Telomerase activity detection kit and detection method
A technology of activity detection and telomerase, applied in measurement devices, instruments, scientific instruments, etc., can solve the problem of inability to detect telomerase activity
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[0051] II. Preparation and Characterization of Quantum Dot Ratiometric Fluorescent Probes
[0052] 1. Preparation
[0053] According to previous literature (Mao G., Q. Cai, F. Wang, C. Luo, X. Ji & Z. He, One-Step Synthesis of Rox-DNA Functionalized CdZnTeS Quantum Dots for the VisualDetection of Hydrogen Peroxide and Blood Glucose . Anal Chem 89, 11628- 11635(2017)), Preparation of CdZnTeS quantum dot ratiometric fluorescent probes by a one-pot hydrothermal method. The specific preparation steps are as follows.
[0054] A. Design and synthesis of DNA sequence modified by Rox dye and phosphorothioate
[0055] The DNA sequence for the design of Rox dyes and phosphorothioate co-modification is as follows: G*G*G*G*G*G*G*G*AAAAAAAACCTTCCTCCGCAATACTCCCCCAGGTAAA-Rox (5'→ 3', where * indicates phosphorothioate modification) . That is, the modified DNA sequence is modified with phosphorothioate at the 5' end and Rox dye at the 3' end, or it can be modified with phosphorothioate ...
Embodiment 1
[0086] In this example, the telomerase sample obtained from HeLa cells is used to illustrate the quantitative detection method of telomerase activity of the present invention, wherein the color reaction is carried out in Tris buffer solution of Rox-CdZnTeS quantum dot ratio fluorescent probe.
[0087] (1) Obtaining telomerase samples
[0088] HeLa cells were cultured in DMEM medium containing 10% heat-inactivated FBS. After harvesting cells with trypsin, 1 x 10 6 The cells were collected into 1.5mL EP tubes and centrifuged at 1000rpm for 10 minutes. Cells were washed once with phosphate-buffered saline (pH=7.4), centrifuged again, and dispensed in 200 µL of ice-cold 1× CHAPS lysis buffer. Then, the cells were incubated on ice for 30 minutes, centrifuged at 12,000 rpm for 20 minutes at 4ºC, and the resulting supernatant was the telomerase sample. Transfer the supernatant to a fresh tube and store at -80ºC.
[0089] (2) Formation of hemin / G-quadruplex
[0090] First, add 1 ...
Embodiment 2
[0098] Embodiment 2: test paper sensor method
[0099] Steps (1) to (3) of this example are the same as those of Example 1, but in step (4) the color reaction is carried out with a test paper sensor. The differences from Example 1 are mainly described below.
[0100] Specifically, prior to step (4), the Rox-CdZnTeS quantum dot ratio fluorescent probe was dissolved in Tris buffer (10 uL) to form a Rox-CdZnTeS quantum dot ratio fluorescent probe solution (200 nM), and passed The test paper was immersed in the Rox-CdZnTeS quantum dot ratio fluorescent probe solution, and air-dried at room temperature for 5 minutes to form a test paper sensor.
[0101] Step (4): Use a microburette to take 10 uL of the mixed system after the reaction in step (3), drop it on the test paper sensor, and react with the Rox-CdZnTeS quantum dot ratio fluorescent probe on the test paper sensor for 5 minutes to display the fluorescent color.
[0102] Step (5): Production of standard color cards
[0103] ...
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