Alkaline phosphatase detection kit and detection method based on double emission lanthanide mof
A detection kit and dual emission technology, applied in the field of analytical chemistry and clinical testing, can solve the problems of difficult high-sensitivity naked-eye visual detection, single fluorescence emission peak, poor monochromaticity, etc., to achieve excellent selectivity, accurate detection results, high accuracy effect
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Embodiment 1
[0043] Application of prepared Tb-BPTC to detect ALP activity in human serum (detection by fluorescence spectrometer)
[0044] Method: Take 50 μL serum, mix 4 μL 50 mM AAP and 5 μL 20 mM MgSO 4 Mixed with it, plus a certain amount of buffer (pH=9.5, 10mM Tris-glycine buffer) to make the total volume reach 0.5mL, put the mixture in a thermostatic shaker (1000rpm), and react at 37°C 60 minutes. After the reaction is complete, add 450 μL of water and 50 μL of Tb-BPTC (2 mgmL -1 ). Using a fluorescence spectrometer, the reaction solution was mixed for 10 minutes and transferred to a cuvette to monitor the fluorescence spectrum.
[0045] In drawing the standard curve, the colorimetric ratio-based assay and the dual-wavelength ratiometric fluorescence-based assay were compared:
[0046] The standard curve of the response of Tb-BPTC to ALP activity directly with the dual wavelength ratio fluorescence as the ordinate is as follows Figure 5 shown.
[0047] Convert spectral data ...
Embodiment 2
[0058] The Tb-BPTC prepared by application, combined with the kit provided by the invention ( Figure 4 a) Detection of ALP activity in human serum (visual detection)
[0059] The procedure is as follows: 4 μL 50 mM AAP, 5 μL 20 mM MgSO 4 , 466 μL of buffer (pH=9.5, 10 mM Tris-glycine buffer) and 25 μL of serum were mixed in a centrifuge tube, placed in a constant temperature shaker (1000 rpm), and reacted at 37° C. for 60 minutes. After the reaction is complete, add 250 μL of water and 250 μL of Tb-BPTC solution (2 mg mL -1 ). The obtained reaction solution can be quickly percolated through the capillary, and the glass fiber test paper in the inner tube of the ultrafiltration can be transfected. After 10 minutes, the glass fiber test paper is taken out with tweezers and transferred to the UV lamp. Under the UV lamp, high sensitivity can be achieved. Detection of naked eye identification (color changes from green to blue as alkaline phosphatase activity changes from 0-8mU m...
Embodiment 3
[0063] The selectivity and anti-interference ability of Tb-BPTC for the determination of ALP activity were tested by fluorescence spectrometer detection method and dual-wavelength ratiometric fluorescence analysis. The specific test parameters are AAP 200μM, Mg 2+ Concentration 100μM, ALP 0mU mL -1 or 10mU mL -1 , pH=9.5, 10mM Tris-glycine buffer, react at 37°C for 60 minutes, Tb-BPTC concentration 100μg mL -1 . Interfering species concentrations of anions and cations were 100 μM. The concentrations of amino acids and glucose interfering substances were 100 μM. The concentration of BSA interfering substances is 2 μg mL -1 . The activity of the interfering enzyme is 100mU mL -1 . Test results such as Figure 7 shown.
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