DNA biped walker signal amplifiers, nanoelectrode-based biosensors, using method thereof, and application thereof
A signal amplifier and biosensor technology, applied in the field of biosensors, can solve problems such as insufficient sensitivity, large sample demand, and complicated methods, and achieve the effects of excellent signal amplification capability, small sample demand, and high detection sensitivity
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Embodiment 1
[0112] This example is used to exemplarily illustrate the preparation process of the biosensor provided by the present invention and verify its selectivity and stability when miRNA-21 is used as the target sequence.
[0113] (1) Preparation of biosensors
[0114] Table 1
[0115]
[0116] *In the nucleic acid probe FS, the underlined ones with methylene blue attached at the 3' end A base.
[0117] **In the nucleic acid probe HP, the 5' end has a thiol modification.
[0118] (1) Dissolve DNA probes W1, W2, and FS (see Table 1 for specific sequences) into 50 μM solutions with 10 mM Tris-HCl (pH 7.4) buffer; min) for 5 minutes, and then slowly cooled to room temperature (cooling rate of 4°C / min) for annealing. After annealing, the probe W1 and W2 solutions were diluted to 100 nM with Tris-HCl (pH 7.4) buffer and mixed, and then added to the probe FS solution (final concentration 2 μM) and mixed.
[0119] (2) Dissolve the DNA probe HP (see Table 1 for the specific sequence...
Embodiment 2
[0133] This example is used to illustrate the application of the biosensor provided by the present invention in the detection of target miRNA.
[0134] miRNA-21 was selected as the target sequence, and it was formulated into solutions with different concentration gradients using 10mM Tris-HCl (pH7.4) buffer solution, and the biosensor obtained in experiment (1) in Example 1 contained 100nM W1 probe, Incubate for 2 hours in a mixed solution of 100 nM W2 probe, 2 μM FS probe and different concentrations of the target sequence. Then in PBS buffer (10mM, 100mM NaCl, 5mM MgCl 2 ,pH7.4) to scan the SWV figure, the results are as follows Figure 7A shown. When the concentration of miRNA-21 increased from 0.1fM to 50pM, the peak current of MB also gradually increased, and the relationship between the peak current and the concentration of miRNA-21 was as follows Figure 7B As shown, the logarithm of peak current and concentration is linear, and the linear equation is I(pA)=10.59log ...
Embodiment 3
[0136] This example is used to illustrate the application of the biosensor provided by the present invention in the detection of samples containing target miRNA.
[0137] Human cervical cancer cells (Hela, purchased from the Cell Bank of the Chinese Academy of Sciences, Shanghai) and breast cancer cells (MCF-7, purchased from the Cell Bank of the Chinese Academy of Sciences, Shanghai) were selected as test samples. Place sample cells at 37 °C, 5% CO 2 The medium is cultured and amplified under certain conditions, and the medium is DMEM medium containing 10% by weight of fetal bovine serum, 100 μg / mL penicillin, and 100 μg / mL streptomycin. Total RNA was extracted using Trizol reagent (purchased from Sangon Bioengineering (Shanghai) Co., Ltd.).
[0138] The specific extraction method is: the cells (amount of about 1 × 10 7 -5×10 7 1), transfer to a 1.5ml centrifuge tube, add 1ml Trizol, mix well, let stand at room temperature for 5min, add 0.2ml chloroform, shake for 15s, let...
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