Determination Of Dna Methyltransferase 1 In Serum By Fluorescence Immunoassay Based On Quantum Dots

DNA methyltransferase 1（DNMT1）is a crucial enzyme to DNA methylation,more and more researches showed that DNA methylation was closely related to cancer and a variety of age-related chronic diseases.The current studies of DNMT1detection mostly have been concerned in activity but rarely in content detection.Compared with conventional organic fluorescent dyes,quantum dots with wide and continuous excitation spectrum,narrow and symmetric emission spectrum and high quantum yield have been widely used in biochemical analysis.In view of this,a fluorescence immunoassay based on quantum dots was established in this thesis for DNMT1 detection,and further used for the quantitative analysis of DNMT1 in serum samples.The main studies of this dissertation are summarized as follows.（1）Synthesis and surface modification of Fe₃O₄ nanoparticles and quantum dotsOil-soluble Fe₃O₄ has been prepared via high temperature decomposing of iron acetylacetonate,and then successfully transferred to aqueous phase by NaClO through a two-step oxidation of oleic acid ligand on the surface of oil-soluble Fe₃O₄.The synthetic nanoparticals of water-soluble carboxy Fe₃O₄ were monodisperse spherical particales with a diameter of 5 nm,and the Zeta potential was-12.14 mV.The efficiency of Fe₃O₄ nanoparticals covalently coupling with human IgG was80.07%;ELISA results showed that the immobilized IgG still retained their immune activity.Orthogonal test and response surface methodology have been used to optimized the reaction conditions of two CdTe QDs with different emission wavelengths.For QDs1 with a calculated diameter of 2.84 nm,the fluorescence excitation（Ex）and emission wavelength（Em）were 310 nm and 520 nm,respectively,and the Zeta potential was-22.56 mV,and the quantum yield was 4.02%.For QDs2 with a calculated diameter of 3.40 nm,the corresponding parameters were 240 nm,570 nm,-17.88 mV,15.04%,respectively.The goat anti-human IgG remained activity after being conjugated with QDs.（2）The establishment and optimization of fluorescence immunoassay based on QDs for the detection of DNMT1Fe₃O₄@McAb and QDs@PcAb were synthesized and characterized.The optimum conditions of immune reaction obtained by single factor optimization were as follows:the dilution ratio of Fe₃O₄@McAb was 1:50,and that of QDs@PcAb was 1:30;the immune reaction buffer was 0.01mol/L pH 7.4 PBST;it took 60 min for Fe₃O₄@McAb to capture objective antigen,then the affinity reactions between QDs@PcAb and above-mentioned antigen were carried out for 120 min.The linear range of FLISA could been divided into two sections,the standard curve equation of0.1⁵.0 ng/mL was Y=0.02779X+1.29858(X was LogC_DNMT1,Y was LogRFU)with the correlation coefficient r as 0.9877;while the standard curve equation of 5.0¹500 ng/mL was Y=0.00775X+20.77161(X was C_DNMT1,Y was RFU)with the correlation coefficient r as 0.9873.The detection limit was 0.1 ng/mL,the recoveries of intra and inter-assays were from 91.67%to 106.50%and from 92.18%to 108.50%,respectively,the relative standard deviations（RSD）were from 5.45%to 11.29%and from 7.03%to 11.25%（n=3）,the cross-reactivity rates with other two DNMTs（DNMT3a and DNMT3b）in serum were 4.0%and 9.4%,respectively.（3）Comparison of FLISA with ELISA and MELISAELISA and MELISA were developed for the detection of DNMT1.The comparison results of FLISA with ELISA and MELISA were as follows:○1 The detection limits of the three methods were 0.1 ng/mL,0.1 ng/mL,1.0ng/mL,respectively.○2 The intra-assays recoveries of the three methods were 91.67%¹06.50%,91.95%¹06.24%and 95.13%¹05.66%,respectively;the inter-assays recoveries were 92.18%¹08.50%,102.45%¹12.02%and 92.56%¹09.05%,respectively.○3 The intra-assays RSD of three methods were 8.56%,6.33%and 8.47%,respectively;the inter-assays RSD were 9.59%,6.29%and 10.05%,respectively.○4 The detection time of three methods were 3 h,18 h and 5 h,respectively.FLISA,ELISA and MELISA were applied for quantitative detection of DNMT1in serum samples of 30 cases,14 and 6 cases were detected as positive for FLISA and ELISA,respectively,none cases could be detected by MELISA due to its high limit of detection.The results of FLISA and ELISA were compared with commercial DNMT1 ELISA kits.Paired sample t test of the results revealed that no significant difference among FLISA,ELISA and commercial ELISA kits for the detection of DNMT1 content in serum samples.The recoveries of standard addition of DNMT1were 71.92%⁸9.74%and 94.30%¹21.27%（n=3）,respectively;the corresponding range of RSD were 4.62%⁹.34%and 3.90⁹.68%（n=3）,respectively.The results showed that the sensitivities of FLISA and ELISA were higher than MELISA,the FLISA established in this study could be used for the rapid detection of DNMT1 in serum with minimum detection time,high precision,high accuracy and good specificity.