Rate fluorescent probe for detecting PTK7 and preparation method of rate fluorescent probe for detecting PTK7

A ratiometric fluorescent probe and fluorescence technology, which is applied in the field of nanomaterials, fluorescence ratio technology and biological analysis and detection, can solve the problems of lack of selectivity and limit the development of fluorescence measurement

Pending Publication Date: 2020-06-05
XUZHOU MEDICAL UNIV
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  • Abstract
  • Description
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However, despite the excellent optical properties, the lack

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  • Rate fluorescent probe for detecting PTK7 and preparation method of rate fluorescent probe for detecting PTK7
  • Rate fluorescent probe for detecting PTK7 and preparation method of rate fluorescent probe for detecting PTK7
  • Rate fluorescent probe for detecting PTK7 and preparation method of rate fluorescent probe for detecting PTK7

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Preparation of y-CDs, b-CDs and Fe 3 o 4 Nanoparticles, the steps are as follows:

[0044] For b-CDs, 1.2 g of citric acid monohydrate and 600 μL of diethylenetriamine were dissolved in 20 mL of deionized water, and ultrasonicated for 20 minutes to fully dissolve; the above solution was transferred to a 30 mL autoclave, and reacted at 180 °C for 4 hours; After the reaction is completed, cool it down to room temperature, take out the product and add ethanol and acetone alternately until it precipitates, centrifuge at 8000 rpm for 10 min, discard the supernatant, and dry the solid in vacuum at 60°C to obtain a light yellow powder, which is sealed and stored for later use to obtain b-CDs . Its TEM picture is as follows figure 2 As shown, its fluorescence and UV absorption diagrams are shown in figure 2 .

[0045] y-CDs was made from o-phenylenediamine (0.32 g) and 4-aminobutyric acid (0.31 g) as raw materials, added 20 mL of deionized water, and dissolved by ultraso...

Embodiment 2

[0048] prepared in Fe 3 o 4 The complementary strand (cDNA) of the cross-linked PTK7 aptamer (APT) on the surface of the nanoparticle is used to specifically bind the probe of APT labeled with yellow fluorescent emission carbon dots (y-CDs), and the steps are as follows:

[0049] Step 1. Preparation of y-CDs-labeled APT (y-CDs-APT)

[0050] Step (1), measure 500 μL of the yellow carbon dot solution (y-CDs) prepared in Example 1 and 500 μL of PBS (pH7.2-7.4) buffer.

[0051] In step (2), add 0.0193 g of EDC and 0.0226 g of NHS, adjust the pH to 7.5 with 1M NaOH after shaking at room temperature for 30 min;

[0052] Step (3): After centrifuging APT at 4000 rpm for 1 min, add 25 μL of PBS (pH7.2-7.4) buffer solution to make it 100 μM, then heat 100 μM cDNA at 95 °C for 4 min, and cool in an ice bath for 4 min. Leave it at room temperature for a while.

[0053] Step (4), add 5 μL of 100 μM APT to the solution in step (2) and shake at room temperature overnight to obtain y-CDs-...

Embodiment 3

[0056] Example 3 Preparation of Standard Curve PTK7:

[0057] The steps are as follows: Take 600 μL of different concentrations of PTK7 (0, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10, 20, 50, 100, 200 ng mL -1 ), each added 20 U of DNase I, and the reaction system was incubated at 37 °C for 30 min. Add 20 μL of b-CDs and mix well by shaking. Each concentration was operated in triplicate in parallel, the excitation wavelength of fluorescence measurement was 380 nm, the emission wavelength of y-CDs was 580 nm, and the emission wavelength of b-CDs was 460 nm. 580 ) and b-CDs (I460 ) fluorescence ratio I 580 / I 460 The concentration correlation with PTK7 quantifies its concentration. see results Figure 6 , 7 shown.

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Abstract

The invention relates to a rate fluorescent probe for detecting PTK7 and a preparation method of the rate fluorescent probe for detecting PTK7. The probe adopts Fe3O4 nanoparticle as a quenching agentand a magnetic separation agent, a complementary chain(cDNA) of a surface-crosslinked PTK7 aptamer(APT) is specially bound with APT labelled with yellow fluorescent emission carbon points(y-CDs), fluorescent is quenched, PTK7 and cDNA are competitively bound with the APT to enable the APT to be divorced from the Fe3O4 nanoparticles, fluorescent is restored, and a fluorescent inner filter effect is used for quenching blue fluorescent launching carbon dots(b-CDs), so as to obtain rate fluorescence detection PTK7.Exonuclease I is used for increasing the fluorescent rate of the y-CDs to the b-CDs, and the ratio I580/I460 of the two fluorescence intensity and the concentration log10 of a substance to be detected show linear relationship. According to the ratio of the two fluorescence intensity, the PTK7 biomolecule is quantitatively determined, and the method is high in sensitivity, and simple to detect, and has high selectivity and high affinity for immune reaction.

Description

technical field [0001] The invention belongs to the fields of nanomaterials, fluorescence ratio technology and biological analysis and detection. Specifically, based on the fluorescence inner filter effect and enzymatic cycle amplification technology, yellow fluorescent carbon dots and blue fluorescent carbon dots are mixed to generate inner filter effect and make the The blue fluorescence is quenched to form a ratiometric fluorescent probe to quantify biomolecules. The ratiometric fluorescent probe used to detect PTK7 is more sensitive than UV absorption and has good selectivity. Background technique [0002] According to statistics, lung cancer has become the first cancer incidence list in China, and its high incidence has put forward new requirements for the innovation of early diagnosis technology. The detection of tumor markers in serum plays an important role in early diagnosis due to its characteristics of less trauma and high sensitivity. As a simple and sensitive d...

Claims

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Application Information

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IPC IPC(8): C12Q1/6818G01N21/64G01N21/31
CPCC12Q1/6818G01N21/6428G01N21/314G01N2021/6432C12Q2525/205C12Q2563/107C12Q2563/137C12Q2521/319
Inventor 马云苏王园刘永杰杨冬芝
Owner XUZHOU MEDICAL UNIV
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