 1366cm -1 The intensity of the characteristic peaks increases significantly. When the concentration reaches 35 μM, the intensity of the characteri...

Embodiment 3

[0081] Embodiment 3 Stability experiment of dual-mode nanoprobe

[0082] (1) Characterization of salt stability of dual-mode nanoprobes by UV-vis spectroscopy

[0083] The prepared dual-mode nanoprobe was centrifuged and resuspended in PBS buffer containing different NaCl concentrations (200mM, 400mM, 600mM, 1000mM), vortexed and mixed, and after standing for 10min, the dual-mode probe was detected by UV-visible absorption spectrometer. Mode nanoprobe spectrum changes, results as Image 6 As shown in A.

[0084] (2) Evaluation of ribozyme and glutathione stability of dual-mode nanoprobes by fluorescence experiments

[0085] There are a variety of enzymes and sulfhydryl compounds in cells, which can cut nucleic acids or compete for thiolated nucleic acids. Therefore, the stability of probes is an important prerequisite for ensuring accurate experimental results in cells. Four groups of nanoprobes were placed in a 96-well fluorescent plate at 37°C. After the sample solution w...

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Abstract

The invention discloses a Raman-fluorescent dual-mode nano-probe for detecting calcium ions in vitro and in living cells or imaging. The detection method is based on the "on-off" conversion of surface-enhanced Raman scattering and fluorescence resonance energy transfer, by assembling Ca on nano-venus 2+ The specific enzyme chain and its partially complementary substrate chain modified with fluorescent groups construct dual-mode nanoprobes. This method combines the advantages of SERS ultra-sensitivity and fluorescence visualization, has good specificity and sensitivity, and has good versatility in various cell lines.

Description

technical field [0001] The invention relates to the fields of nanomaterials and life sciences, in particular to a method for Raman-fluorescence dual-mode detection of calcium ions based on a dual-mode nanoprobe. Background technique [0002] Ca 2+ It not only plays a pivotal role among many second messengers, but also participates in or coordinates the metabolism of other second messengers and the regulation of cell physiological functions. Numerous studies have found that intracellular Ca 2+ Dysregulation of homeostasis is a conserved biochemical event in apoptosis, suggesting that Ca 2+ Involved in the signal transduction process in apoptosis. When a cell is stimulated, the cellular calcium store Ca 2+ Released into the cytosol, the Ca in the cytosol 2+ Increased concentration stimulates related pathways and induces apoptosis. About Ca 2+ There are also many problems to be solved urgently in the understanding of the relationship with apoptosis, so tracking intracell...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64G01N21/65C12Q1/25
CPCG01N21/658G01N21/6458G01N21/6428C12Q1/25G01N2333/9005
Inventor 马小媛李晨彪王周平
Owner JIANGNAN UNIV
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