Sensitive Electrochemical Detection of Human Methyltransferase Based on a Dual Signal Amplification Strategy Coupling Gold Nanoparticle–DNA Complexes with Ru(III) Redox Recycling

Abstract

Effective detection of DNA methyltransferase (DNMT) activity is significant for cancer research. Herein, we developed a sensitive electroanalytical method to detect human DNA (cytosine-5)-methyltransferase 1 (DNMT1) from crude lysates of cancer cells. In this assay, capture DNA having a preferred DNMT1 methylation site was immobilized on a gold electrode and then hybridized with gold nanoparticle (Au NP)-DNA complexes. The modified electrodes were equilibrated with the lysate and then incubated with methylation-sensitive restriction enzyme. If the lysate was negative for DNMT1 activity, the Au NP-DNA complexes would be cut by the restriction enzyme and released from the electrode. Conversely, restriction enzyme cleavage would be blocked by the fully methylated duplexes, and the Au NP-DNA complexes would remain on the electrode. Electroactive Ru(NH) was used as the signal reporter, because of its electrostatic attraction to DNA, resulting in an electrochemical signal. Since the electrochemical signal reflects the amount of Ru(III) redox and the amount of Ru(III) redox is correlated with the activity of DNMT1, the activity of DNMT1 is proportional to the electrochemical signal. The signal could be amplified by the numerous DNAs on the Au NPs and further amplified by Ru(III) redox recycling. With this method, a detection limit down to 0.3 U/mL for pure DNMT1 and 8 MCF-7 cells was achieved. DNMT1 activities of different cell lines were also successfully evaluated.

Single-ribonucleotide repair-mediated ligation-dependent cycling signal amplification for sensitive and specific detection of DNA methyltransferase

“…This method is extremely simple, easily manipulated and highly sensitive, and it can be performed in a homogeneous format without the involvement of any thermal cycling, washing and separation steps. Taking advantage of the high specificity of RNase HII-catalyzed single-ribonucleotide excision, the high amplification efficiency of cyclic ligation-dependent SDA, and the low background signal resulting from the inhibition of nonspecific amplification by single-ribonucleotide excision and specific ligation-dependent strand displacement polymerase extension, this method exhibits the highest sensitivity reported so far with a detection limit of as low as 4.8 × 10 –6 U mL –1 and a large dynamic range of 5 orders of magnitude, which is more sensitive than the reported colorimetric (0.25 U mL –1 ), 15 chemiluminescence (1.29 × 10 –4 U mL –1 ), 25 electrochemical (0.3 U mL –1 ) 18 and fluorescence assays (0.01, 0.06 and 0.002 U mL –1 ). 21 – 23 Moreover, this method can be applied for the discrimination of Dam MTase from other DNA MTases, screening of potential Dam MTase inhibitors, and accurate quantification of Dam MTase activity in real complex samples.…”

“…To the best of our knowledge, this is the most sensitive method for DNA MTase assay reported so far. In comparison with the reported DNA MTase assays, 15 , 18 , 21 – 23 , 25 the sensitivity of the proposed method has been improved by 5 orders of magnitude as compared with that of methylation-responsive DNAzyme-based colorimetric assay (0.25 U mL –1 ), 15 2 orders of magnitude as compared with that of rolling circle amplification (RCA)- and G-quadruplex DNAzyme-based chemiluminescence assay (1.29 × 10 –4 U mL –1 ), 25 5 orders of magnitude as compared with AuNP–DNA and Ru( iii )-mediated dual signal amplification-based electrochemical assay (0.3 U mL –1 ), 18 4 orders of magnitude as compared with that of nicking enzyme-assisted signal amplification-based fluorescence assay (0.06 U mL –1 ), 23 4 orders of magnitude as compared with that of exonuclease III-mediated target recycling-based fluorescence assay (0.01 U mL –1 ), 22 and 3 orders of magnitude as compared with that of QD-mediated FRET assay (0.002 U mL –1 ). 21 The enhanced sensitivity can be ascribed to (1) the high specificity of RNase HII-catalyzed single-ribonucleotide excision, (2) the high amplification efficiency of cyclic ligation-dependent SDA, and (3) the low background signal resulting from the inhibition of nonspecific amplification by single-ribonucleotide excision and specific ligation-dependent strand displacement polymerase extension.…”

“… 15 Electrochemical assays combine a series of nanomaterials ( e.g. graphene oxide (GO), 16 silver nanoparticles (AgNPs) 17 and gold nanoparticles (AuNPs) 18 , 19 ) with the methylation-sensitive restriction endonuclease to monitor the DNA MTase activity. Fluorescence assays use GO- 20 and quantum dot (QD) 21 -based fluorescence resonance energy transfer (FRET) to quantify DNA MTase activity.…”

Cytosine-5 methylation-directed construction of a Au nanoparticle-based nanosensor for simultaneous detection of multiple DNA methyltransferases at the single-molecule level

“…The conventional methods include radioactive labelingbased gel electrophoresis, 15 enzyme-linked immunoassay, 16 and high-performance liquid chromatography, 17 but they suffer from intrinsic drawbacks of hazardous radiation labeling, 15 costly protein antibodies, 16 tedious sample preparation, 17 low detection sensitivity, 15,16 and time-and labor-consuming experimental procedures. [15][16][17] Alternatively, some new approaches including colorimetric, 18,19 luminescence, 20 electrochemical, [21][22][23][24] and uorescence assays 25,26 have been developed to overcome the limitations. For example, the colorimetric assay takes advantage of methylation-responsive DNA-gold nanoparticle (AuNP) assembly 18 and terminal protectiondirected DNA-AuNP diffusion 19 for visualized detection of Dam MTase and DNA cytosine-5 methyltransferase (Dnmt 1), but it exhibits relatively poor sensitivity.…”

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

“…Among the studies on the determination of DNA MTase, only some have focused on human DNMT1, 34,35 and the current methods are limited to laboratory study and have not been applied in the detection of DNMT1 level in serum samples, and hence cannot achieve the high-throughput screening in clinical practice. Therefore, it is necessary to establish another efficient, simple, rapid, and high-throughput method for the detection of DNMT1 level in serum samples to meet the future clinical needs.…”