Preparation method of DNA nanowire cluster loaded with phenanthroline ruthenium

A technology of nanowires and ruthenium pyroxoline, applied in the field of analytical chemistry, can solve the problems of few biosensing applications, and achieve the effects of good reproducibility, simple synthesis method, and high sensitivity

Inactive Publication Date: 2019-10-01
FUZHOU UNIV
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Problems solved by technology

At present, RCA-based functional nanotechnology has been used in biodetection, drug delivery and bioelectronics (Ouyang et al...

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  • Preparation method of DNA nanowire cluster loaded with phenanthroline ruthenium
  • Preparation method of DNA nanowire cluster loaded with phenanthroline ruthenium
  • Preparation method of DNA nanowire cluster loaded with phenanthroline ruthenium

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specific Embodiment approach

[0029] 1. Design and synthesize the palindromic sequence-encoded rolling circle amplification (RCA) reaction template strand and the corresponding primer strand. RCA amplification template strand sequence from left to right 5' to 3': phosphorylation-GGGCACGGGCAGCTTGCCGGTGGAAGCTAGATGCATCTAGCAAGCGCCGCCACTGATTTCACCGCTTCAAGCTAGATGCATCTAGCAAT; RCA amplification The primer strand sequence is from left to right 5' to 3': GTGGCGGCGC. .

[0030] 2. The ligation and cyclization process of the RCA template strands, the specific steps are as follows: 40 μL of the solution contains RCA template strands with a final concentration of 0.50 μM, 5.0 U μL -1 CircLigase II ssDNA Ligase, 2.5 mM Manganese Chloride (MnCl 2 ) and 1 M betaine, and incubated at 60 °C for 1 hour to form a circularized template strand.

[0031] 3. Start the RCA amplification reaction to self-assemble and synthesize three-dimensional spherical DNA nanowire clusters (DNA NCs). The specific steps are as follows: add 2 μL...

Embodiment 1

[0037] 1. Prepare different sizes of DNA NCs-Ru(phen) 3 2+ The operation process of ECL analysis, the specific steps are as follows:

[0038] (1) DNA NCs of different sizes were synthesized by adjusting different RCA reaction times (3, 10, and 17 hours, respectively).

[0039] (2) The phenanthroline ruthenium (Ru(phen) 3 2+ ) loaded into the double-stranded structure of DNA NCs of different sizes to prepare DNANCs-Ru(phen) 3 2+ :

[0040] A: Take 10 uL DNA NCs and 1.0 uL phenanthroline ruthenium (Ru(phen) 3 2+ ) (concentration of 10 mM) was mixed and added to 380 μL buffer 1, and incubated at 4 °C for 3 hours;

[0041] B: Transfer the above solution to an ultrafiltration centrifuge tube (3 K), and centrifuge at 10700 rpm for 20 minutes in a refrigerated centrifuge;

[0042] C: Collect the prepared DNA NCs-Ru(phen) 3 2+ Beacons were stored in clean centrifuge tubes at 4°C until use.

[0043] (3) Take 10 uL of DNA NCs-Ru(phen) of different sizes prepared above 3 2+...

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Abstract

The invention discloses a preparation method of a DNA nanowire cluster loaded with phenanthroline ruthenium. The preparation method comprises the following steps that 1, a rolling circle amplification(RCA) reaction template chain coded by a palindromic sequence and a corresponding primer chain are designed; 2, the RCA template chain is subjected to connection cyclization by using a DNA ligase; 3,an RCA amplification reaction is started, and products of the RCA amplification reaction are self-assembled into the three-dimensional spherical DNA nanowire cluster (DNA NCs); 4, the phenanthrolineruthenium (Ru(phen)32+) is subjected to high-efficiency loading into a double-chain structure of the DNA NCs to prepare an ECL beacon molecule. The method has the advantages of simplicity in operation, low cost and good reproducibility. The three-dimensional spherical DNA NCs can be used for high-efficiency loading of Ru(phen) 32+ as the ECL beacon molecule, and signal amplification is realized; meanwhile, the stability is high, the biocompatibility is good, and the method is expected to be widely applied to the fields of life science, medical clinical detection and the like.

Description

technical field [0001] The invention belongs to the field of analytical chemistry, and in particular relates to a preparation method of a DNA nanowire cluster loaded with phenanthroline ruthenium. Background technique [0002] Rolling circle amplification (RCA) is a simple and effective in vitro amplification technique, which is essentially an isothermal enzymatic reaction process mediated by certain DNA polymerases. Using a 5' phosphorylated single-stranded DNA (ssDNA) padlock probe as a template, the end fragments are designed to be complementary to the target DNA or RNA. When the target exists, it can hybridize with the end of the probe so that the 5' and 3' ends are infinitely close to each other, and then closed to form a circle under the action of DNA ligase. The circularized padlock probes are then amplified using a DNA polymerase, generating ssDNA or RNA containing tens to hundreds of repeats complementary to the circular template (Zhao et al., Angew. Chem., Int. E...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844C12Q1/6806
CPCC12Q1/6806C12Q1/6844C12Q2531/125
Inventor 林振宇张莹罗芳郭隆华邱彬
Owner FUZHOU UNIV
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