Nanomaterial-based biosensors for DNA methyltransferase assay

Fei Ma , Qian Zhang and Chun-yang Zhang *
College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan 250014, China. E-mail: cyzhang@sdnu.edu.cn; Fax: +86 0531-82615258; Tel: +86 0531-86186033

Received 3rd November 2019 , Accepted 28th January 2020

First published on 28th January 2020


DNA methyltransferases are responsible for catalyzing the methylation of adenine/cytosine residues in specific regions of the genome, and they participate in the establishment of epigenetic modification patterns. Deregulation of DNA methyltransferase activity will disturb DNA methylation systems, leading to the occurrence of various human diseases including cancers. Moreover, DNA methyltransferases may serve as promising therapeutic targets, and DNA methyltransferase inhibitors have been used for disease treatment. Therefore, the detection of DNA methyltransferases and screening of their inhibitors are crucial for both fundamental biomedical research studies and clinical practice. Due to their excellent size-dependent optical, chemical, electronic, and mechanical features, nanomaterials have been widely used as powerful building materials to construct efficient biosensors for DNA methyltransferase assay with high sensitivity and good selectivity. In this review, we summarize the recent progress in the development of nanomaterial-based biosensors for DNA methyltransferase assay including the strategies, features and applications, and highlight the future direction and challenges in this area as well.


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Fei Ma

Fei Ma obtained his PhD degree from Shandong Normal University in 2019. He worked at the Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences during 2013–2015. Currently, he is a lecturer at Shandong Normal University. His research focuses on biosensor development and single-molecule detection.

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Qian Zhang

Qian Zhang obtained her BS degree from Shandong Normal University in 2018. Currently, she is a research assistant at Shandong Normal University. Her research focuses on bioanalytical chemistry and single-molecule detection.

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Chun-yang Zhang

Chun-yang Zhang obtained his PhD degree from Peking University, China, in 1999. During 1999–2008, he worked in Tsinghua University, Emory University, Johns Hopkins University and The City University of New York. In 2009, he joined as a professor Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, China. In 2015, he relocated to Shandong Normal University, China, and he is a professor of chemistry now. He is the recipient of the Hundred Talent Program of the Chinese Academy of Sciences (2011), the China National Fund for Distinguished Young Scientists (2013), and the Hundred Talent Program of Nanyue of Guangdong Province, China (2015). His research focuses on bioanalytical chemistry, bionanotechnology and single-molecule detection.


1. Introduction

DNA methyltransferases catalyze the transfer of the methyl group from S-adenosyl-l-methionine to adenine1/cytosine2 to cause DNA methylation at a specific location for the establishment of a special DNA methylation pattern.3–5 As crucial epigenetic gene regulators, DNA methyltransferases participate in many physiological processes such as cell development,6 differentiation,7 aging,8 memory formation,9 and paternal and maternal imprinting.10 Abnormal DNA methyltransferase activities will disturb the normal DNA methylation pattern, leading to the occurrence of a variety of human diseases,11–13 such as immunodeficiency–centromeric instability–facial anomaly (ICF) syndrome,14 leukaemia,15 Alzheimer's disease,16 autoimmune disease,17 and multiple types of cancers including lung,18 colon,19 prostate,20 breast,21 and endometrium22 cancers. Moreover, DNA methyltransferases may serve as potential therapeutic targets,23–25 and DNA methyltransferase inhibitors can be used for disease treatment.26–28 Therefore, sensitive detection of DNA methyltransferase is of great importance in biomedical research studies, clinical diagnosis and drug discovery.

Traditional analytical methods including gel electrophoresis,29 capillary electrophoresis,30 high-performance liquid chromatography,31 and polymerase chain reaction32 have been successfully adopted for DNA methyltransferase assay, but they either involve radioactive materials or suffer from large sample consumption, poor sensitivity, and laborious and time-consuming procedures.33,34 Alternative simple and efficient methods with high sensitivity and good selectivity are highly desirable.

With the development of material science and nanotechnology, a series of new nanomaterials has been successfully designed and synthesized, including gold nanoparticles,35 carbon nanomaterials,36 semiconductor quantum dots,37 and metal nanoclusters.38 Due to their unique sizes and shapes, nanomaterials possess some attractive optical, chemical, electronic, and mechanical properties, and they may function as optical emitters, electronic conductors, catalytic species, and carrier platforms.39–41 Moreover, nanomaterials can be easily biofunctionalized through physical adsorption, electrostatic binding and covalent coupling, making them powerful building materials for the construction of efficient biosensors.41,42 Nanomaterial-based biosensors have been widely applied for the sensitive and selective detection of nucleic acids,43–47 proteins,48–50 enzymes,51–53 and various biomolecules.42,54,55 However, to the best of our knowledge, there has been no review article focusing on nanomaterial-based biosensors for DNA methyltransferase assays so far. In this review, we summarize the recent advances in the development of functional nanomaterial-based biosensors for DNA methyltransferase assay. We will review the strategies, features and applications of these biosensors, and discuss the future direction and challenges in this area.

2. Gold nanoparticle-based DNA methyltransferase biosensors

Gold nanoparticles (AuNPs) are among the most popular nanomaterials because they can be easily synthesized. AuNPs have a large surface area that can be readily modified using different ligands they and possess unique optical and electronic properties.60–62 AuNPs can be applied in colorimetry,56 circular dichroism,57 electrochemistry,58 and fluorescence59 detection of DNA methyltransferase.

Gold nanoparticles (AuNPs) have been widely used for colorimetric detection due to their unique properties of high extinction coefficients and distance- and size-dependent color changes.44,63,64 Ren et al. developed a AuNP-based biosensor for colorimetric detection of DNA methyltransferase.56 As shown in Fig. 1A, the AuNPs are labelled with short oligonucleotide linkers that can specifically hybridize with the double-stranded detection probes to induce the assembly of AuNPs. When the target DNA methyltransferase is absent, the detection probes will be cleaved by Dpn II, resulting in the disassembly of AuNPs. In contrast, when the target DNA methyltransferase Dam is present, the detection probes are methylated, protecting the detection probes from cleavage by Dpn II endonuclease. As a result, the AuNPs remain in the aggregation state, and a color change can be observed (Fig. 1B), and the ratio of absorbance at 524/700 nm can be used to quantify the presence of the target Dam (Fig. 1C). This biosensor can detect as low as 2.5 U mL−1 Dam, and it can screen two DNA methyltransferase inhibitors including cyclopentaquinoline carboxylic acid and ethidium bromide. Moreover, this biosensor involves only a single type of DNA-modified AuNP, with distinct advantages of low cost and simplicity.


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Fig. 1 Gold nanoparticle-based DNA methyltransferase biosensors. (A) Schematic illustration of the AuNP-based biosensor for the colorimetric detection of DNA methyltransferase Dam. (B) Color changes of AuNP probes in response to Dam. (C) Measurement of absorbance in response to different-concentration Dam. Reproduced from ref. 56 with permission from the American Chemical Society. (D) Schematic illustration of AuNP-based CD spectroscopy for the detection of DNA methyltransferase M.SssI. (E) Screening of DNA methyltransferase inhibitors including 5-Aza and procaine. Reproduced from ref. 57 with permission from The Royal Society of Chemistry. (F) Schematic illustration of the AuNP-based electrochemical biosensor for the detection of DNA methyltransferase Dam. (G) Variance of the current signal in response to different-concentration Dam. Reproduced from ref. 58 with permission from Elsevier B. V. (H) Schematic illustration of the AuNR-based fluorescent biosensor for the detection of DNA methyltransferase. (I) Variance of the fluorescence intensity in response to different-concentration Dam. Reproduced from ref. 59 with permission from The Royal Society of Chemistry.

Because of their unique chiroptical characteristics, self-assembled chiral AuNPs produce strong circular dichroism (CD) signals with a high anisotropy factor and a tunable plasmonic chiroptical response.65–67 Wei et al. demonstrated the use of AuNP-based CD spectroscopy for DNA methyltransferase assay.57 As shown in Fig. 1D, two DNA-labelled AuNPs can be linked together to form AuNP dimers through specific DNA hybridization. In the absence of the target DNA methyltransferase M.SssI, the DNA link will be cleaved by HpaII restriction enzyme, leading to the dissociation of AuNP dimers and consequently no distinct CD signal. In contrast, when the target DNA methyltransferase M.SssI is present, the DNA link is methylated and cannot be digested by HpaII enzyme. As a result, the AuNP dimers remain intact and generate a distinct CD signal. This method enables simple and sensitive detection of DNA methyltransferase M.SssI with a linear range from 0.5 to 150 U mL−1 and a detection limit of 0.27 U mL−1. Moreover, it can be used for the screening of two DNA methyltransferase inhibitors, 5-Aza and procaine (Fig. 1E), and it exhibits good accuracy even in human serum samples with a recovery rate of 93–104.6%.

Since AuNPs have a large surface area, one AuNP can bind to a large number of signal probes to achieve efficient signal amplification.68 Xie et al. developed a AuNP-based electrochemical biosensor for the detection of DNA methyltransferase.58 As shown in Fig. 1F, the thiolated DNA S1 is assembled on the surface of the gold electrode (Au electrode), which enables subsequent capture of DNA S3-AuNPs through the formation of the S1/S2/S3 sandwiched hybrid. Consequently, many DNA strands are close to the Au electrode, resulting in the adsorption of abundant redox-active methylene blue molecules for the generation of an amplified electrochemical signal. In the absence of the target DNA methyltransferase Dam, the S1/S2 duplex is cleaved by Mbo I endonuclease, leading to the release of DNA S3-AuNPs from the Au electrode and the decrease of electrochemical signal. In the presence of the target DNA methyltransferase Dam, the S1/S2 duplex is methylated and cannot be cleaved by Mbo I endonuclease, and thus a high electrochemical signal is detected. This electrochemical biosensor exhibits a linear range from 0.075 to 30 U mL−1 and a low detection limit of 0.02 U mL−1 (Fig. 1G), and it can be further applied for the screening of the DNA methyltransferase inhibitor 5-fluorouracil.

Gold nanorods (AuNRs) are rod-shaped gold nanoparticles and they possess unique shape-dependent plasmonic properties.69–71 Li et al. developed a AuNR-based fluorescent biosensor for the detection of DNA methyltransferase.59 As shown in Fig. 1H, the positively charged AuNRs can bind with the negatively charged double-stranded detection probes containing FAM-labelled DNAs (FAM-DNAs) and complementary DNAs (cDNAs), resulting in the quenching of FAM signal due to FRET from FAM to the AuNR. In the presence of the target DNA methyltransferase Dam, the detection probes are methylated and cleaved by DpnI to produce small DNA fragments. The small DNA fragments exhibit much lower affinity towards the AuNRs than the intact detection probes due to the weaker electrostatic interaction, inducing the recovery of the FAM signal. In contrast, when target DNA methyltransferase Dam is absent, the detection probe cannot be cleaved by DpnI, and the FAM signal is completely quenched by AuNRs. This fluorescent biosensor is very simple and cost-effective, enabling sensitive detection of DNA methyltransferase with a linear range from 0.5 to 20 U mL−1 and a low detection limit of 0.25 U mL−1 (Fig. 1I), and it can be further used to screen the DNA methyltransferase inhibitor 5-fluorouracil.

3. Carbon nanomaterial-based DNA methyltransferase biosensors

Carbon nanomaterials including one-dimensional carbon nanotubes and two-dimensional graphene have distinct features of high electrochemical stability, extremely large surface area, and brilliant mechanical and electrical properties,36,76,77 and they can be used for fluorescence polarization,72 light scattering,73 and fluorescence74,75 detection of DNA methyltransferase.

Carbon nanotubes (CNTs) are one-dimensional carbon nanomaterials with widespread applications in biosensing due to their unique structural, mechanical and optoelectronic properties.78–80 Zhao et al. developed a multiwalled carbon nanotube (MWCNT)-based fluorescence polarization biosensor for the detection of DNA methyltransferase.72 As shown in Fig. 2A, the single-stranded region of FAM-labelled detection probes can bind with MWCNTs through π–π stacking interaction, resulting in efficient quenching of FAM fluorescence by MWCNTs. In the absence of the target DNA methyltransferase Dam, the double-stranded region of the detection probes can be digested by EcoRI endonuclease, inducing the release of FAM. The free FAM in solution will generate a low fluorescence polarization signal due to the increased rotational rate. In contrast, in the presence of the target DNA methyltransferase Dam, the detection probes are methylated and cannot be digested by EcoRI. As a result, FAM is still adsorbed on the MWCNTs, leading to a high fluorescence polarization signal due to the decrease of the rotational rate (Fig. 2B). This fluorescence polarization biosensor can achieve an extremely low detection limit of 1 × 10−4 U mL−1. It can be applied for the screening of four DNA methyltransferase inhibitors, 5-fluorouracil, cisplatin, 5-chloro-7-iodo-8-hydroxyquinoline, and oxoglaucine, and it even exhibits good performance in human serum samples with a recovery rate of 92.8–97.6%. Moreover, this biosensor can be further applied for the detection of other DNA methyltransferases such as EcoRI.


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Fig. 2 Carbon nanomaterial-based DNA methyltransferase biosensors. (A) Schematic illustration of the MWCNT-based fluorescence polarization biosensor for the detection of DNA methyltransferase Dam. (B) Measurement of fluorescence polarization in response to different-concentration Dam. Reproduced from ref. 72 with permission from Elsevier B. V. (C) Schematic illustration of the SWNT-based light scattering biosensor for homogeneous and label-free detection of DNA methyltransferase EcoRI. (D) Variance of the LS intensity in response to different-concentration EcoRI. Reproduced from ref. 73 with permission from WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. (E) Schematic illustration of the GO-based fluorescent biosensor for the detection of DNA methyltransferase HaeIII. (F) Measurement of the fluorescence images of the reaction mixtures in response to different-concentration HaeIII. Reproduced from ref. 74 with permission from the American Chemical Society. (G) Schematic illustration of the GQD-based fluorescent biosensor for the detection of DNA methyltransferase M.SssI. (H) Variance of the fluorescence intensity in response to different-concentration M.SssI. Reproduced from ref. 75 with permission from Elsevier B.V.

Qu et al. developed a single-walled carbon nanotube (SWNT)-based light scattering (LS) biosensor for the label-free detection of DNA methyltransferase.73 As shown in Fig. 2C, in the absence of the target DNA methyltransferase EcoRI, the hairpin DNA (HP-DNA) detection probes are cleaved by EcoRI endonuclease to generate single-stranded DNAs, which can efficiently wrap SWNTs via π–π stacking interaction to ensure the suspension of SWNTs in the salt medium. After centrifugation, the remaining SWNTs can produce a distinct light scattering signal. In the presence of the target DNA methyltransferase EcoRI, the HP-DNAs are methylated and cannot be digested by EcoRI endonuclease. The intact double-stranded HP-DNAs are unable to wrap the SWNTs, leading to the aggregation of SWNTs in the salt medium. After the centrifugation, no SWNT remained in the supernatant and thus no light scattering can be detected. This light scattering biosensor enables the homogeneous and label-free detection of DNA methyltransferase with a linear range from 0 to 100 U mL−1 (Fig. 2D) and a detection limit of 10 U mL−1.

Graphene is a kind of carbon nanomaterial with a one-atom-thick and two-dimensional structure, and it possesses highly attractive electronic and mechanical features.81,82 Graphene oxide (GO) is prepared by the oxidation of graphite, and it can serve as an excellent FRET acceptor for biosensing applications.43 Min et al. developed a GO-based fluorescent biosensor for the detection of DNA methyltransferase.74 As shown in Fig. 2E, the partially complementary detection probes labelled with FAM can bind with the GO through the π-stacking interaction between the single-stranded regions of detection probes and the GO surface, inducing efficient quenching of the FAM signal. When target DNA methyltransferase HaeIII is absent, the double-stranded regions of the detection probes are cleaved, leading to the release of FAM from the GO surface and the recovery of FAM fluorescence (Fig. 2F). When the target DNA methyltransferase HaeIII is present, the double-stranded regions of the detection probes are methylated and cannot be digested by EcoRV, and thus the FAM signal is still quenched by GO. This fluorescent biosensor employs GO as the efficient quencher, greatly reducing the assay cost. This fluorescent biosensor enables real-time detection of DNA methyltransferase with a detection limit of 0.1 U mL−1, and it can further be used for the screening of DNA methyltransferase inhibitor S-adenosylhomocysteine.

Graphene quantum dots (GQDs) are a class of graphene nanomaterials consisting of a π-conjugated single sheet or disk, and they can serve as promising fluorophores due to their superior properties of high quantum yield, low toxicity, resistance to photobleaching, and good biocompatibility.83–85 Hosseini et al. developed a GQD-based fluorescent biosensor for the detection of DNA methyltransferase.75 As shown in Fig. 2G, the 5′-amine-labelled double-stranded DNA detection probes can bind with GQDs through electrostatic interaction, resulting in a decrease of GQD fluorescence, because the negatively charged DNAs may influence the surface passivation layer of GQDs and induce efficient fluorescence quenching. In the absence of the target DNA methyltransferase M.SssI, the detection probes will be cleaved by HpaII enzyme, leading to the release of detection probes from the surface of the GQDs and the recovery of GQD fluorescence (Fig. 2H). In contrast, in the presence of the target DNA methyltransferase M.SssI, the detection probes are methylated and cannot be cleaved by HpaII, and thus the GQD is still quenched by the remaining detection probes. This fluorescent biosensor enables sensitive detection of DNA methyltransferase with a linear range from 2 to 30 U mL−1 and a detection limit of 0.7 U Ml−1, and it exhibits good assay performance in 10% human serum samples with a recovery rate of 90.2–102%.

4. Semiconductor quantum dot-based DNA methyltransferase biosensors

Semiconductor quantum dots (QDs) exhibit unique optical properties including a high quantum yield, broad absorption spectra, narrow emission spectra, and excellent photo-stability, making them ideal alternatives to organic dyes for biosensing applications.88–90 Zhang et al. developed a semiconductor QD-based fluorescent biosensor for sensitive detection of DNA methyltransferase Dam.86 As shown in Fig. 3A, a rationally designed hairpin probe contains a biotin at the 3′ end, a BHQ2 at the 5′ end, and a Cy5 at the opposite strand of the stem. The hairpin probe can be assembled on a streptavidin-coated 605QD surface through specific biotin–streptavidin binding. Because of the significant spectral overlap between 605QD emission and Cy5 absorption, efficient fluorescence resonance energy transfer (FRET) from 605QD to Cy5 can occur, but the FRET signal will subsequently be quenched by the nearby BHQ2. In the presence of the target DNA methyltransferase Dam, the hairpin probes are methylated and subsequently cleaved by DpnI enzyme, inducing the release of BHQ2 from the hairpin probes and consequently the recovery of the FRET signal and the emission of Cy5. This fluorescent biosensor is very simple with the involvement of only a single probe. Due to the introduction of single-molecule detection technology,91 this biosensor is very sensitive with a detection limit as low as 0.008 U mL−1. It can be further applied for the screening of the DNA methyltransferase inhibitor gentamicin (Fig. 3B) and accurate measurement of DNA methyltransferase Dam in human serum samples with a recovery rate of 98.55–102.77%.
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Fig. 3 Semiconductor quantum dot-based DNA methyltransferase biosensors. (A) Schematic illustration of the semiconductor QD-based fluorescent biosensor for the detection of DNA methyltransferase Dam. (B) Measurement of the inhibition effect of gentamicin upon DAM activity. Reproduced from ref. 86 with permission from The Royal Society of Chemistry. (C) Schematic illustration of the semiconductor QD-based electrochemical biosensor for the detection of DNA methyltransferase Dam. (D) Variance of the current signal in response to different-concentration Dam. Reproduced from ref. 87 with permission from Elsevier B. V.

In addition, the semiconductor QDs may function as promising photoelectrochemical materials for the construction of electrochemical biosensors.92–94 Park et al. developed a semiconductor QD-based electrochemical biosensor for the detection of DNA methyltransferase Dam.87 As shown in Fig. 3C, the double-stranded detection probe is immobilized on the gold matrix surface, with one strand being labelled with biotin at the 5′ end. The streptavidin-conjugated QD can bind with the detection probes via streptavidin–biotin interaction, leading to the formation of the QD-modified gold matrix. In the presence of the target DNA methyltransferase Dam, the detection probes are methylated and subsequently cleaved by DpnI enzyme, resulting in the release of QDs from the gold matrix surface. The free QDs can move to the glassy carbon electrode via convective transport, promoting the generation of an electrochemical signal (Fig. 3D). In contrast, when target DNA methyltransferase Dam is absent, the detection probe remains intact and no QDs can be released, and thus no electrochemical signal can be detected. This electrochemical biosensor exhibits a detection limit of 0.79 U mL−1 and a linear range from 1 to 128 U mL−1, and it can be used to screen the DNA methyltransferase inhibitor 5-fluorouracil. Moreover, this biosensor is very simple and convenient, and it enables one-step detection of DNA methyltransferase activity without the involvement of tedious washing steps, making it suitable for applications in facility-limited environments.

5. Metal nanocluster-based DNA methyltransferase biosensors

Metal nanoclusters possess some molecule-like properties such as size-dependent fluorescence and discrete electronic states because their size is similar to the electron Fermi wavelength.38,97,98 The florescent metal nanoclusters have been widely used for bioanalysis applications due to their attractive features such as ease of synthesis, ultrasmall size, and excellent photostability.99–101 Zhou et al. developed a AgNC-based fluorescent biosensor for the detection of DNA methyltransferase Dam.95 As shown in Fig. 4A, a hairpin detection probe is used for target recognition, in which the AgNCs can be captured by the C-rich tail and their fluorescence is significantly enhanced by the complementary G-rich tail. Upon addition of the target DNA methyltransferase Dam, the detection probes are methylated and cleaved by DpnI enzyme, leading to the release of the stem duplex. The melting temperature of the free stem duplex containing a C-rich tail and a G-rich tail is 22 °C, much lower than the reaction temperature of 33 °C. As a result, the C-rich tail and the G-rich tail are separated from each other, inducing a sharp decrease of AgNC fluorescence. This fluorescent biosensor eliminates the use of expensive fluorescence labelling, enabling simple detection of DNA methyltransferase with a detection limit of 1 U mL−1, and it can be further applied for the screening of the DNA methyltransferase inhibitor 5-fluorouracil (Fig. 4B).
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Fig. 4 Metal nanocluster-based DNA methyltransferase biosensors. (A) Schematic illustration of the AgNC-based fluorescent biosensor for the detection of DNA methyltransferase Dam. (B) Measurement of the inhibition effect of 5-fluorouracil upon Dam activity. Reproduced from ref. 95 with permission from Elsevier B. V. (C) Schematic illustration of the CuNP-based fluorescent biosensor for the detection of DNA methyltransferase M.SSsI. (D) Measurement of M.SssI activity in 1% cell extracts. Reproduced from ref. 96 with permission from Elsevier B. V.

In contrast to AgNCs, copper nanoclusters (CuNCs) exhibit a fast synthetic speed, large Stokes shift, and low toxicity.102 Liu et al. developed a CuNP-based fluorescent biosensor for the detection of DNA methyltransferase M.SssI.96 As shown in Fig. 4C, in the presence of the target DNA methyltransferase M.SssI, the dumbbell detection probe is methylated in its stem region, and it cannot be digested by T5 and Exo I exonuclease. The intact dumbbell probes can serve as templates to synthetize highly fluorescent CuNCs. In contrast, the dumbbell probes are completely digested by T5 and Exo I exonuclease when the target DNA methyltransferase is absent, and thus no CuNCs can be synthetized and no fluorescence signal is detected. This fluorescent biosensor is very sensitive with a detection limit of 0.16 U mL−1, and it can be used to screen two DNA methyltransferase inhibitors, SGI-1027 and 5-Aza. Moreover, it performs well even in complex biological samples such as 1% human serum and 1% cancer cell lysate (Fig. 4D).

6. DNA methyltransferase biosensors based on the integration of different nanomaterials

Besides the use of a single type of nanomaterial for the development of DNA methyltransferase assay, the integration of different nanomaterials can be applied for methyltransferase assay103–107 through the combination of different functional nanomaterials in a single biosensor (e.g., both MSNs and AuNPs as carriers,105 AuNPs as the carrier and CNNS as the ECL emitter106) or the introduction of new signal transduction between different nanomaterials (e.g., exciton energy transfer between QDs and AuNPs,103 the enhanced chemiluminescence between AuNPs and QDs104).

There is a significant overlap between the semiconductor QD exciton band and the AuNP plasmon band, and thus efficient exciton energy transfer (EET) from the semiconductor QDs to the AuNPs may occur in the photoelectrochemical system.108–110 Shen et al. developed a semiconductor QD and a AuNP-based photoelectrochemical biosensor for the detection of DNA methyltransferase M.SssI based on the EET effect.103 As shown in Fig. 5A, the indium tin oxide (ITO) electrode is functionalized with TiO2 film and the COOH-modified CdSe QDs. Then, the 5′-NH2-detection probe-labelled AuNPs are assembled on the surface of the ITO electrode through the coupling reaction between the COOH and NH2 group, resulting in a decrease of the photoelectrochemical signal due to efficient EET from the QD to the AuNP. In the absence of the target DNA methyltransferase M.SssI, the pDNAs can be cleaved by HpaII endonuclease, leading to the release of AuNPs from the CdSe QDs and the increase of the photoelectrochemical signal. With the addition of the target DNA methyltransferase M.SssI, the pDNAs are methylated and cannot be digested by HpaII, and a low photoelectrochemical signal is detected as a result of the EET effect. This photoelectrochemical biosensor exhibits excellent reproducibility and good stability even after storing in PBS solution for one month. It shows a linear range from 0.01 to 150 U mL−1 and a low detection limit of 0.0042 U mL−1 (Fig. 5B), and it can be further used to screen two DNA methyltransferase inhibitors, fisetin and 5-fluorouracil.


image file: c9tb02458a-f5.tif
Fig. 5 (A) Schematic illustration of the integration of semiconductor QDs with AuNPs for the development of photoelectrochemical biosensors for the detection of DNA methyltransferase M.SssI. (B) Variance of the photocurrent signal in response to different-concentration M.SssI. Reproduced from ref. 103 with permission from Elsevier B. V. (C) Schematic illustration of the integration of AuNPs with semiconductor QDs for the development of a chemiluminescent biosensor for the detection of DNA methyltransferase M.SssI. (D) Measurement of the ECL intensity in response to different-concentration M.SssI. Reproduced from ref. 104 with permission from the American Chemical Society. (E) Schematic illustration of the integration of MSNs with AuNPs for the development of a SERS biosensor for the detection of DNA methyltransferase Dam. (F) Measurement of the SERS spectra in response to different-concentration Dam. Reproduced from ref. 105 with permission from The Royal Society of Chemistry.

AuNPs exhibit similar activity to that of glucose oxidase, and they can catalyze the oxidation of glucose to produce hydrogen peroxide (H2O2).111 Based on the glucose oxidase-like catalytic activity, Zhang et al. developed a AuNP-based electrochemiluminescent biosensor for the sensitive detection of DNA methyltransferase M.SssI.104 As shown in Fig. 5C, the double-stranded detection probes containing the target recognition sequence are immobilized on the surface of the semiconductor QD-modified glassy carbon electrode (GCE) through NH2–COOH condensation. The target DNA methyltransferase M.SssI can catalyze the methylation of the detection probes, protecting the detection probes from cleavage by HpaII enzyme. AuNPs are then linked with the intact detection probes via the Au–S reaction to catalyse the oxidation of glucose to produce H2O2, generating an enhanced chemiluminescence signal of CdS QDs via energy transfer. In contrast, when the target DNA methyltransferase M.SssI is absent, the unmethylated detection probes will be cleaved by HpaII enzyme, and thus no AuNPs can be linked to the GCE surface and no chemiluminescence signal is detected. This electrochemiluminescent biosensor exhibits a significantly reduced background and high sensitivity with a detection limit of 0.05 U mL−1 and a linear range from 1 to 120 U mL−1 (Fig. 5D), and it performs well in human serum samples with a recovery rate of 96.0–100.5%.

Mesoporous silica nanoparticles (MSNs) have a highly ordered tunable pore structure, large loading capacity, and good biocompatibility, making them promising carrier vehicles for drug delivery and biosensing.112–114 Zhang et al. developed a MSN and AuNP-based surface-enhanced Raman scattering (SERS) biosensor for the detection of DNA methyltransferase Dam.105 As shown in Fig. 5E, the loading DNAs are introduced to MSNs to form MSN–DNA complexes, and then the channels of the MSN–DNA complexes are capped with DNA 1/2 to form MSN-loading DNA–DNA 1/2 complexes. In the presence of the target DNA methyltransferase Dam, the hairpin DNA detection probes are methylated and cleaved by DpnI enzyme to generate short single-stranded trigger DNAs. The trigger DNAs can bind the toehold of DNA 1 and DNA 2 to induce the cleavage of DNA 1/2 by Nb.BbvCI enzyme in a cyclic manner. As a result, the DNAs 1/2 are detached from the MSNs, leading to the unlocking of MSN channels and the subsequent release of loaded DNAs. The released loaded DNA may serve as the capture probe to enrich the Rox-modified AuNP signal probes through magnetic separation, generating a distinct SERS signal (Fig. 5F). In contrast, when the target DNA methyltransferase Dam is absent, the DNAs 1/2 remain intact, and no loaded DNA is released from the MSNs and thus no SERS signal can be detected. In this biosensor, the MSN serves as both a carrier and a signal amplifier, enabling cost-effective and sensitive detection of DNA methyltransferase with a detection limit of 0.02 U mL−1.

Polymeric carbon nitride exhibits controllable band-gap luminescence and high biocompatibility, and it may function as a novel luminophore in the development of electrochemiluminescence (ECL) biosensors.115–117 Zhang et al. developed a carbon nitride nanosheet (CNNS) and AuNP-based electrochemiluminescent biosensor for the sensitive detection of DNA methyltransferase Dam.106 As shown in Fig. 6A, the AuNPs are immobilized on the CNNS, enabling the assembly of hairpin DNA detection probes onto the surface of the CNNS. In the presence of the target DNA methyltransferase Dam, the detection probes are methylated and subsequently cleaved by DpnI enzyme. The remaining single-stranded DNAs can capture the additional CNNSs due to their stronger noncovalent interaction with the CNNSs than the double-stranded DNA in the detection probe. Consequently, the CNNSs in both the bottom and top layers of the glassy carbon electrode can serve as efficient luminophores to generate an enhanced ECL signal. In contrast, when the target DNA methyltransferase Dam is absent, the detection probes remain intact and are unable to capture the CNNSs, resulting in a decreased ECL signal. This electrochemiluminescent biosensor exhibits a linear range from 0.05 to 80 U mL−1 (Fig. 6B) and high sensitivity with a detection limit of 0.043 U mL−1, superior to the nucleic acid amplification-based DNA methyltransferase assay.118 Moreover, this biosensor can be applied for the screening of the DNA methyltransferase inhibitor 5-fluorouracil and the detection of DNA methyltransferase in 10% human serum with a recovery rate of 97.4–113%.


image file: c9tb02458a-f6.tif
Fig. 6 (A) Schematic illustration of the integration of CNNSs with AuNPs for the development of electrochemiluminescent biosensors for the detection of DNA methyltransferase Dam. (B) Variance of the ECL intensity in response to different-concentration Dam. Reproduced from ref. 106 with permission from the American Chemical Society. (C) Schematic illustration of the integration of semiconductor QDs with g-C3N4 for the development of photoelectrochemical biosensors for the detection of DNA methyltransferase M.SssI. (D) Measurement of the inhibition effect of atrazine (a) and azamethipos (b) upon M.SssI activity. Reproduced from ref. 107 with permission from Elsevier B. V.

Graphite-like C3N4 (g-C3N4) is a metal-free semiconductor material with similar energy band features to metal semiconductors, and it can be used in photoelectrochemical biosensing.119–121 Yin et al. developed a g-C3N4 and semiconductor QD-based photoelectrochemical biosensor for the detection of DNA methyltransferase M.SssI.107 As shown in Fig. 6C, g-C3N4 serves as the photoelectric material and is modified on the ITO electrode surface. The probed DNA can bind with g-C3N4 through the reaction between the COOH group of the probed DNA and the NH2 group of g-C3N4. Subsequently, target DNA will hybridize with the probed DNA to form a recognition double-stranded DNA detection probe. The target DNA methyltransferase M.SssI can catalyse the methylation of detection probes and protects them from digestion by HpaII endonuclease. The CdS QDs can bind to the remaining detection probes via the coupling reaction, bringing the CdS QDs and the electrode together to generate a distinct photoelectrochemical signal. In the absence of the target DNA methyltransferase M.SssI, the detection probes are cleaved by HpaII, and thus no CdS QDs can approach the electrode and no photoelectrochemical signal is detected. This photoelectrochemical biosensor shows a linear range from 1 to 80 U mL−1 and a detection limit of 0.316 U mL−1, and it can be used to screen two DNA methyltransferase inhibitors, atrazine and azamethipos (Fig. 6D).

7. Summary and outlook

DNA methyltransferases are important epigenetic regulators for gene regulation and cellular functions, and the development of efficient biosensors for DNA methyltransferase assay is essential for fundamental biological and biomedical research studies, disease diagnosis, and drug discovery. Nanomaterials possess unique size-dependent optical, chemical, electronic, and mechanical properties, making them attractive for the construction of biosensors with high sensitivity and good selectivity. A variety of DNA methyltransferase biosensors based on AuNPs,56–59 carbon nanomaterials,72–75 semiconductor QDs,86,87 metal nanoclusters,95,96 and the integration of different nanomaterials103–107 as building materials were rationally designed. These biosensors have been successfully applied for the quantification of DNA methyltransferase activity and the screening of DNA methyltransferase inhibitors, and they exhibit high sensitivity and good selectivity even in complex biological matrix. Despite the progress in the development of efficient biosensors, there are still some issues that need to be solved. First, most of the conventional assays are available for the detection of bacterial DNA methyltransferase (e.g. M.SssI and Dam, Table 1), and more efforts should be devoted to developing biosensors suitable for the detection of human DNA methyltransferases such as human DNA methyltransferases 1, 3a and 3b (Dnmt1, Dnmt3a and Dnmt3b).122–124 Second, further improvement of detection sensitivity will enable efficient detection of trace amounts of DNA methyltransferase, greatly benefiting early disease diagnosis. Nucleic acid-based amplification is the most effective way to amplify the target signal for the improvement of detection sensitivity. The introduction of appropriate nucleic acid-based amplification approaches such as polymerase-dependent amplification,125–130 ligase amplification reaction,53,131,132 nuclease-assisted signal amplification,133–135 and enzyme-free signal amplification136–138 into the biosensor system may greatly enhance the detection sensitivity. Third, the imaging of DNA methyltransferases in living cells is of great importance for real-time monitoring of their roles in cellular physiological and pathological processes. However, the applications of nanomaterial-based biosensors for the imaging of DNA methyltransferase in living cells have not been explored so far. This can be ascribed to the fact that an additional nuclease is frequently required for the recognition of the methylated DNA products, but it is difficult to enter living cells without altering their normal function. Therefore, the development of novel DNA methyltransferase assays without the involvement of nuclease for cell imaging applications is urgently needed. Fourth, one particular cellular function may rely on the cooperation of a variety of different DNA methyltransferases,139–141 and the simultaneous detection of multiple DNA methyltransferases is highly desired, which can be achieved by functionalizing various nanomaterials with specific probes towards different DNA methyltransferases. Through these efforts, we believe that nanomaterial-based DNA methyltransferase biosensors may find more and more applications in biological and biomedical research studies, clinical diagnosis and drug discovery in the near future.
Table 1 Summary of nanomaterial-based DNA methyltransferase biosensorsa
Target Type of nanomaterials Type of signal LOD (U mL−1) Linear range (U mL−1) Ref.
a Abbreviations: AgNC, silver nanocluster; AuNP, gold nanoparticle; AuNR, gold nanorod; CD, circular dichroism; CNNS, carbon nitride nanosheets; CuNC, copper nanocluster; ECL, electrochemiluminescence; FP, fluorescence polarization; g-C3N4, graphite-like C3N4; GO, graphene oxide; GQD, graphene quantum dot; MS, mesoporous silica; MWCNT, multiwalled carbon nanotube; QD, quantum dot; SERS, surface-enhanced Raman scattering; SWNT, single-walled carbon nanotube.
Dam AuNP Absorbance 2.5 56
M.SssI AuNP CD 0.27 0.5 to 150 57
Dam AuNP Current 0.02 0.075 to 30 58
Dam AuNR Fluorescence 0.25 0.5 to 20 59
Dam MWCNT FP 0.0001 72
EcoRI SWNT Light scattering 10 0 to 100 73
HaeIII GO Fluorescence 0.1 74
M.SssI GQD Fluorescence 0.7 2 to 30 75
Dam QD Fluorescence 0.008 0.004 to 2 86
Dam QD Current 0.79 1 to 128 87
Dam AgNC Fluorescence 1 1 to 100 95
M.SssI CuNC Fluorescence 0.16 100 to 200 96
M.SssI AuNP, QD Current 0.0042 0.01 to 150 103
M.SssI AuNP, QD Chemiluminescence 0.05 1 to 120 104
Dam MS, AuNP SERS 0.02 0.1 to 10 105
Dam CNNS, AuNP ECL 0.043 0.05 to 80 106
M.SssI QD, g-C3N4 Current 0.316 1 to 80 107


Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (grant no. 21735003, 21527811 and 21705096) and the Award for Team Leader Program of Taishan Scholars of Shandong Province, China.

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Footnote

These authors contributed equally to this work.

This journal is © The Royal Society of Chemistry 2020