Magnetic immunoassay using CdSe/ZnS quantum dots as fluorescent probes to detect the level of DNA methyltransferase 1 in human serum sample

Abstract

Background: DNA methyltransferase 1 (DNMT1), a dominant enzyme responsible for the transfer of a methyl group from the universal methyl donor to the 5-position of cytosine residues in DNA, is essential for mammalian development and closely related to cancer and a variety of age-related chronic diseases. DNMT1 has become a useful biomarker in early disease diagnosis and a potential therapeutic target in cancer therapy and drug development. However, till now, most of the studies on DNA methyltransferase (MTase) detection have focused on the prokaryote MTase and its activity.

Methods: A magnetic fluorescence-linked immunosorbent assay (FLISA) using CdSe/ZnS quantum dots as fluorescent probes was proposed for the rapid and sensitive detection of the DNMT1 level in this study. Key factors that affect the precision and accuracy of the determination of DNMT1 were optimized.

Results: Under the optimal conditions, the limit of detection was 0.1 ng/mL, the linear range was 0.1-1,500 ng/mL, the recovery was 91.67%-106.50%, and the relative standard deviations of intra- and inter-assays were respectively 5.45%-11.29% and 7.03%-11.25%. The cross-reactivity rates with DNA methyltransferases 3a and 3b were only 4.0% and 9.4%, respectively. Furthermore, FLISA was successfully used to detect the levels of DNMT1 in human serum samples, and compared with commercial enzyme-linked immunosorbent assay (ELISA) kits. The results revealed that there was a good correlation between FLISA and commercial ELISA kits (correlation coefficient r=0.866, p=0.001). The linear scope of FLISA was broader than ELISA, and the measurement time was much shorter than ELISA kits.

Conclusion: These indicated that the proposed FLISA method was sensitive and high throughput and can quickly screen the level of DNMT1 in serum samples.

Keywords: DNA methyltransferase 1; fluorescence immunoassay; high throughput; magnetic carboxyl beads; quantum dots; serum sample.

Figure 1

(A) TEM image of the bare MBs. (B) TEM image of MBs coated with DNMT1 McAb (MBs@McAb). (C) Characterization of biological activity of MBs@McAb. (D) Characterization of biological activity of QDs@PcAb. Abbreviations: TEM, transmission electron microscopy; MBs, magnetic beads; DNMT1, DNA methyltransferase 1; McAb, monoclonal antibody; QDs, quantum dots; PcAb, polyclonal antibody; OD, optical density; RFU, relative fluorescence units.

Figure 2

(A) The effect of MBs@McAb dilution ratio on fluorescence intensity. (B) The effect of QDs@PcAb dilution ratio on fluorescence intensity. (C) The effect of different immune reaction buffers on fluorescence intensity. (D) The best reaction time of MBs@McAb and QDs@PcAb with DNMT1 in serum sample. Abbreviations: MBs, magnetic beads; McAb, monoclonal antibody; QDs, quantum dots; PcAb, polyclonal antibody; DNMT1, DNA methyltransferase 1; RFU, relative fluorescence units; PBS, phosphate buffer saline; PBST, phosphate buffer saline with 0.05% Tween-20; CB, carbonate buffer; BS, borate saline.

Figure 3

(A) The relationship between fluorescence intensity and DNMT1 concentration (0.1–1,500 ng/mL). (B) The relationship between fluorescence intensity and logCDNMT1 (0.1–10 ng/mL). Abbreviations: DNMT1, DNA methyltransferase 1; RFU, relative fluorescence units.

Scheme 1

The principle of FLISA. Abbreviations: FLISA, fluorescence-linked immunosorbent assay; EDC, 3-dimethylaminopropyl-N′-ethylcarbo-diimide hydrochloride; NHS, N-hydroxysuccinimide; MBs, magnetic beads; DNMT1, DNA methyltransferase 1; McAb, monoclonal antibody; PcAb, polyclonal antibody; RFU, relative fluorescence units.