Method for determining contents of biological marker and DNA through direct-reading portable glucometer
A biomarker and blood glucose meter technology, which is applied in measuring devices, biological testing, instruments, etc., can solve the problem of less blood collection, and achieve the effects of sensitive detection, low analysis cost, and simple operation
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Embodiment 1
[0028] Determination of phosphorylated p53 protein (p53 15 )Proceed as follows:
[0029] (1) Immunological reaction: Add 100 μL of magnetic nanocomposite Fe to nine 0.5 mL plastic centrifuge tubes 3 o 4 -p53 15 Ab 1 , and then add 100 μL concentration of 0.2ng / mL, 0.5ng / mL, 1ng / mL, 2ng / mL, 5ng / mL, 10ng / mL, 20ng / mL, 50ng / mL, 100ng / mL to the above nine centrifuge tubes respectively mL of p53 15 , after incubation for 50 minutes, magnetic separation; after washing with phosphate buffer solution for more than 2 times, add 100 μL liposome complex marker p53 15 Ab 2 -liposome, incubated for 40 minutes, magnetically separated;
[0030] (2) Determination: Add 10 μL of 10 mg / mL Triton X-100 to the centrifuge tube to release the glucose molecules from the lumen of the captured liposomes; then remove the immune complex by magnetic separation, and take 20 μL of supernatant droplets On the test strip of the blood glucose meter, directly read the reading of the blood glucose meter,...
Embodiment 2
[0032] Fe used in embodiment 1 3 o 4 -p53 15 Ab 1 preparation of
[0033] 1), 200μL 5.0mg / mL carboxylated magnetic nanoparticles Fe 3 o 4 -COOH, dispersed in 1.0 mL of pH 5.2 sulfo fatty acid methylate containing 400 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 100 mM N-hydroxysuccinimide In ester sodium salt buffer solution, magnetically separate after activation for 30min, and wash with phosphate buffer solution several times to remove excess 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N - hydroxysuccinimide;
[0034] 2), the Fe 3 o 4 -COOH dispersed in 1.0 mL of 20 μg / mL p53 15 Ab 1 Reacted for 12 h in the medium, washed 3 times with phosphate buffer solution after magnetic separation, and the obtained magnetic nanocomposite Fe 3 o 4 -p53 15 Ab 1 Disperse in phosphate buffer solution containing 1% bovine serum albumin and store at 4°C for later use.
Embodiment 3
[0036] p53 used in Example 1 15 Ab 2 - Preparation of liposome composite markers
[0037] The first step is to prepare biotin-labeled liposomes for embedding glucose: Weigh 124 mg of hydrogenated soybean lecithin, 25 mg of cholesterol, and 6 mg of distearylethanolamine-polyethylene glycol-biotin into a 50 mL round-bottomed flask. Stearyl ethanolamine-polyethylene glycol-biotin molar ratio 159:64:2, dissolved in 15mL mixed solvent, mixed solvent is a mixture of chloroform, isopropyl ether and methanol, chloroform:isopropyl ether:methanol volume ratio=6: 6:1, sonicate under nitrogen protection at 45°C until the mixture is evenly dispersed, then add 5mL of 5mg / mL glucose solution at 45°C to the above mixture and sonicate for 5 minutes to form an emulsion with uniformly dispersed particles; then at 45°C Rotary evaporation under reduced pressure to remove the organic solvent to obtain a gel-like dispersion; the resulting liposome solution was incubated in a water bath at 45°C for...
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