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Polypeptide Raman probe for targeted recognition of collagen and preparation method of polypeptide Raman probe

A collagen and target recognition technology, applied in Raman scattering, measuring devices, instruments, etc., can solve the problems of inaccurate positioning, non-specific adsorption, etc., and achieve narrow Raman slit width, good stability, and anti-photobleaching ability strong effect

Active Publication Date: 2020-05-19
LANZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Through specific design and ratio screening, the probe solves the problem of non-specific adsorption and inaccurate positioning of nanoprobes in tissue imaging, and provides a new tool for Raman imaging of tissues in vitro and in vivo. In-depth study of collagen-related diseases provides a competitive assay

Method used

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  • Polypeptide Raman probe for targeted recognition of collagen and preparation method of polypeptide Raman probe
  • Polypeptide Raman probe for targeted recognition of collagen and preparation method of polypeptide Raman probe
  • Polypeptide Raman probe for targeted recognition of collagen and preparation method of polypeptide Raman probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Polypeptide Raman probe Cys-Ahx-(Gly-Pro-Hyp) 7 - Preparation of Ag nanoparticles-4-MBN

[0045] The designed peptide Raman probe is: Cys-Ahx-(Gly-Pro-Hyp) 7 -Ag nanoparticles-4-MBN.

[0046] Preparation of peptide Raman probes:

[0047] (1) Solid phase synthesis of collagen targeting polypeptide Cys-Ahx-(Gly-Pro-Hyp) 7 :

[0048] ① Add 100mg of Rink ammonia resin to a reactor with a sieve plate, and use 5mL of dichloromethane to swell the resin;

[0049] ②The N-terminal Fmoc protecting group was removed by 20% piperidine / N,N-dimethylformamide (DMF) solution, and the protecting group was completely removed by color reaction detection;

[0050] ③ Dissolve the amino acid (4eq) protected by Fmoc at the N-terminal, HOBt (4eq) and HBTU (4eq) in DMF, activate at low temperature for 20 minutes, add DIEA (6eq) dropwise to the solution, mix the solution and add it to the reactor, Response 3hrs.

[0051] ④ After the reaction, the reaction solution was extracted f...

Embodiment 2

[0057] Example 2 Preparation of Polypeptide Raman Probe Cys-Ahx-LRELHLNNNG-Ag Nanoparticle-R1

[0058] The designed polypeptide Raman probe is: Cys-Ahx-LRELHLNNNG-Ag nanoparticle-R1, and the R1 is S-(4-trimethylsilylethynyl-phenyl) thioacetate.

[0059] Preparation of peptide Raman probes:

[0060] (1) Solid phase synthesis of collagen targeting polypeptide Cys-Ahx-LRELHLNNNG:

[0061] ① Add 100mg of Rink ammonia resin to a reactor with a sieve plate, and use 5mL of dichloromethane to swell the resin;

[0062] ②The N-terminal Fmoc protecting group was removed by 20% piperidine / N,N-dimethylformamide (DMF) solution, and the protecting group was completely removed by color reaction detection;

[0063] ③ Dissolve the amino acid (4eq) protected by Fmoc at the N-terminal, HOBt (4eq) and HBTU (4eq) in DMF, activate at low temperature for 20 minutes, add DIEA (6eq) dropwise to the solution, mix the solution and add it to the reactor, Response 3hrs.

[0064] ④ After the reaction, t...

Embodiment 4

[0083] Example 4 In vitro tissue imaging of two kinds of polypeptide Raman probes

[0084] 1. Raman probe Cys-Ahx-(Gly-Pro-Hyp) 7 -Ag nanoparticles-4-MBN tissue imaging in vitro

[0085] Add 100 μL, 400 pM polypeptide Raman probe Cys-Ahx-(Gly-Pro-Hyp) prepared in Example 1 to the frozen section of rat tail tissue 7 -Ag nanoparticles-4-MBN solution and the random probe Cys-Ahx-PGGOOPPGGOOPPGGOOPPGOG-Ag nanoparticles-4-MBN prepared in Comparative Example 1 were stained, and incubated at 4° C. for 2 hrs. After aspirating the staining solution, wash 5 times with 1×PBS to wash away unbound probes. Using a Raman microscope to observe and take pictures, the results are as follows figure 1 Shown, where a is the bright field image of the random probe Cys-Ahx-PGGOOPPGGOOPPGGOOPPGOG-Ag nanoparticles-4-MBN on rat tail tissue staining; b is the random probe Cys-Ahx-PGGOOPPGGOOPPGGOOPPGOG-Ag nanoparticles-4- Dark field image of rat tail tissue stained by MBN; c is polypeptide Raman prob...

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Abstract

According to the invention, Raman characteristics and a probe for accurate detection are combined through a specific method for the first time; the Raman probe Cys-Ahx-(Gly-Pro-Hyp) 7-Ag nanoparticles-4-MBN and the Raman probe Cys-Ahx-LRELHLNNG-Ag nanoparticles-R1 for targeted detection of collagen are prepared, and the Raman probe Cys-Ahx-(Gly-Pro-Hyp) 7-Ag nanoparticles-4-MBN and the Raman probeCys-Ahx-LRELHLNNG-Ag nanoparticles-R1 are prepared, wherein R1 and 4-MBN are signal molecules, and R1 is thioacetic acid S-(4-trimethylsilyl ethynyl-phenyl) ester; the invention also provides a preparation method of the polypeptide Raman probe. The preparation method comprises the following steps: synthesizing collagen targeting polypeptide through a solid phase, and simultaneously modifying Ag nanoparticles with signal molecules and the collagen targeting polypeptide. The polypeptide Raman probe provided by the invention has remarkably enhanced Raman signals, has high specific binding capacity to collagen, is simple to prepare and convenient to detect, can be used for in-vitro and in-vivo tissue imaging, and provides a potential detection tool for research of collagen-related diseases.

Description

technical field [0001] The invention relates to a polypeptide Raman probe for targeted recognition of collagen and its preparation and imaging methods, belonging to the technical field of biological detection. Background technique [0002] Collagen is a biological polymer, the main component of animal connective tissue, and the most abundant and widely distributed functional protein in mammals, accounting for 25%-30% of the total protein, and even up to 80% in some organisms . There are many types of collagen, and the common types are type Ⅰ, type Ⅱ, type Ⅲ, type Ⅴ and type Ⅺ. Collagen has been widely used in food, medicine, tissue engineering, cosmetics and other fields due to its good biocompatibility, biodegradability and biological activity. [0003] Collagen is a key structural and functional protein of the human body. It is the main component of connective tissues such as skin, bones, and tendons. It plays an important role in basic life activities such as cell migra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/65
CPCG01N21/658
Inventor 肖建喜粘琳格
Owner LANZHOU UNIVERSITY
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