Alkaline phosphatase detection kit based on dual-emission lanthanide MOF and detection method
A detection kit and dual emission technology, applied in the field of analytical chemistry and clinical testing, can solve the problems of poor monochromaticity, wide spectrum, single fluorescence emission peak, etc.
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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 50mM AAP and 5 μL 20mM MgSO 4 Mix it with it, add a certain amount of buffer (pH=9.5, 10mM Tris-glycine buffer) to make the total volume 0.5mL, place the mixture in a constant temperature 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 ). Measured with a fluorescence spectrometer, the reaction solution was mixed for 10 minutes and transferred to a cuvette to monitor the fluorescence spectrum.
[0045] When drawing the standard curve, the analysis method based on colorimetric ratio and the analysis method based on dual-wavelength ratiometric fluorescence were compared:
[0046] The standard curve of the Tb-BPTC response to ALP activity directly with the dual-wavelength ratio fluorescence as the ordinate is as follows: Figure 5 sho...
Embodiment 2
[0058] Application prepared Tb-BPTC, 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 50mM AAP, 5 μL 20mM 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 was complete, 250 μL of water and 250 μL of Tb-BPTC solution (2 mg mL -1 ). The resulting reaction solution can be quickly diafiltered through the capillary pores, and the glass fiber test paper in the ultrafiltration inner tube can be transfected. After 10 minutes, the glass fiber test paper is taken out with tweezers and transferred to a UV lamp. High sensitivity can be achieved under the UV lamp. Naked eye detection (color changes from green to blue as alkaline phosphatase activity changes from 0-8mU mL). Under the excitation of 254...
Embodiment 3
[0063] The selectivity and anti-interference ability of Tb-BPTC to the determination of ALP activity were tested by fluorescence spectrometer detection method and dual-wavelength ratio fluorescence analysis method. 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 degrees Celsius for 60 minutes, Tb-BPTC concentration 100μg mL -1 . The concentration of interfering substances for anions and cations was 100 μM. The concentration of amino acid and glucose interfering substance was 100 μM. The concentration of BSA interfering substances is 2 μg mL -1 . The activity of interfering enzymes is 100mU mL -1 . Test results such as Figure 7 shown.
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