Dual-enzyme-mediated cascade-signal-amplified ampicillin detecting aptasensor
An aptamer sensor, ampicillin technology, applied in the direction of determination/inspection of microorganisms, instruments, biological tests, etc., can solve the problems of expensive reagents, precision instruments and technicians, low sensitivity and specificity, and cumbersome analysis process. Strong scalability, high sensitivity and specificity
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Embodiment 1
[0046] The preparation of embodiment 1 probe
[0047] Take 50 μL of 10 μM DNA1, bio-Apt, and H-DNA priming strands respectively, place them in a metal constant temperature oscillator, and activate at 70 °C for 10 min. After mixing evenly, incubate at 4°C for 2 h to complete the preparation of the probe, and store at 4°C for use. Then 10 μL of SYBR Green I with a final concentration of 5× was added to it, incubated at 4 °C for 30 min, and the fluorescence intensity at 520 nm of the sample was detected under the irradiation of 490 nm excitation light. In order to verify the hybridization between DNA1, bio-Apt, and H-DNA. Select the following samples: a) DNA1 and bio-Apt of equal volume and concentration (10 μL, 10 μM), and 10 μL of water; b) DNA1 and H-DNA of equal volume and concentration (10 μL, 10 μM), and 10 μL of water. The mixture was incubated at 4°C for 2 h. Then, 10 μL of SYBR Green I with a final concentration of 5× was added to the two systems, incubated at 4 °C f...
Embodiment 2T4
[0049] Activity verification of embodiment 2T4 ligase and Exo III
[0050] Test procedure: a) Mix 10 μL of 100 U / mL T4 ligase with 100 μL of detection probe, add 90 μL of PBS buffer solution, incubate at 37 °C for 30 min, and then inactivate at 70 °C for 20 min. After cooling, 10 μL of SYBR Green I with a final concentration of 5× was added, and incubated at 4 °C for 30 min. b) Mix 10 μL of 100 U / mL Exo III with 100 μL of detection probe, add 90 μL of PBS buffer solution, incubate at 37 °C for 30 min, and then inactivate at 70 °C for 20 min. After cooling, 10 μL of SYBR Green I with a final concentration of 5× was added, and incubated at 4 °C for 30 min. c) Take 10 μL of 100 U / mL T4 ligase and 100 μL of detection probe, and incubate at 16 °C for 30 min. Add 10 μL of 100 U / mL Exo III and 80 μL of PBS buffer solution, incubate at 37 °C for 30 min, and then inactivate at 70 °C for 20 min. After cooling, 10 μL of SYBR Green I with a final concentration of 5× was added, and incu...
Embodiment 3
[0052] The optimization of the DNA degradation reaction experimental condition of embodiment 3 double enzyme mediation
[0053] Take 5, 10, 20, 30, and 40 μL of 100 U / mL T4 ligase, 60 μL of detection probe, and incubate at 16 °C for 30 min. Add 10 μL of 100 U / mL Exo III and mix with 80 μL of PBS buffer, incubate at 37 °C for 30 min, and then inactivate at 70 °C for 20 min. After cooling, 10 μL of SYBR Green I with a final concentration of 5× was added, and incubated at 4 °C for 30 min. Detect the fluorescence intensity value of the solution at 520 nm.
[0054] see image 3 A, When the volume of T4 ligase is 10 μL, the fluorescence intensity of the solution reaches a plateau, which is the optimal reaction volume.
[0055] Take 10 μL of 100 U / mL T4 ligase and 60 μL of detection probe, and incubate at 16 °C for 10, 30, 50, 70 and 90 min, respectively. Add 10 μL of 100 U / mL Exo III and mix with 80 μL of PBS buffer, incubate at 37 °C for 30 min, and then inactivate at 70 °C for...
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