Fluorescent probe capable of simultaneously detecting two kinds of HIVDNA sequences based on triple-helix molecular beacons
A fluorescent probe and fluorescence spectroscopy technology, applied in the field of molecular detection, can solve problems such as application limitations, long synthesis time, and harsh synthesis conditions
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Embodiment 1
[0024] Principle design of a fluorescent probe based on HIV gene detection
[0025] like figure 1 As shown, the probe consists of two nucleic acid strands: a complementary binding strand to the target HIV-DNA, designated as "cDNA" in this work. The other is the synthetic chain of the fluorescent probe, which is named "P". Part of the poly-AT dsDNA contains a rich guanine sequence. + It can be folded into a G-quadruplex under the same action. At first, the cDNA and the G-rich sequence cannot be folded due to base mismatch, and the sequence is in a locked state. In the absence of target, the G-quadruplex does not function, so the synthesized copper nanoparticles exhibit weaker fluorescence signals. When the target is added, the target HIV-DNA can compete with the cDNA, and the released G-rich sequence can be folded into a G-quadruplex, and the G-quadruplex has the ability to light up the fluorescence of copper nanoparticles. As more and more targets are added, more and more ...
Embodiment 2
[0027] Feasibility determination of a fluorescent probe based on detection of HIV gene
[0028] (1) Fluorescence spectrum test
[0029] The test environment was carried out in 10 mM MOPS buffer (150 mM NaCl, pH 7.5). Under optimal optimized conditions, 40 μL of dsDNA (0.5 μM) and 40 μL of cDNA (1 μM) were mixed together. After heating in a water bath at 90° C. for 10 minutes, cool to room temperature, and start incubation after adding AA (2 mM) and copper sulfate (500 μM). Subsequently, various concentrations of HIV-DNA entered the solution and reacted for 30 minutes, and 20 μL of K + Add at the end to bring the final volume to 200 μL. Finally, the fluorescence intensity was recorded after 60 minutes of reaction. Detection conditions: excitation wavelength: 340nm, emission wavelength: 550nm; excitation slit: 5.0nm, emission slit: 10.0nm. like figure 2 As shown, from bottom to top, when only the fluorescence of dsDNA-CuNPs was analyzed, a weak fluorescence intensity was ...
Embodiment 3
[0035] An optimization experiment based on fluorescent probe for detecting HIV gene
[0036] like Figure 5 As shown, in order to achieve the best sensing performance of the designed experimental scheme, the experiment has to adjust the pH value of the reaction, reaction time, AA concentration, Cu 2+ Concentration, K + Concentration etc. were optimized. Under acidic conditions, only weak fluorescence intensity was detected, while the best fluorescence intensity of dsDNA-CuNPs was obtained at pH 7.5. As a reducing agent for the synthesis of copper nanoparticles, AA is a key factor in the whole synthesis process. The results show that with the increase of AA concentration, the fluorescence intensity of copper nanoparticles is significantly enhanced, and reaches the highest value when the concentration of AA is 2mM. Therefore, the use of 2nM AA was optimal in this study. In addition, Cu 2+ Concentration is also an important factor in the synthesis of fluorescent copper nanop...
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