Utilizing Gold Nanoparticle Probes to Visually Detect DNA Methylation

Nanoscale Research Letters, Jun 2016

The surface plasmon resonance (SPR) effect endows gold nanoparticles (GNPs) with the ability to visualize biomolecules. In the present study, we designed and constructed a GNP probe to allow the semi-quantitative analysis of methylated tumor suppressor genes in cultured cells. To construct the probe, the GNP surfaces were coated with single-stranded DNA (ssDNA) by forming Au–S bonds. The ssDNA contains a thiolated 5′-end, a regulatory domain of 12 adenine nucleotides, and a functional domain with absolute pairing with methylated p16 sequence (Met-p16). The probe, paired with Met-p16, clearly changed the color of aggregating GNPs probe in 5 mol/L NaCl solution. Utilizing the probe, p16 gene methylation in HCT116 cells was semi-quantified. Further, the methylation of E-cadherin, p15, and p16 gene in Caco2, HepG2, and HCT116 cell lines were detected by the corresponding probes, constructed with three domains. This simple and cost-effective method was useful for the diagnosis of DNA methylation-related diseases.

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Utilizing Gold Nanoparticle Probes to Visually Detect DNA Methylation

Chen et al. Nanoscale Research Letters Utilizing Gold Nanoparticle Probes to Visually Detect DNA Methylation Kui Chen 0 1 Mingyi Zhang 1 Ya-Nan Chang 1 Lin Xia 1 Weihong Gu 1 Yanxia Qin 1 Juan Li 1 Suxia Cui 0 Gengmei Xing 1 0 School of Life Sciences, Capital Normal University , Beijing 100048 , China 1 CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Science (CAS) , Beijing 100049 , China The surface plasmon resonance (SPR) effect endows gold nanoparticles (GNPs) with the ability to visualize biomolecules. In the present study, we designed and constructed a GNP probe to allow the semi-quantitative analysis of methylated tumor suppressor genes in cultured cells. To construct the probe, the GNP surfaces were coated with single-stranded DNA (ssDNA) by forming Au-S bonds. The ssDNA contains a thiolated 5′-end, a regulatory domain of 12 adenine nucleotides, and a functional domain with absolute pairing with methylated p16 sequence (Met-p16). The probe, paired with Met-p16, clearly changed the color of aggregating GNPs probe in 5 mol/L NaCl solution. Utilizing the probe, p16 gene methylation in HCT116 cells was semi-quantified. Further, the methylation of E-cadherin, p15, and p16 gene in Caco2, HepG2, and HCT116 cell lines were detected by the corresponding probes, constructed with three domains. This simple and cost-effective method was useful for the diagnosis of DNA methylation-related diseases. DNA methylation; Gold nanoparticles probe; Surface plasmon resonance; Visual detection; Semi-quantitative assay Background DNA methylation is an important regulator of gene expression, and its role in tumorigenesis has been a central topic in the last few decades [ 1 ]. Many studies have indicated that hypermethylated CpG islands in tumor suppressor gene promoter sites can increase chromosome coiling and gene silencing, and this process occurs prior to malignant cell growth [ 2, 3 ]. As site-specific methylation occurs early and can be detected even in body fluid, it is regarded as a potential biomarker for early tumor detection and determining prognosis [ 4–6 ]. To date, numerous techniques have been developed for DNA methylation detection [ 7, 8 ]. Due to the particular physicochemical property of nanoscale materials, nano-based DNA methylation detection has emerged as an important option. The specific optical properties of nanomaterials change the very foundation of traditional DNA methylation sensing [ 9–11 ]. Among the abundant types of nanomaterials, gold nanoparticles (GNPs) are most extensively applied due to their unique chemical and physical properties that are strongly dependent on their size, shape, and degree of aggregation [12]. Colorimetric assays based on GNP surface plasmon resonance are more applicable as clinical markers because they only require a UV/vis spectrometer. Most studies have concentrated on indirect methods to detect DNA methyltransferases or DNA methylases based on GNPs [ 13–15 ]. Zeng’s group used antibody-conjugated magnetic microspheres to capture methylated DNA [ 16 ]. After their release from the microsphere by heat denaturation, methylated DNA was added to unmodified GNPs to prevent GNPs from aggregating in the salt solution, whereas the non-methylated group cannot be captured, and no DNA was released into the GNP solution. One limitation of this method is the need of an antibody to recognize the methylated DNA sequence. In this work, we tried to construct a highly sensitive single-stranded DNA (ssDNA)-GNP probe to detect DNA methylation in cultured cells. p16 are tumor suppressor genes and their transcription activities can be inhibited by hypermethylation in the promoter site. Therefore, the small CpG region in the promoter site was selected as the target to test the probe and achieve semi-quantitative detection of DNA methylation. We designed an ssDNA-GNP probe to target and visually detect the CpG region of the tumor suppressor genes by introducing a colorimetric method to modify an existing bisulfite-based method that measures CpG region methylation. After the sequence was treated with bisulfate, the 5′-ends were C-CH3 and U in the methylated and non-methylated DNA sequences, respectively. Their polymerase chain reaction (PCR) products (Met-p16 and Dem-p16, respectively) were used as standard sequences in the following experiment to test the probe and calculate a standard curve for semi-quantitative detection of intracellular DNA methylation (Table 1). We applied the aggregation principle reported by Sato et al. [ 17 ] and Liu and Lu [ 18 ] to construct an ssDNA-GNP probe. The probe contained a sequence that absolutely paired with Met-p16 but mismatched Dem-p16 at the terminal base. This assay consisted of two steps (Fig. 1a, b): (a) the target sequences (Met-p16 and Dem-p16) were added to the ssDNA-GNP probe solution and incubated on ice for 1 h, and then Tris-acetate buf (...truncated)


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Kui Chen, Mingyi Zhang, Ya-Nan Chang, Lin Xia, Weihong Gu, Yanxia Qin, Juan Li, Suxia Cui, Gengmei Xing. Utilizing Gold Nanoparticle Probes to Visually Detect DNA Methylation, Nanoscale Research Letters, 2016, pp. 304, Volume 11, Issue 1, DOI: 10.1186/s11671-016-1487-5