A simple colorimetric detection of DNA methylation Chenchen Ge, ab Zhiyuan Fang, b Junhua Chen, b Jie Liu, b Xuewen Lu b and Lingwen Zeng * b Received 10th January 2012, Accepted 5th March 2012 DOI: 10.1039/c2an35043b In this work, we describe a simple colorimetric method to detect DNA methylation. Adenomatous polyposis coli (APC) with a small CpG region containing methylated cytosine (methylated APC) was synthesized and tested. Methylated APC was first captured and enriched by anti-5-methylcytosine monoclonal antibody conjugated magnetic microspheres (MMPs). Then a probe partly complemen- tary to the APC sequence was added, resulting in the formation of DNA duplexes. The microsphere-captured probe was then released by heat denaturation and added into unmodified gold nanoparticle (AuNP) solution. Colorimetric detection was performed by salt- induced aggregation. The limit of detection is 80 fmol. Semi-quan- titative analysis was done with a UV/Vis spectrophotometer by recording the absorbance of AuNP solution at 520 nm. Thus, this method provides a simple, rapid and quantitative tool for DNA methylation detection. Introduction DNA methylation is a crucial epigenetic modification of the genome, which refers to adding a methyl group to the 5 0 -position of the cytosine nucleotide and usually occurs in the CpG dinucleotide in the mammal genome. The genome regions which contain a high occur- rence of CpG dinucleotides are called CpG islands. In normal cells, the promoter-associated CpG islands are generally unmethylated, but in tumor cells, promoter region CpG islands are likely to be hyper- methylated, 1 especially for some tumor suppressor genes (TSGs), which will effectively silence transcription. 2 Consequently, DNA methylation at the CpG islands in gene promoter regions often causes the silence of TSGs which protect against tumor formation, and promoter hypermethylation in CpG islands is usually considered as an early sign of tumorigenesis. 3 Therefore, accurate analysis of the DNA methylation status at CpG islands is one way to report gene transcriptional activity and may help early cancer diagnosis. The extensively used methods to detect DNA methylation are methylation-specific PCR (MSP) 4 and methylation-specific restriction enzyme (MS-RE)-PCR/Southern blot. 5 The former method is bisul- fite-based, and the later is enzyme-based, both of which require long manipulating time. Recently, some new methods have been developed for the detection of DNA methylation, such as methylation-specific microarray 6 and methylation-specific fluorescence resonant energy transfer (MS-FRET). 7 But they are timing-consuming and need complex operations. Other methods such as nanowire-transistor and surface plasmon resonance (SPR) biosensors do not need bisulfite treatment or PCR amplification. 8 However, the requirement of professional design and operations still restricts their application. So it is necessary to explore a simple method to detect DNA methylation. Adenomatous polyposis coli (APC) is a TSG that regulates important events in cell growth, such as cell adhesion, migration, and signaling. 9 Previous findings indicate that the CpG island in the APC gene promoter 1A are hypermethylated in colorectal, gastric cancer cells, breast, and lung cancer cells. 10,11 Meanwhile, there are 31 CpG dinucleotides in promoter 1A of the APC gene. We selected a relatively small CpG region in the APC promoter 1A sequence as a target. 12 In this report, we introduce a new colorimetric method to detect DNA methylation based on the enrichment of magnetic micro- spheres (MMPs), using an unmodified gold nanoparticle (AuNP) solution (Scheme 1). A small CpG region in the APC promoter 1A sequence containing methylated cytosine was synthesized as the target. This assay consisted of five steps: (a) the methylated APC (target) was captured by MMPs conjugated with anti-5-methyl- cytosine monoclonal antibody, (b) a probe partly complementary to the target was added to form a partial duplex region, (c) the hybrid- conjugated microspheres were magnetically separated and the captured probe was released from the hybrid-conjugated micro- spheres by heat denaturation, (d) the released probe was added into AuNP solution. Since negatively charged single-stranded DNA can adsorb on the surface of AuNPs through electrostatic force and increases the repulsive force between AuNPs, which prevents the AuNPs in the solution from effective aggregation in the presence of NaCl, thus the color of the solution changed slightly but remains red. (e) In contrast, unmethylated APC did not contain methylated cytosine and could not be captured by the antibody, thus no probe was released into the AuNP solution. The color of the solution changed from red to purple immediately upon the addition of NaCl, which can be observed visually. Experimental Materials Carboxylated magnetic microspheres with a diameter of 4–5 mm were purchased from BaseLine ChromTech Research Centre. a University of Science and Technology of China, Hefei, 230026, China b Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China. E-mail: zeng6@yahoo.com; Fax: +86 20 32015245; Tel: +86 20 32015312 2032 | Analyst, 2012, 137, 2032–2035 This journal is ª The Royal Society of Chemistry 2012 Dynamic Article Links C < Analyst Cite this: Analyst, 2012, 137, 2032 www.rsc.org/analyst COMMUNICATION Published on 05 March 2012. Downloaded by University of Queensland on 9/24/2022 4:05:42 PM. View Article Online / Journal Homepage / Table of Contents for this issue