•A novel electrogenerated chemiluminescence biosensing method has been successfully developed for detection of 5-hmC.•Subtly making the signal materials to covalently bond to the electrode surface by using APBA served as the bridging reagent.•The ECL biosensor shows good analytical performance for quantitative detection of DNA hydroxymethylation.We developed a novel electrogenerated chemiluminescence (ECL) biosensing method for quantitative detection of DNA hydroxymethylation (5-hydroxymethylcytosine, 5-hmC) using T4 β-glucosyltransferase (β-GT) and graphene oxide (GO) for enzymatic and chemical modification of the 5-hmC. β-GT selectively glucosylated the hydroxymethyl group of 5-hmC to improve selectivity, whereas GO, which loads substantial amounts of the ECL reagent, was used as the signal amplification element to enhance assay sensitivity. The ECL bioassay involved the capture of DNA containing 5-hmC, which was chemically modified with a glucose moiety, through hybridization with complimentary 5′-thiol-modified DNA on a glassy carbon electrode (GCE) under the catalysis of β-GT. The dichlorotris (1,10-phenanthroline) ruthenium-assembled GO (Ru(phen)32+/GO) composite was coupled to the electrode surface via a bridging agent, and provided excellent ECL signal. The bridging agent used was 3-aminophenylboronic acid (APBA), boronic acid and its derivatives can interact with 1,2- or 1,3-diols (such as glucose) to generate 5- or 6-membered cyclic boronate esters. The obtained ECL intensity was positively correlated with the concentration of DNA containing 5-hmC, the linear range was from 0.01 pM to 1.00 nM, with a low limit of detection of 3.84 fM. Our results demonstrate that use of enzymatic and chemical modification in combination with highly sensitive ECL is a facile and promising approach for DNA hydroxymethylation analysis.

•A signal-off ECL biosensing method was developed for the quantification of 5-hmC.•The method is based on the glycosylation of 5-hmC.•The method has a high detection sensitivity with a low LOD of 1.63×10–11 M for 5-hmC DNA.•The method exhibited excellent stability and reproducibility.In this study, a new and sensitive signal-off electrogenerated chemiluminescence (ECL) biosensing method for the quantification of 5-hydroxymethylcytosine in DNA (5-hmC-DNA) was developed. The method achieved simple and sensitive detection of 5-hmC-DNA based on the glycosylation of 5-hmC, combining both the amplification function of gold nanoparticles (AuNPs) and the high quenching efficiency of the tris(2, 2′-ripyridine) dichlororuthenium(II) (Ru(bpy)32+)-ferrocene (Fc) system. First, the electrode modified with a mixture of Nafion and AuNPs was utilized as the platform for electrostatically adsorbing Ru(bpy)32+(an ECL-emitting species) and assembling 5-hmC-DNA. The 5-hmC-DNA was glycosylated by T4 β-glucosyltransferase, yielding β-glucosyl-5-hydroxymethyl-cytosine in DNA (5-ghmC-DNA). Finally, quencher-FcBA was further covalently bound to 5-ghmC-DNA through formation of boronate ester covalent bonds between boronic acid and cis-diols of 5-ghmC, resulting in a decrease in ECL intensity. The results indicated that the decreased ECL intensity was directly linear to the concentration of 5-hmC-DNA in the range from 1.0×10−8 to 5.0×10–11 M with a low detection limit of 1.63×10–11 M. In addition, this ECL method was demonstrated to be useful for the quantification of 5-hmC in clinical serum samples. Moreover, the method allowed good discrimination among cytosine (5-C), 5-methylcytosine (5-mC), and 5-hmC in DNA.Download high-res image (236KB)Download full-size image

DNA methylation is used to dynamically reprogram cells in the course of early embryonic development in mammals. 5-Hydroxymethylcytosine in DNA (5-hmC-DNA) plays essential roles in the demethylation processes. 5-Methylcytosine in DNA (5-mC-DNA) is oxidized to 5-hmC-DNA by 10–11 translocation proteins, which are relatively high abundance in embryonic stem cells and neurons. A new method was developed herein to quantify 5-hmC-DNA based on selective electrogenerated chemiluminescence (ECL) labeling with the specific oxidation of 5-hmC to 5-fC by KRuO4. A thiolated capture probe (ssDNA, 35-mer) for the target DNA containing 5-hmC was self-assembled on a gold surface. The 5-hmC in the target DNA was selectively transformed to 5-fC via oxidation by KRuO4 and then subsequently labeled with N-(4-aminobutyl)-N-ethylisoluminol (ABEI). The ABEI-labeled target DNA was hybridized with the capture probe on the electrode, resulting in a strong ECL emission. An extremely low detection limit of 1.4 × 10–13 M was achieved for the detection of 5-hmC-DNA. In addition, this ECL method was useful for the quantification of 5-hmC in serum samples. This work demonstrates that selective 5-hmC oxidation in combination with an inherently sensitive ECL method is a promising tactic for 5-hmC biosensing.

•An ECL biosensing method for discrimination of DNA hydroxymethylation was developed.•An ECL biosensing method for assay of the β-GT activity was developed.•The method is based on the β-GT-MspI interaction system and the quenching of ECL.•The method is PCR-free and sensitive, achieved a lower DL of 0.04 U/mL β-GT.An electrogenerated chemiluminescence (ECL) biosensing method for highly sensitive discrimination of DNA hydroxymethylation and assay of the β-glucosyltransferase (β-GT) activity was developed. The ECL biosensing electrode was fabricated by gold nanoparticles (AuNPs)/Nafion film, and then, tris(2, 2′-ripyridine) dichlororuthenium(II) (Ru(bpy)32+) was electrostatically adsorbed into the AuNPs/Nafion film, finally, the hydroxymethylated double-stranded DNA (ds-DNA)-tagged with ferrocene was self-assembled onto the surface of the AuNPs. When β-GT and uridine diphosphoglucose (UDP-Glu) were introduced, the hydroxymethylcytosine residues within 5′-CCGG-3′ of ds-DNA on the biosensing electrode were glucosylated. After the glucosylated biosensing electrode was treated by MspI endonuclease, the unglucosylated hydroxymethylcytosine was cleaved, leading to the quencher leaving the electrode, resulting in an increased ECL signal. For the ECL biosensing method, it showed an extremely low detection limit of 0.04 U/mL for β-GT, and offered a good discrimination toward cytosine, 5-methylcytosine, and 5-hydroxymethylcytosine. This work demonstrates that the combination of the enzyme-linkage reactions with the highly sensitive ECL method is a promising strategy for the discrimination of DNA hydroxymethylation, assay of the activity of β-GT, and evaluation of the capability of inhibitors for the β-GT.

An electrogenerated chemiluminescence (ECL) biosensing method for the determination of DNA methyltransferase activity is developed by the quenching of tris(2,2′-bipyridine)ruthenium ECL by ferrocene, and it is demonstrated that the ECL biosensing method measures DNA adenine methylation methyltransferase over a dynamic concentration range (0.1 U mL−1–100 U mL−1) with an extremely low detection limit of 0.03 U mL−1, using gold nanoparticles and a quenching ECL signal produced by a chemical quencher such as ferrocene.

•Label-free ECL biosensing method for DNA MTase was developed.•The ECL biosensing method is based on Ru(phen)32+-assembled graphene oxide served as an ECL signal compound.•The biosensing method showed high sensitivity.A novel label-free electrogenerated chemiluminescence (ECL) biosensing method for the determination of DNA methyltransferase (MTase) activity was developed on base of enzyme-linkage reactions and tris(1, 10-phenanthroline) ruthenium-assembled graphene oxide (GO) served as an ECL signal compound. The ECL biosensing electrode was fabricated by self-assembling 5′-thiol modified hairpin-capture DNA probe containing methylation recognition site 5′-GATC-3′ on the surface of a gold electrode. When DNA adenine methylation (Dam) MTase and S-adenosyl-L-methionine were introduced, all adenine residues within 5′-GATC-3′ of hairpin-capture DNA probe on the biosensing electrode were methylated. After the methylated biosensing electrode was treated by the methylation-sensitive restriction endonuclease Dpn I, the methylated adenines were cleaved, methylation-induced scission of hairpin-capture DNA probe would displace the hairpin section and remain the “capture DNA probe” section on the gold electrode, then a long ssDNA was immobilized via the partial hybridization reaction between long ssDNA and hairpin-capture DNA probe remained section, the more binding site allow tris(1, 10-phenanthroline) ruthenium-assembled GO to be more bound to the long ssDNA on the electrode surface through both hydrophobic and π–π stacking interaction, in conjunction with the generation of a increased ECL signal. The ECL intensity versus the concentration of Dam MTase was linear in the range from 0.02 unit/mL to 10 unit/mL. The detection limit was 0.01 unit/mL. This work demonstrates that using the different affinities of GO for ssDNA and dsDNA for the fabrication of the label-free ECL biosensing method for DNA MTase activity is promising approach.

•A novel ECL biosensing method for DNA MTase was developed.•The method was based on graphene oxide-assisted amplification.•The screening of the inhibitors of DNA MTase could be achieved based on the assay.A novel electrogenerated chemiluminescence (ECL) biosensing method for highly sensitive detection of DNA methylation and assay of the methyltransferase (MTase) activity was developed by using the methylation-sensitive restriction endonuclease Dpn I and methylation-responsive hairpin-capture DNA probe to improve selectivity and by employing signal amplification of graphene oxide (GO) to enhance the assay sensitivity. The ECL biosensing electrode was fabricated by self-assembling a design of 5′-thiol modified methylation-responsive hairpin-capture DNA probe on the surface of a gold electrode. When DNA adenine methylation (Dam) MTase and S-adenosylmethionine were introduced, all adenines in the symmetric tetranucleotide 5′-GATC-3′ of hairpin-capture DNA probe on the biosensing electrode were methylated. After the methylated biosensing electrode was treated by Dpn I endonuclease, the methylated adenines were cleaved, methylation-induced scission of hairpin-capture DNA probe would displace the hairpin section and remain the “capture DNA probe” section on the gold electrode. Subsequently, the remained “capture DNA probe” on the gold electrode can hybridize with the 5′-amino modified DNA probe. Finally, tris(1, 10-phenanthroline) ruthenium (Ru(phen)32+)-assembled GO composites were conjugated to the electrode surface via EDC–NHS coupling, a strong ECL response was electrochemically generated. The increased ECL intensity was proportion to Dam MTase activity in the range from 0.05U/mL to 40 U/mL with a detection limit of 0.02 U/mL. The present work demonstrates that the combination of the enzyme-linkage and GO as a platform for signal probe is a great promising approach for MTase activity and evaluation of the capability of inhibitors for the MTase.

A method based on electrogenerated chemiluminescence (ECL) for detection of DNA methylation and assay of the methyltransferase activity is developed, and it is demonstrated that the label-free ECL method is capable of detecting methyltransferase with a detection limit of 3 × 10−6 U mL−1, using a supersandwich amplification technique.

A novel label-free electrogenerated chemiluminescence (ECL) DNA-based biosensing method for the determination of trace bleomycin (BLM) was developed on basis of Fe(II)·BLM-mediated DNA strand scission and Ru(phen)32+ as an ECL probe. A thiolated ss-DNA, as substrate for BLMs, was self-assembled onto surface of a gold electrode to form a hairpin structure. Ru(phen)32+ was intercalated into the hairpin DNA structure. In the presence of Fe(II)·BLM, the hairpin DNA sequence undergoes the irreversible cleavage event under the oxidative effect of BLM with Fe(II) as a cofactor and the intercalated Ru(phen)32+ released from the gold electrode, which can be transduced into a significant decrease in ECL intensity. The ECL intensity versus the concentration of BLMs was linear in the range from 0.1 pM to 50 pM. The detection limit was 0.03 pM. This work demonstrates that using the sequence selectivity of DNA cleavage strategy for the fabrication of the label-free ECL biosensing method is a promising approach for the determination of antitumor drugs.Highlights► Label-free ECL DNA-based biosensing method for bleomycin was developed. ► The ECL biosensing method is based on Fe(II)·BLMs-mediated DNA strand scission. ► The biosensing method showed high sensitivity and selectivity.

An ultrasensitive electrogenerated chemiluminescent (ECL) DNA-based biosensing switch for the determination of bleomycin (BLM) was developed based on Fe(II) ·BLM-mediated hairpin DNA strand cleavage and a structure-switching ECL-dequenching mechanism. A thiolated ss-DNA was used as a substrate for BLMs: one terminus was tethered onto an electrode surface, and the other terminus was labelled with the ECL quencher ferrocene to form a hairpin structure. This thiolated ss-DNA self-assembled on to the tris(2,2′-bipyridine)ruthenium-gold nanoparticle composite modified gold electrode. In the presence of Fe(II) ·BLM, the ECL DNA biosensing switch undergoes an irreversible cleavage event that can trigger a significant increase in ECL intensity. The relationship of ECL intensity and the concentration of BLMs was found to be linear in the range of 5 fM – 5000 fM with a detection limit of 2 fM. This work demonstrates that the design of a highly sensitive ECL DNA-based biosensing switch that uses the sequence selectivity of DNA cleavage mediated by the antitumor drug BLM in combination with a chemical quencher, such as ferrocene, to quench ECL signal(s), offers a promising approach for the determination of ultratrace amounts of antitumor drugs.Highlights► A novel ECL DNA-based biosensing switch for bleomycin was developed. ► The biosensing switch can achieve extremely high sensitivity. ► A promising strategy for the determination of antitumor drugs was provided. ► The biosensing switch is based on Fe(II) ·BLM-mediated hairpin DNA strand cleavage.

A novel electrogenerated chemiluminescence (ECL) biosensing method for highly sensitive detection of DNA methylation and assay of the CpG methyltransferase (M. SssI) activity was developed on basis of enzyme-linkage reactions and ruthenium complex served as an ECL tag. The ECL biosensing electrode was fabricated by self-assembling 5′-thiol modified 32-mer single-strand DNA (ss-DNA)-tagged with ruthenium bis (2,2′-bipyridine) (2,2′-bipyridine-4,4′-dicarboxylic acid)-ethylenediamine on the surface of a gold electrode, and then hybridized with complementary ss-DNA to form duplex DNA (ds-DNA). When M. SssI and S-adenosylmethionine were introduced, all cytosine residues within 5′–CG-3′ of ds-DNA on the biosensing electrode were methylated. After the methylated biosensing electrode was treated by HpaII endonuclease, the un-methylated cytosines were cleaved, thus led to decrease ECL signal. The ECL intensity of ECL biosensing electrode is related to the methylation level and M. SssI activity in a fixed concentration HpaII endonuclease. The increased ECL intensity was direct proportion to M. SssI activity in the range from 0.05 to 100 U/mL with a detection limit of 0.02 U/mL. This work demonstrates that the combination of the enzyme-linkage reactions with a highly sensitive ECL technique is a great promising approach for the detection of DNA methylation level, assay of the activity of MTase, and evaluation of the capability of inhibitors for the methyltransferase.Highlights► A novel ECL biosensing method for highly sensitive detection of DNA methylation and assay of the methyltransferase activity was developed. ► The ECL biosensing method is based on enzyme-linkage reactions and ruthenium complex served as an ECL label. ► Compared with the traditional method, the ECL biosensing method is PCR-free, nonradioactive, simple and sensitive, achieved a lower detection limit of 0.02 U/mL.

A novel electrochemical aptasensor for the detection of thrombin was developed on basis of the thrombin-binding aptamer (TBA) as a molecular recognition element and multi-walled carbon nanotubes (MWCNTs) as a carrier of the electrochemical capture probe. Amine-modified capture probe (12-mer) was covalently conjugated to the MWCNTs modified glassy carbon electrode (GCE). The target aptamer probe (21-mer) contains TBA (15-mer) labeled with ferrocene (Fc), which is designed to hybridize with capture probe and specifically recognize thrombin, is immobilized on the electrode surface by hybridization reaction. Introduction of the analyte thrombin triggered the dissociation of the aptamer probe labeled with Fc from the biosensors, led to a significant decrease in peak current intensity. Differential pulse voltammetry (DPV) was employed to detect the target analyte with different concentrations. The decreased peak current was in proportion to the concentration of thrombin in a range from 1.0 × 10−12 to 5.0 × 10−10 M with a detection limit of 5 × 10−13 M. The present work demonstrates that using MWCNTs as a carrier for electrochemical capture probe is a promising way to amplify the electrochemical signal and to improve the sensitivity of the electrochemical aptasensor.

An electrogenerated chemiluminescence (ECL) biosensing method for the determination of DNA methyltransferase activity is developed by the quenching of tris(2,2′-bipyridine)ruthenium ECL by ferrocene, and it is demonstrated that the ECL biosensing method measures DNA adenine methylation methyltransferase over a dynamic concentration range (0.1 U mL−1–100 U mL−1) with an extremely low detection limit of 0.03 U mL−1, using gold nanoparticles and a quenching ECL signal produced by a chemical quencher such as ferrocene.

A method based on electrogenerated chemiluminescence (ECL) for detection of DNA methylation and assay of the methyltransferase activity is developed, and it is demonstrated that the label-free ECL method is capable of detecting methyltransferase with a detection limit of 3 × 10−6 U mL−1, using a supersandwich amplification technique.