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A detection method and sample technology are applied in the visual detection field of PSA to achieve the effects of low price, convenient operation and high sensitivity
Inactive Publication Date: 2018-06-01
ZHEJIANG UNIV OF TECH
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Problems solved by technology
The method of measuring PSA by observing the color change with the naked eye has not been reported so far, and it belongs to a new type of PSA detection technology
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Embodiment 1
[0020] Embodiment 1: the study of the critical value of hydrogen peroxide that changes the color of nano-gold colloid
[0021] A 0.1 mM chloroauric acid solution was prepared by dissolving chloroauric acid trihydrate in 0.2 mM, pH 6.5 sucrose aqueous solution.
[0022] Firstly, different concentrations (40, 80, 120, 160, 200, 240 μM) of H were added to the microtiter plate. 2 o 2 Aqueous solution, 100 microliters per well, the difference between each adjacent well is 40 μM. Then add 100 microliters of 0.1 mM chloroauric acid solution to each well to make H 2 o 2 The final concentrations were 0, 20, 40, 60, 80, 100, 120 μM, respectively. Let it stand at room temperature for 30 minutes, and observe the color change with the naked eye.
[0023] The critical value is identified as: H in the detection hole 2 o 2 The final concentration (1-fold dilution after adding an equal volume of chloroauric acid), rather than the amount added.
[0024] Such as figure 1 show that when ...
Embodiment 2
[0025] Embodiment 2: Visual detection of PSA
[0026] The steps are consistent with the ELISA double antibody sandwich method. At the final signal amplification, when the test sample has PSA, the H at the critical value 2 o 2 Will be consumed by streptavidin-HRP, with H 2 o 2When the particle size of gold nanoparticles closely related to the concentration changes, the color will change from red to blue, realizing the visual detection of PSA.
[0027] The operation method is as follows: add 100 μL of mouse anti-PSA monoclonal antibody to a 96-well microtiter plate, dilute it with PBS at a volume ratio of 1:1000, and embed overnight at 4°C. After washing the plate 3 times with wash buffer (0.01M PBS+1‰TWEEN 20), block with blocking buffer (1 mg / mL BSA (bovine serum albumin) dissolved in PBS) for 1 hour at room temperature. The plate was washed 3 times with wash buffer, and different concentrations (10 -8 g / mL, 10 -9 g / mL, 10 -10 g / mL, 10 -11 g / mL, 10 -12 g / mL) PSA stan...
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Abstract
The invention discloses a visual inspection method for PSA. According to the method, only the color development system of ELISA needs to be replaced and 100 [mu]L of H2O2 with a concentration of 200 [mu]M and 100 [mu]L of a chloroauric acid solution with a concentration of 0.1 [mu]M are added into a color development step; when the concentration of PSA in a sample is higher than 10<-10> g/mL, a reaction solution is blue and a positive detection result is obtained; and when the concentration of PSA in a sample is lower than 10<-10> g/mL, a reaction solution is red and a negative detection result is obtained. The lowest concentration for acquirement of the positive detection result is lower compared with conventional ELISA methods. The detection results can be obtained through judgment withthe naked eyes without usage of ELIASA and reference substances. The visual inspection method for PSA in the invention is high in sensitivity and simple to operate and is of great significance to diagnosis, treatment and prognosis of the prostatic cancer.
Description
(1) Technical field [0001] The invention relates to a PSA with a minimum detection limit of 10 -10 The g / mL determination method is characterized by the use of naked eyes to observe the color change to judge the test result. (2) Background technology [0002] PSA (Prostate specific antigen) is a single-chain glycoprotein encoded by hKLK3 gene, which is secreted by prostate epithelial cells. It is mainly secreted into prostatic fluid, hydrolyzes semen coagulation protein, and liquefies semen. PSA is higher in the prostate and less in the serum. Slightly higher than normal levels of PSA can be detected in the serum of prostate cancer patients. As a tumor marker of prostate cancer, PSA is monitored clinically and plays an important role in the prevention, treatment and prognosis of prostate cancer. Using the double-body sandwich ELISA detection technology, 0-4ng / mL PSA can be detected from male serum at present. When the PSA concentration is lower, it will not be detected du...
Claims
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