Fluorescent probe CP-DNA without quenching agent and preparation method and application thereof, and method for detecting trace Pb2+
A CP-DNA and fluorescent probe technology, applied in the field of biological detection, can solve the problems of low reliability and high cost, and achieve the effect of high reliability, low cost and high sensitivity
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Embodiment 1
[0052]This example provides a quencher-free fluorescent probe CP-DNA, the structural formula of which is formula (I):
[0053] , the average molecular weight of the probe is 13633.
[0054] The preparation method of this probe is as follows:
[0055] (1) Activate the carboxyl group of polyfluorene CP (average molecular weight 7168, n>1) represented by formula (II) by using NHS and DCC, the molar ratio of polyfluorene CP, NHS and DCC is 1:4:8
[0056] (2) Design a probe molecule whose DNA sequence is GGAAGGTGTGGAAGG, and modify the amino group at the 5' end to obtain a modified DNA as shown in formula (III).
[0057] (3) The activated polyfluorene CP and the modified DNA were reacted at a molar ratio of 1:3 at a temperature of 25 ℃ to 30 ℃ and stirred for 24 hours. The reaction product was purified by reverse high-performance liquid chromatography, and the finally obtained The product is a fluorescent probe molecule represented by formula (I).
[0058] If you want to obtai...
Embodiment 2
[0060] Taking the non-quencher fluorescent probe CP-DNA provided in Example 1 as an example, it detects Pb 2+ effect is measured.
[0061] Commercially purchased Pb(NO 3 ) 2 For the analyte, prepare a 1 mM solution. Take two 1.5 mL centrifuge tubes, add the non-quencher fluorescent probe CP-DNA and PBS buffer solution to one of the centrifuge tubes; add the non-quencher fluorescent probe CP-DNA, Pb(NO 3 ) 2 aqueous solution and PBS buffer solution. The total reaction volume in the two centrifuge tubes was 50 μL, the concentration of the non-quencher fluorescent probe CP-DNA was 250 nM / L, and the fluorescence value was measured immediately after reacting at 30 °C for 10 minutes. The measurement conditions of the fluorescence spectrum are: the voltage is 600 v, the excitation wavelength is 340 nm, and the emission scanning range is: 390~520 nm.
[0062] Step 2: Take eight 1.5 mL centrifuge tubes, and add the same concentration of quencher-free fluorescent probe CP-DNA, di...
Embodiment 3
[0069] Taking the non-quencher fluorescent probe CP-DNA provided in Example 1 as an example, Pb in environmental water samples 2+ content was measured.
[0070] Step 1: Take five 1 L glass bottles, add water from the Yangtze River, Yellow River, South China Sea, East Lake, and tap water respectively, and let stand at 25 ℃ to 30 ℃ for 2 weeks. The supernatant was taken by centrifugation (10000 rpm / min), and after centrifugation for 5 minutes, the supernatant was taken and filtered with a 13 μm filter head.
[0071] Step 2: Take six 1.5 mL centrifuge tubes and add equal concentrations of quencher-free fluorescent probe CP-DNA and Pb(NO 3 ) 2 The Pb(NO 3 ) 2 River water solution, the Pb(NO 3 ) 2 Seawater solution, the Pb(NO 3 ) 2 Lake water solution, the Pb(NO 3 ) 2 Tap water solution, Pb(NO 3 ) 2 Ultrapure aqueous solution and PBS solution. Reaction condition and test condition are identical with reaction condition and test condition in example 1 step one.
[0072]...
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