Homogeneous phase biological analysis method for detecting platelet derived growth factor BB and application of homogeneous phase biological analysis method
A platelet-derived and growth factor technology, applied in the field of biological analysis, can solve problems such as poor repeatability, high analysis cost, and low technical requirements, achieve high analytical sensitivity, overcome cumbersome operations, and ensure repeatability
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Embodiment 1
[0025] A homogeneous bioanalysis method for detecting platelet-derived growth factor BB in this embodiment comprises the following steps:
[0026] (1) High temperature annealing treatment of DNA strands
[0027] Take 5 PV tubes and number them as #1, #2, #3, #4 and #5 respectively, add 190 µL tris buffer solution with a concentration of 20 mM pH=7.4 to each PV tube, the The buffer solution contains 100mM NaCl, 10mMKCl and 10mMMgCl 2 ;
[0028] Next, add 10 µL of 10 µM end-modified Mg to tube #1PV 2+ Nuclease 1 / 2 cleaves the PDGF-BB nucleic acid aptamer S1 of the base series, and the base sequence of the nucleic acid aptamer S1 is 5'-CAG GCT ACG GCA CGT AGAGCA TCA CCA TGA TCC TGT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT AGAGTA AGC ACC CAT GTT AGA CCT C-3';
[0029] Add 10 µL of 10 µM end-modified Mg to #2 PV tube 2+ Nuclease 1 / 2 cleaves the PDGF-BB nucleic acid aptamer S2 of the base series, and the base sequence of the nucleic acid aptamer S2 is 5'-CCT GCT CAG CGA ...
Embodiment 2
[0038] Example 2 Detection of PDGF-BB content in human serum
[0039] Take 500µL of clinical serum samples, dilute 50 times with 20mM pH=7.4 Tris-HCl buffer solution to obtain diluted sample solution, the buffer solution contains 100mM NaCl, 10mM MgCl 2 and 10 mM KCl.
[0040] Take 10 µL of the diluted sample solution, add 10 µL of single-stranded DNA solution S1, 10 µL of single-stranded DNA solution S2, 10 µL of ligated DNA solution, 10 µL of DNA solution H1 and 10 µL of DNA solution after high-temperature annealing in step (1) of Example 1 H2, vortex and mix at 37°C for 120min; then add 25mM pH=7.4 HEPES buffer solution containing 10µL 20µM hemin, the HEPES buffer solution also contains 100mM NaCl, 10mM MgCl 2 , 10mM KCl, 0.05% TritonX-10 and 1% DMSO, vortexed and mixed at 37°C for 50min; then added 20µL containing 20mM ABTS, 40mMH 2 o 2 20mM pH=7.4 Tris-HCl buffer solution, the Tris-HCl buffer solution also contains 100mM NaCl, 10mMMgCl 2 and 10mM KCl, after the color ...
Embodiment 3
[0043] The standard solution of platelet-derived growth factor BB was added to the serum sample solution in Example 2, and the standard addition recovery experiment was carried out, and 5 groups of parallel experiments were performed for each concentration. The sample processing method of the standard addition recovery experiment is the same as the standard solution, and the absorbance value of the treated solution is measured by a UV-visible spectrophotometer. According to the difference between the absorbance obtained in step (2) of Example 1 and the PDGF-BB concentration Quantitative relationship, calculated the content of platelet-derived growth factor BB in the spiked and recovered sample solution, and the obtained experimental results are shown in Table 2 below.
[0044] Table 2 The results of the recovery experiment of the serum sample solution
[0045]
[0046] As can be seen from the above table 2, the relative standard deviation (RSD) of the test results of the pr...
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