Electrogenerated Chemiluminescence Biosensing Method Research Articles

A sensitive and noninvasive cyclic peptide-based electrogenerated chemiluminescence biosensing method for the determination of sweat glucose.

A sensitive and noninvasive cyclic peptide-based electrogenerated chemiluminescence biosensing method for the determination of sweat glucose was developed. Glucose can be quantified in sweat samples with a recovery of 93%-113% via one-step recognition, which is promising for the determination of sweat glucose.

Sensitive and non-separation 5-hydroxymethylcytosine double-stranded DNA sensing in different mice tissues: Electrogenerated chemiluminescence biosensing method incorporating DNA nanomachine amplification and nanocomposite magnetic separation

Herein, a sensitive and non-separation electrogenerated chemiluminescence (ECL) biosensing method that combines poly (diallyl dimethyl ammonium chloride)/ferrosoferric oxide/molybdenum disulfide (PDDA/Fe3O4/MoS2) nanocomposite with DNA nanomachine was developed to determine 5-hydroxymethylcytosine modified double-strand DNA (5-hmC-dsDNA). A positively charged and magnetic PDDA/Fe3O4/MoS2 nanocomposite was proposed, which can be used to fix negatively charged 5-hmC-dsDNA to overcome the steric resistance problem and is available for magnetic fields separation. In addition, the DNA nanomachine with three elements was fabricated: a DNA walking probe for signal amplification, an ECL reagent labeled S1 DNA (Ru-S1), 5-hmC antibody (Ab) for specifically recognizing 5-hmC modification. In the presence of 5-hmC-dsDNA, sandwich-type conjugates were generated to incubate PDDA/Fe3O4/MoS2 and DNA nanomachine. After magnetic separation and the DNA walking driven by enzyme cleavage, a lot of ECL reagents were released and increased the ECL signal. ECL measurement was performed in a separation-free format. The method showed a broader linear range (0.0090–0.5761 %) and a low limit of detection (0.0024 %). Furthermore, the determination of 5-hmC-dsDNA in different mice tissues using the ECL biosensing method was also carried out, demonstrating that the established strategy may pave a novel avenue for the early clinical diagnosis of disease.

Electrogenerated chemiluminescence biosensing method for DNA hydroxymethylation detection via glycosylation and a new multi-functional ECL signal compound

A simple electrogenerated chemiluminescence (ECL) biosensing method was proposed for specific detection of 5-hydroxymethylcytosine (5-hmC) in double-stranded DNA (5-hmC-dsDNA) on the basis of glycosylation and a new multi-functional ECL signal compound (N-(phthalhydrazide-4-yl)-thiophen-2-amino-5-boronic acid, PTAB). 5-hmC-dsDNA was chosen as the target, T4-β-Glucosyltransferase (β-GT) was as a molecular recognition element, and PTAB was as ECL signal reagent. PTAB includes the three main modules: 4-aminophthalhydrazide (isoluminol module), borinic acid module, and thiophene module. 5-hmC-dsDNA was glycosylated to glucosyl-5-hydroxymethylcytosine dsDNA (5-ghmC-dsDNA) by β-glucosyltransferase (β-GT); then, PTAB (boronic acid module) acted as a bridging agent to couple with 5-ghmC-dsDNA, yielding PTAB-5-ghmC-dsDNA; finally, the PTAB-5-ghmC-dsDNA (thiophene module) anchored to the gold surface via Au-S bond, the ECL signal parts (isoluminol module) was brought close to the biosensing electrode interface and the ECL enhanced simultaneously. The ECL intensity positively correlated with 5-hmC-dsDNA content in the range of 0.0090 % to 0.5761 %, with a detection limit of 0.0058 %. The proposed method was verified by detecting 5-hmC-dsDNA content in mice tissues. In addition, due to the multi-functional ECL signal compound, the presented strategy could be easily extended to ECL detection of various analytes with 1, 2- or 1, 3-diols sample, which provides a great potential for clinical applications.

Electrogenerated chemiluminescence biosensing method based on 5-hydroxymethylcytosine antibody and PDDA-CNTs nanocomposites for the determination of 5-hydroxymethylcytosine double-stranded DNA

A highly sensitive electrogenerated chemiluminescence (ECL) method was developed for trace analysis of 5-hydroxymethylcytosine double-stranded DNA (5-hmC-dsDNA). The poly-(dimethyldiallyl ammonium chloride)/multiwalled carbon nanotubes composite was assembled on a bare glassy carbon electrode (GCE) to provide high specific surface area on which the loadable capacity of 5-hmC-dsDNA and 5-hmC antibody can be greatly increased. The derivative of ruthenium (II) bibyridine, Ru (bpy)2 (dcbpy)NHS, coupled with 5-hmC antibody to activate an ECL reaction when the applied potential was biased at 1.4 V vs. Ag/AgCl. The loading ratio of substrates were optimized to enhance the detection sensitivity of 5-hmC-dsDNA. It was found that the ECL intensity was a piecewise linear function of the concentration of 5-hmC-dsDNA over the range of 1.0 × 10−11–2.0 × 10−9 M. A linear relationship of I = 6850.3 C(nM) + 863.8 (R = 0.9954) was obtained from 0.01 to 0.2 nM, while the fitting equation of I = 3840.0 C(nM) + 1392.4 (R = 0.9974) is for the concentration range of 0.2 – 2.0 nM. The detectable low limit can reach to 2.3 × 10−12 M. Formation of the antigen-antibody immunocomplex in highly concentrated solutions should undertake most of the responsibility for a decrease in slope. Furthermore, reliability, reproducibility and practicability of the ECL method have been proved to perform well, even in real bio-tissues, suggesting promising prospect in early diagnosis of cancer.

Signal-Off Electrogenerated Chemiluminescence Biosensing Platform Based on the Quenching Effect between Ferrocene and Ru(bpy)32+-Functionalized Metal-Organic Frameworks for the Detection of Methylated RNA.

N6-methyladenine (m6A), one of the most common chemical modifications of eukaryotic RNA, participates in many important biological processes. An effective strategy for the quantitative determination of m6A is of great significance. Herein, we used methylated microRNA-21 (miRNA21) as the model target to propose a simple and sensitive electrogenerated chemiluminescence (ECL) biosensing platform to detect a specific m6A RNA sequence. This strategy is based on the fact that the anti-m6A-antibody can specifically recognize and bind to the m6A site in the RNA sequence, resulting in a quenching effect between Ru(bpy)32+-functionalized metal-organic frameworks and ferrocene. Luminescent metal-organic frameworks (Ru@MOFs) not only act as ECL indicators but also serve as nanoreactors for the relative ECL reactions owing to their porous or multichannel structure, which overcomes the fact that Ru(bpy)32+ is easily released when used for aqueous-phase detection, thus enhancing the ECL efficiency. Moreover, the ECL method has fewer modification steps and uses only one antibody to recognize the target RNA sequence, which simplifies the operation process and reduces the detection time, presenting a wide linear range (0.001-10 nM) for m6A RNA determination with a low detection limit (0.0003 nM). Additionally, this developed strategy was validated for m6A RNA detection in human serum. Thus, the ECL biosensing method provides a new method for m6A RNA determination that is simple, highly specific, and sensitive.

A convenient electrogenerated chemiluminescence biosensing method for selective detection of 5-hydroxymethylcytosine in genomic DNA

Herein, a novel and convenient electrogenerated chemiluminescence (ECL) biosensing method for the quantitative detection of 5-hydroxymethylcytosine (5-hmC) in genomic DNA was developed by employing thiolated anchor probe DNA to immobilize 5-hydroxymethylcytosine double-stranded DNA (5-hmC-dsDNA) and a ruthenium(II) complex-tagged reporter probe as an ECL probe. Briefly, the 5-hmC-dsDNA was firstly oxidized to 5-formylcytosine dsDNA (5-fC-dsDNA) by KRuO4 in high yields. Subsequently, the thiolated anchor probe DNA is employed to help immobilize the 5-fC-dsDNA onto the gold electrode surface via the Au-S bond. An ECL probe DNA, labeled with a signal transducer − Ru(bpy)2(cbpy)NHS, hybridized with the anchor probe DNA on the electrode, resulting in an intensive ECL emission. The obtained ECL intensity was linearly proportional to the content of 5-hmC in the range from 0.0090% to 0.5761%, with the limit of detection calculated to be 0.0036% (S/N = 3). Finally, we applied the developed 5-hmC detection strategy in various mice tissues. It demonstrates that the content of 5-hmC measured by this method in different mice tissues vary significantly: 5-hmC is highest in brain and lowest in spleen. The success in assaying the mice tissues samples signifies its great promise in a broad range of practical applications for the quantitative detection of 5-hmC in genomic DNA.

Sensitive electrogenerated chemiluminescence biosensing method for the determination of DNA hydroxymethylation based on Ru(bpy)32+-doped silica nanoparticles labeling and MoS2-poly(acrylic acid) nanosheets modified electrode

5-Hydroxymethylcytosine (5-hmC), an oxidation product of 5-mC (5-methylcytosine), is presented in DNA of most mammalian cells and play an important role in the alteration of cancer-related genes. Herein, a sensitive electrogenerated chemiluminescence (ECL) biosensing method for the determination of 5-hmC in DNA (5-hmC DNA) was established on the basis of chemical modification and nanomaterial amplification. First, electrochemically reduced molybdenum disulfide-poly(acrylic acid) (rMoS2-PAA) nanosheets were used to modify glassy carbon electrode (GCE) to form an ECL biosensing electrode (rMoS2-PAA/GCE) which has large accessible surface area to immobilize more DNA. Then, a capture probe with amino group was hybridized with the target 5-hmC DNA and immobilized on the surface of rMoS2-PAA/GCE via amido bond. When cysteamine was introduced, the M.HhaI methyltransferase (M.HhaI) was used as specific recognition element to replace the hydroxyl group of 5-hmC by thiol and generated the amine-derivated DNA. Finally, surface chemically activated Ru(bpy)32+-doped silica (Ru@SiO2) nanoparticles, carriers of ECL reagents, were employed as signal amplification unit which covalently bonded to the amine-derivated DNA resulting in an increased ECL intensity. The increased ECL intensity was linearity to the 5-hmC DNA concentration in a range from 5.0 × 10−14 M to 1.0 × 10−11 M, with a lower detection limit of 1.2 × 10−14 M. Besides, the proposed method also displayed a good selectivity to 5-hmC in the presence of 5-C and 5-mC. Moreover, the developed biosensing method was successfully employed to monitor human urine sample.

Electrogenerated chemiluminescence biosensing method for highly sensitive detection of DNA hydroxymethylation: Combining glycosylation with Ru(phen)32+-assembled graphene oxide

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 0.01pM to 1.00nM, with a low limit of detection of 3.84fM. 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 sensitive signal-off electrogenerated chemiluminescence biosensing method for the discrimination of DNA hydroxymethylation based on glycosylation modification and signal quenching from ferroceneboronic acid

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 5.0×10–11M with a low detection limit of 1.63×10–11M. 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.

Electrogenerated chemiluminescence biosensing method for the detection of DNA demethylase activity: Combining MoS2 nanocomposite with DNA supersandwich

We report an ultrasensitive electrogenerated chemiluminescence (ECL) biosening method for the detection of DNA demethylase activity based on the site-specific cleavage of the HpaII endonuclease in conjunction with signal amplification by a Thionin-MoS2 (Th-MoS2) nanocomposite and supersandwich DNA. The Thionin (Th) served as an effective mediator for the immobilization of the capture probe onto the surface of MoS2, after which the long concatamers containing the methylated site 5′-CmCGG-3′ were hybridized with the capture probe, creating a supersandwich DNA structure. Ru(phen)32+ ECL reagents can be intercalated into the grooves of the formed supersandwich DNA structure with high affinity leading to enhanced signaling. At the biosensing electrode (Ru(phen)32+/supersandwich DNA/Th-MoS2/glassy carbon electrode) after demethylation with demethylase, the demethylated supersandwich DNA could be specifically cleaved using HpaII, resulting in a decreased ECL signal from Ru(phen)32+. An extremely low detection limit of 0.16pgmL−1 was achieved for the detection of DNA demethylase activity. This work demonstrates that the combination of dual signal amplification using a MoS2-thionin nanocomposite and supersandwich with an inherently sensitive ECL method is a promising tactic for the detection of DNA demethylation and assay of the activity of demethylase.

Electrogenerated chemiluminescence biosensing method for the discrimination of DNA hydroxymethylation and assay of the β-glucosyltransferase activity

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 0.04U/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.

Electrogenerated chemiluminescence biosensing method for methyltransferase activity using tris(1, 10-phenanthroline) ruthenium-assembled graphene oxide

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 40U/mL with a detection limit of 0.02U/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.

Label-free and amplified electrogenerated chemiluminescence biosensing method for the determination of DNA methyltransferase activity using signal reagent-assembled graphene oxide

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.

Label-free electrogenerated chemiluminescence biosensing method for trace bleomycin detection based on a Ru(phen)32+–hairpin DNA composite film electrode

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 0.1pM to 50pM. The detection limit was 0.03pM. 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.

Electrogenerated chemiluminescence biosensing method for the detection of DNA methylation and assay of the methyltransferase activity

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 100U/mL with a detection limit of 0.02U/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.