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)