Enzymatic Signal Amplification Research Articles

Ultrasensitive quantification of tumor mRNAs in extracellular vesicles with an integrated microfluidic digital analysis chip.

Extracellular vesicles (EVs) present a promising liquid biopsy for cancer diagnosis. However, it remains a daunting challenge to quantitatively measure molecular contents of EVs including tumor-associated mRNAs. Herein, we report a configurable microwell-patterned microfluidic digital analysis platform combined with a dual-probe hybridization assay for PCR-free, single-molecule detection of specific mRNAs in EVs. The microwell array in our device is configurable between the flow-through assay mode for enhanced hybridization capture and tagging of mRNAs and the digital detection mode based on femtoliter-scale enzymatic signal amplification for single-molecule counting of surface-bound targets. Furthermore, a dual-probe hybridization assay has been developed to enhance the sensitivity of the digital single-molecule detection of EV mRNAs. Combining the merits of the chip design and the dual-probe digital mRNA hybridization assay, the integrated microfluidic system has been demonstrated to afford quantitative detection of synthetic GAPDH mRNA with a LOD as low as 20 aM. Using this technology, we quantified the level of GAPDH and EWS-FLI1 mRNAs in EVs derived from two cell lines of peripheral primitive neuroectodermal tumor (PNET), CHLA-9 and CHLA-258. Our measurements detected 64.6 and 43.5 copies of GAPDH mRNA and 6.5 and 0.277 copies of EWS-FLI1 fusion transcripts per 105 EVs derived from CHLA-9 and CHLA-258 cells, respectively. To our knowledge, this is the first demonstration of quantitative measurement of EWS-FLI1 mRNA copy numbers in Ewing Sarcoma (EWS)-derived EVs. These results highlight the ultralow frequency of tumor-specific mRNA markers in EVs and the necessity of developing highly sensitive methods for analysis of EV mRNAs. The microfluidic digital mRNA analysis platform presented here would provide a useful tool to facilitate quantitative analysis of tumor-associated EV mRNAs for liquid biopsy-based cancer diagnosis and monitoring.

Ultrasensitive Detection of Cancer Cells Combining Enzymatic Signal Amplification with an Aerolysin Nanopore.

Sensitive detection of cancer cells at extremely low concentrations would greatly facilitate the screening and early diagnosis of cancer. Herein, we present a novel nanopore-based strategy for ultrasensitive detection of Ramos cells (human Burkitt's lymphoma cells), by combining the enzymatic signal amplification with an aerolysin nanopore sensor. In this assay, an aptamer for Ramos cells was prehybridized with a short complementary DNA. The presence of target cells causes the target-aptamer complex to unwind to free the complementary DNA, which would subsequently trigger the enzymatic cycling amplification. This process eventually generated a large number of output DNA, which could quantitatively produce characteristic current events when translocated through aerolysin. The proposed method exhibits excellent sensitivity, and as few as 5 Ramos cells could be detected. With good selectivity, the approach can allow for the determination of cancer cells in human serum, offering a powerful tool for biomedical research and clinical diagnosis.

Ultrasensitive SPR detection of miRNA-93 using antibody-enhanced and enzymatic signal amplification.

MiRNAs are endogenous noncoding RNAmolecules. They play important gene-regulatory roles by binding to the mRNA of target genes thereby leading to either transcript degradation or translational repression. In virtually all diseases, distinct alterations of miRNA expression profiles have been found thus suggesting miRNAs as interesting biomarkers. Here, we present an SPR biosensor that utilizes disposable, injection-molded sensor chip/microfluidic hybrids combined with a lateral imaging optical system for parallel analysis of three one-dimensional spot arrays to detect miRNA-93. To increase the sensitivity of the biosensor we used two different amplification strategies. By adding an RNA-DNA-hybrid antibody for primary signal amplification, a limit of detection of 10pmol/L was achieved. Based on that method we demonstrate the detection of miRNA-93 in total RNA lysate from HEK-293 cells. Utilizing an enzymatic signal amplification with Poly(A) polymerase, the sensitivity could be increased even further leading to a limit of detection of 1fmol/L.

Erratum to: Amperometric biosensor for 5-hydroxymethylcytosine based on enzymatic catalysis and using spherical poly(acrylic acid) brushes

5-Hydroxymethylcytosine in DNA (5hmC-DNA) plays an important biological role in sculpting the epigenetic landscape. Its presence is linked to diseases, especially cancers. The authors describe an amperometric biosensor for the determination of 5hmC. It is based on a chemical modification of the hydroxy group of 5hmC in the DNA sequence. Enzymatic signal amplification is accomplished by using DNA methyltransferase (M.HhaI-DNA-cytosine-5-methyltransferase) to achieve chemical modification. A graphene-perylenetetracarboxylic acid nanocomposite was used to modify a glassy carbon electrode (GCE) that acts as a substrate electrode. A composite consisting of horseradish peroxidase on silica/poly(acrylic acid) brushes is employed as the signal amplification unit. Under the optimized conditions, there is a linear response to the logarithm of the 5hmC-DNA concentration in the range from 0.5 to 30 nM, with a 0.23 nM detection limit (at an S/N ratio of 3) in the potential range from −0.3 V to -0.8 V at 100 mV/s. The bioassay has excellent specificity and can even discriminate the similar base 5mC.

Sequence specific recognition of HIV-1 dsDNA in the large amount of normal dsDNA based upon nicking enzyme signal amplification and triplex DNA

A sensitive fluorescent strategy for sequence specific recognition of HIV dsDNA was established based upon Nicking Enzyme Signal Amplification (NESA) and triplex formation. dsDNA sequence from the site 7960 to site 7991 of the HIV1 dsDNA gene was designed as target dsDNA, which was composed of two complementary strands Oligonucleotide 1 with the sequence of 3′-CTT CCT TAT CTT CTT CTT CCA CCT CTC TCT CT-5′ (Oligo-1) and Oligonucleotide 2 with the sequence of 5′-GAA GGA ATA GAA GAA GAA GGT GGA GAG AGA GA-3′ (Oligo-2). As a proof of concept, Oligonucleotide 5′-6-FAM-GAG GTG GAG CTG CGC GAC TCC TCC TCT CTC TCT CTC CAC CTC-BHQ-1–3′(Oligo-4) acted as molecular beacon(MB) probe, Oligonucleotide 5′-CTT CCT TAT CTT CTT CTT CCA AAA GGA GTC GCG-3′ (Oligo-7) acted as assistant probe. In the presence of target dsDNA, Oligo-4 and Oligo-7 hybridized with target dsDNA through triplex formation and formed Y-shaped structure, NESA occurred with further addition of Nt.BbvCI, accompanied with the release of fluorescent DNA fragment circularly, resulted in the increase of fluorescence intensity. Under the optimum conditions, the fluorescence intensity was linear with the concentration of target dsDNA over the range from 100pM to 200nM, the linear regression equation was I = 1.266 C + 84.3 (C: nmol/L, R2 = 0.991), with a detection limit of 65pM. Moreover, the effect of coexisted other dsDNA was investigated as well, and satisfactory results were obtained.

Ultrasensitive electrochemical biosensor based on the oligonucleotide self-assembled monolayer-mediated immunosensing interface

Highly sensitive and selective quantitation of a variety of proteins over a wide concentration range is highly desirable for increased accuracy of biomarker detection or for multidisease diagnostics. In the present contribution, using human immunoglobulin G (HIgG) as the model target protein, an electrochemical ultrasensitive immunosensing platform was developed based on the oligonucleotide self-assembled monolayer-mediated (OSAM) sensing interface. For this immunosensor, the “signal-on” signaling mechanism and enzymatic signal amplification effect were integrated into one sensing architecture. Moreover, the thiolated flexible single-stranded DNAs immobilized onto gold electrode surface not only performs the wobbling motion to facilitate the electron transfer between the electrode surface and biosensing layer but also fundamentally prohibiting the direct interaction of proteins with gold substrate. Thus, the electrochemical signal could be efficiently enhanced and the unspecific adsorption or cross-reaction might be eliminated. As a result, utilizing the newly-proposed immunosensor, the HIgG can be detected down to 0.5 ng/mL, and the high detection specificity is offered. The successful design of OSAM and the highly desirable detection capability of new immunosensor are expected to provide a perspective for fabricating new robust immunosensing platform and for promising potential of oligonucleotide probe in biological research and biomedical diagnosis.

A novel fluorescent biosensor for Adenosine Triphosphate detection based on the polydopamine nanospheres integrating with enzymatic recycling amplification

Based on the protective performance of polydopamine nanospheres (PDANSs) for DNA against nuclease digestion and the specific recognition characteristic of aptamer, we have developed an enzymatic recycling signal amplification method for highly sensitive and selective detection of adenosine triphosphate (ATP). Fluorescence measurements were carried out to verify the DNA polymerase and exonuclease III (Exo III) assisted target recycling process and fluorescence signal amplification. In the absence of the ATP, initially, the signal DNA-PDANSs complex was in the “off” state due to the efficient fluorescence quenching of 6-carboxyfluorescein (FAM) adjacent to the surface of PDANSs. Due to the binding of the aptamer by ATP, it trigger DNA polymerase and Exo III assisted target recycling process by the product of release, the complex would change into the “on” state as a result of the dissociation of the FAM from the surface of PDANSs, thus providing greatly enhanced fluorescence emission intensity. The method allows quantitative detection of ATP in the range of 20–600nM with a detection limit of 8.32nM. This biosensor requires no complex operations, and is a new high efficiency method for ATP detection.

Electrochemical DNA sandwich biosensor based on enzyme amplified microRNA-21 detection and gold nanoparticles

In this work, a novel electrochemical biosensor for miRNA-21 determination, involving a sandwich hybridization assay onto gold nanoparticles modified pencil graphite electrode (PGE) and enzyme signal amplification was reported.The thiol terminated capture probe 1 (SH-P1) was immobilized on the electrode through AuS interaction. In the presence of target miRNA-21, SH-P1 hybridized with the first part of the target, however, the second part hybridizes with a biotinylated probe P2 (B-P2). Then, a streptavidin-conjugated alkaline phosphatase was immobilized by a specific binding of avidin-B-P2. The enzyme catalyzed the electro-inactive α-naphtyl phosphate to an electro-active α-naphtol. The miRNA-21 detection was achieved through the changes of α-naphtol oxidation signals observed at +0.12V vs Ag/AgCl with Differential Pulse Voltammetry.Under the optimal detection conditions, the biosensor exhibited selective and sensitive detection with a linear range from 200pM to 388nM and the detection limit was 100pM (10fmol in 100μL).

Electrochemical biosensor for hydroxymethylated DNA detection and β-glucosyltransferase activity assay based on enzymatic catalysis triggering signal amplification

A simple and sensitive electrochemical biosensor was fabricated for hydroxymethylated DNA detection and T4 β-glucosyltransferase activity assay based on the glycosylation of hydroxymethylated DNA and HpaII restriction endonuclease system triggering signal amplification. After immobilizing double-stranded DNA (dsDNA) on the surface of carboxyl functionalized Fe3O4 nanoparticle, the glycosylation reaction of hydroxymethylated cytosine can be performed with the catalytic effect of T4 β-glucosyltransferase using uridine diphosphoglucose as glucose donor. Then, 4-mercaptophenylboronic acid (4-MPBA) can be further reacted with glucose and captured on Fe3O4 nanoparticle surface. After the dsDNA was digested by HpaII, the fragment with 4-MPBA can be released from Fe3O4 nanoparticle surface. After magnetic separation, this fragment can be further modified on gold electrode surface through the interaction between Au and −SH, which can result in a decreased electrochemical signal of [Fe(CN)6]3−. According to this detection strategy, the electrochemical biosensor showed high sensitivity with the low detection limits of 0.14nM and 0.27 unit/mL for hydroxymethylated DNA and T4 β-glucosyltransferase. The fabricated biosensor also presented high selectivity and acceptable reproducibility. In addition, the inhibition activity of 4-phenylimidazole towards T4 β-glucosyltransferase was also investigated. This work provides a novel platform for hydroxymethylated DNA detection, T4 β-glucosyltransferase activity assay and its inhibition screening.

Sandwich-type microRNA biosensor based on magnesium oxide nanoflower and graphene oxide–gold nanoparticles hybrids coupling with enzyme signal amplification

An ultrasensitive sandwich-type electrochemical biosensor for microRNA (miRNA) detection is developed based on magnesium oxide (MgO) nanoflower and graphene oxide–gold nanoparticles (GO–AuNPs) hybrids coupling with electrochemical–chemical–chemical (ECC) detection system. In this bioassay system, MgO nanoflowers and AuNPs are modified on electrode to act as sensing platform. The thiolated capture probe is then self-assembled onto AuNPs/MgO substrate via formation of Au-S bonds. Subsequently, a biotinylated DNA signal probe is conjugated to GO–AuNPs hybrids. When miRNA-21 is added, a sandwich complex is formed and a lot of signal indicators streptavidin-conjugated alkaline phosphatases (SA-ALP) are immobilized upon electrode by the specific reaction between avidin and biotin. Finally, ECC reaction is performed in the system to improve detection signal. The proposed sandwich-type assay benefits from advantages of sandwich-type structure for enhanced sensitivity and specificity, MgO nanoflowers/AuNPs as sensing platform and GO–AuNPs hybrids as signal carriers for signal amplification, and ECC as a sensitive detection system for low detection limit. This biosensor exhibits a good dynamic ranging from 0.1 to 100 fM and a low detection limit of 50 aM (S/N=3) toward target miRNA-21.

A label-free and high-efficient GO-based aptasensor for cancer cells based on cyclic enzymatic signal amplification

A label-free and high-efficient graphene oxide (GO)-based aptasensor was developed for the detection of low quantity cancer cells based on cell-triggered cyclic enzymatic signal amplification (CTCESA). In the absence of target cells, hairpin aptamer probes (HAPs) and dye-labeled linker DNAs stably coexisted in solution, and the fluorescence was quenched by the GO-based FÖrster resonance energy transfer (FRET) process. In the presence of target cells, the specific binding of HAPs with the target cells triggered a conformational alternation, which resulted in linker DNA complementary pairing and cleavage by nicking endonuclease-strand scission cycles. Consequently, more cleaved fragments of linker DNAs with more the terminal labeled dyes could show the enhanced fluorescence because these cleaved DNA fragments hardly combine with GOs and prevent the FRET process. Fluorescence analysis demonstrated that this GO-based aptasensor exhibited selective and sensitive response to the presence of target CCRF-CEM cells in the concentration range from 50 to 105 cells. The detection limit of this method was 25 cells, which was approximately 20 times lower than the detection limit of normal fluorescence aptasensors without amplification. With high sensitivity and specificity, it provided a simple and cost-effective approach for early cancer diagnosis.

Au nanoparticles/hollow molybdenum disulfide microcubes based biosensor for microRNA-21 detection coupled with duplex-specific nuclease and enzyme signal amplification

An ultrasensitive electrochemical biosensor for detecting microRNAs is fabricated based on hollow molybdenum disulfide (MoS2) microcubes. Duplex-specific nuclease, enzyme and electrochemical–chemical–chemical redox cycling are used for signal amplification. Hollow MoS2 microcubes constructed by ultrathin nanosheets are synthesized by a facile template-assisted strategy and used as supporting substrate. For biosensor assembling, biotinylated ssDNA capture probes are first immobilized on Au nanoparticles (AuNPs)/MoS2 modified electrode in order to combine with streptavidin-conjugated alkaline phosphatase (SA-ALP). When capture probes hybridize with miRNAs, duplex-specific nuclease cleaves the formative duplexes. At the moment, the biotin group strips from the electrode surface and SA-ALP is incapacitated to attach onto electrode. Then, ascorbic acids induce the electrochemical–chemical–chemical redox cycling to produce electrochemical response in the presence of ferrocene methanol and tris (2-carboxyethyl) phosphine. Under optimum conditions, the proposed biosensor shows a good linear relationship between the current variation and logarithm of the microRNAs concentration ranging from 0.1fM to 0.1pM with a detection limit of 0.086fM (S/N=3). Furthermore, the biosensor is successfully applied to detect target miRNA-21 in human serum samples.

Ultrasensitive electrochemical sensing platform for microRNA based on tungsten oxide-graphene composites coupling with catalyzed hairpin assembly target recycling and enzyme signal amplification

An ultrasensitive electrochemical biosensor for microRNA (miRNA) is developed based on tungsten oxide-graphene composites coupling with catalyzed hairpin assembly target recycling and enzyme signal amplification. WO3-Gr is prepared by a simple hydrothermal method and then coupled with gold nanoparticles to act as a sensing platform. The thiol-terminated capture probe H1 is immobilized on electrode through Au–S interaction. In the presence of target miRNA, H1 opens its hairpin structure by hybridization with target miRNA. This hybridization can be displaced from the structure by another stable biotinylated hairpin DNA (H2), and target miRNA is released back to the sample solution for next cycle. Thus, a large amount of H1-H2 duplex is produced after the cyclic process. At this point, a lot of signal indicators streptavidin-conjugated alkaline phosphatase (SA-ALP) are immobilized on the electrode by the specific binding of avidin-biotin. Then, thousands of ascorbic acid, which is the enzymatic product of ALP, induces the electrochemical-chemical-chemical redox cycling to produce a strongly electrochemical response in the presence of ferrocene methanol and tris (2-carboxyethyl) phosphine. Under the optimal experimental conditions, the established biosensor can detect target miRNA down to 0.05fM (S/N=3) with a linear range from 0.1fM to 100pM, and discriminate target miRNA from mismatched miRNA with a high selectivity.

Highly sensitive electrogenerated chemiluminescence biosensor for galactosyltransferase activity and inhibition detection using gold nanorod and enzymatic dual signal amplification

As one of the glycosyltransferase involved in protein glycosylation, β-1,4-galactosyltransferase (Gal T) plays an important role in the cellular process and progression of cancer. Here, using the bovine serum albumin conjugated N-acetylglucosamine (GlcNAc-BSA) as a receptor to fabricate bioelectrode interface, a sensitive electrochemiluminescence (ECL) biosensor was constructed for Gal T activity analysis based on the recognition between artocarpus integrifolia lectin (AIA) and galactose, integrating with a dual signal amplification strategy from the xanthine oxidase (XOD) and AIA multi-labeled gold nanorod nanoprobes. The gold nanorods promoted the electron transfer on the electrode interface and were also employed as carriers of AIA and XOD due to their large surface area. Furthermore, both the gold nanorods and XOD catalyzed the ECL reaction, which dramatically amplified the ECL signal of luminol in the presence of hypoxanthine (HA) and oxygen. The as-proposed ECL biosensor exhibited high sensitivity on the detection of Gal T activity and a detection limit of 9×10−4UmL−1 was obtained. This assay was successfully applied for the analysis of Gal T activity expression in different cell lines and inhibition detection, showing great potential for glycosyltransferase activity analysis and inhibitors screening in clinic diagnostics.

High-Sensitivity and High-Efficiency Detection of DNA Hydroxymethylation in Genomic DNA by Multiplexing Electrochemical Biosensing

DNA hydroxymethylation (5-hmC) is a kind of new epigenetic modification, which plays key roles in DNA demethylation, genomic reprogramming, and the gene expression in mammals. For further exploring the functions of 5-hmC, it is necessary to develop sensitive and selective methods for detecting 5-hmC. Herein, we developed a novel multiplexing electrochemical (MEC) biosensor for 5-hmC detection based on the glycosylation modification of 5-hmC and enzymatic signal amplification. The 5-hmC was first glycosylated by T4 β-glucosyltransferase and then oxidated by sodium periodate. The resulting glucosyl-modified 5-hmC (5-ghmC) was incubated with ARP-biotin and was bound to avidin-HRP. The 5-hmC can be detected at the subnanogram level. Finally, we performed 5-hmC detection for mouse tissue samples and cancer cell lines. The limit of detection of the MEC biosensor is 20 times lower than that of commercial kits based on optical meaurement. Also, the biosensor presented high detection specificity because the chemical reaction for 5-hmC modification can not happen at any other unhydroxymethylated nucleic acid bases. Importantly, benefited by its multiplexing capacity, the developed MEC biosensor showed excellent high efficiency, which was time-saving and cost less.

A signal amplification strategy via enzymatic cascade reactions for ultrasensitive DNA detection

Event Abstract Back to Event A signal amplification strategy via enzymatic cascade reactions for ultrasensitive DNA detection Eunjung Kim1*, Philip D. Howes1 and Molly M. Stevens1, 2 1 Imperial College London, Materials, United Kingdom 2 Imperial College London, Bioengineering and Institute of Biomedical Engineering, United Kingdom Introduction: The field of molecular diagnostics is in dire need of fast and easy-to-use diagnostic tools that detect and measure the presence of genetic materials or proteins associated with a specific health condition or disease. The development of such techniques would provide a greater depth of information that can be derived from genetic profiles and molecular signatures of disease compared to traditional diagnostic approaches. While some diagnostic tests such as pregnancy tests and diabetes test strips have been successfully implemented into the health care system, the ability to detect DNA, RNA, and other biomolecules has been hampered by lack of sensitivity and selectivity[1]. Herein, we developed a simple mix-and-read fluorescent assay for the detection of pathogen-derived DNA from Neisseria meningitides, which leads to meningococcal diseases, by using highly sensitive and selective fluorescence resonance energy transfer (FRET) signals from a quantum dot (QD)-based detection system. This assay is based on an enzymatic cascade signal amplification composed of flap endonuclease-1 (FEN-1)-assisted massive flap generation in the presence of a target DNA and exonuclease III (Exo III)-mediated flap recycling and exponential signal amplification. The synergistic generation and recycling of flap DNA by combining two enzymatic reactions are interpreted to be a contributing factor for the high detection sensitivity. Therefore, our approach enabled a simple and robust diagnostic tool that operates in complex media, detecting an extremely low abundance of targets. Materials and Methods: The first amplification assay was tested by mixing synthetic probe DNA with a target, followed by incubation with FEN-1 at 40oC for 1 hr, and polyacrylamide gel analysis was carried out to determine the target-dependent flap accumulation in a solution. Next, the Exo III-catalysed sensing was tested using the amplified flaps and a dye-functionalised QD. The kinetics of the flap binding to the capture probe on the surface of QD were also determined by controlling enzyme concentrations and the surface density of DNA. Results and Discussion: We used synthetic targets with different lengths of 50 and 200 nt respectively by selecting a target region of Neisseria meningitides genomic DNA in our assay[2]. In the first cleavage reaction, we showed the positive response of flaps to the concentration of both targets, meaning that our assay has the ability to detect DNA targets of any length including extremely long ones (Fig 1). We then examined whether an increase in the FRET signals could be initiated by the introduction of the flap DNA and a distinct dose-response profile between the concentration of flap and the QD fluorescence was observed. Conclusions: In summary, we designed a novel DNA detection assay by coupling FEN-1-based signal amplification with Exo III-based QD signal enhancement. Our assay does not require the target of interest to have an endonuclease recognition sequence or specific lengths, therefore, there is not any significant restriction in the choice of targets. Furthermore, due to the isothermal reaction and simple process consisting of stepwise addition of reagents into each well in the microplate, our detection method would open up the potential application into in situ simple and rapid diagnosis of infectious diseases. This project was funded by Imperial Confidence in Concept Scheme, Imperial NIHR BRC/Imperial Innovations Funds, and NIHR BRC at The Royal Marsden and The Institute of Cancer Research Funds 2014.

Stellaris® RNA Fluorescence In Situ Hybridization for the Simultaneous Detection of Immature and Mature Long Noncoding RNAs in Adherent Cells.

RNA fluorescence in situ hybridization (FISH), long an indispensable tool for the detection and localization of RNA, is becoming an increasingly important complement to other gene expression analysis methods. Especially important for long noncoding RNAs (lncRNAs), RNA FISH adds the ability to distinguish between primary and mature lncRNA transcripts and thus to segregate the site of synthesis from the site of action.We detail a streamlined RNA FISH protocol for the simultaneous imaging of multiple primary and mature mRNA and lncRNA gene products and RNA variants in fixed mammalian cells. The technique makes use of fluorescently pre-labeled, short DNA oligonucleotides (circa 20 nucleotides in length), pooled into sets of up to 48 individual probes. The overall binding of multiple oligonucleotides to the same RNA target results in fluorescent signals that reveal clusters of RNAs or single RNA molecules as punctate spots without the need for enzymatic signal amplification. Visualization of these punctate signals, through the use of wide-field fluorescence microscopy, enables the counting of single transcripts down to one copy per cell. Additionally, by using probe sets with spectrally distinct fluorophores, multiplex analysis of gene-specific RNAs, or RNA variants, can be achieved. The presented examples illustrate how this method can add temporospatial information between the transcription event and both the location and the endurance of the mature lncRNA. We also briefly discuss post-processing of images and spot counting to demonstrate the capabilities of this method for the statistical analysis of RNA molecules per cell. This information can be utilized to determine both overall gene expression levels and cell-to-cell gene expression variation.

Short review:Electrochemical MicroRNAs Biosensors Based on Enzymatic Signal Amplification

Short review:Electrochemical MicroRNAs Biosensors Based on Enzymatic Signal Amplification

Methyltransferase activity assay based on the use of exonuclease III, the hemin/G-quadruplex system and reduced graphene oxide on a gold electrode, and a study on enzyme inhibition

We describe an electrochemical bioassay for the detection of the activity of methyltransferase (MTase), and for screening this enzyme’s inhibitors. The assay is based on the conjugation of a hemin to a G-quadruplex that enables enzymatic signal amplification with the aid of exonuclease III (ExoIII). In the first step, double-stranded DNA containing the quadruplex-forming oligomer is assembled on the surface of a gold electrode and then methylated by DNA adenine methyltransferase (DAM). After cleaved by endonuclease DpnI, the methylated DNA is digested by ExoIII and the quadruplex-forming oligomers are liberated. This leads to the formation of a hemin/G-quadruplex (in presence of hemin and of potassium ions). The hemin/G-quadruplex catalyzes the oxidization of hydroquinone by H2O2 and the benzoquinone was formed to generate electrochemical signal. Finally, the gold electrode modified with reduced graphene oxide was used as working electrode for performing differential pulse voltammetry. The method has a detection limit of 0.31 unit · mL−1. A study on the inhibition of MTase showed it was inhibited by epicatechin with an IC50 value of 157 μM.

EzyAmp signal amplification cascade enables isothermal detection of nucleic acid and protein targets.

Advancements in molecular biology have improved the ability to characterize disease-related nucleic acids and proteins. Recently, there has been an increasing desire for tests that can be performed outside of centralised laboratories. This study describes a novel isothermal signal amplification cascade called EzyAmp (enzymatic signal amplification) that is being developed for detection of targets at the point of care. EzyAmp exploits the ability of some restriction endonucleases to cleave substrates containing nicks within their recognition sites. EzyAmp uses two oligonucleotide duplexes (partial complexes 1 and 2) which are initially cleavage-resistant as they lack a complete recognition site. The recognition site of partial complex 1 can be completed by hybridization of a triggering oligonucleotide (Driver Fragment 1) that is generated by a target-specific initiation event. Binding of Driver Fragment 1 generates a completed complex 1, which upon cleavage, releases Driver Fragment 2. In turn, binding of Driver Fragment 2 to partial complex 2 creates completed complex 2 which when cleaved releases additional Driver Fragment 1. Each cleavage event separates fluorophore quencher pairs resulting in an increase in fluorescence. At this stage a cascade of signal production becomes independent of further target-specific initiation events. This study demonstrated that the EzyAmp cascade can facilitate detection and quantification of nucleic acid targets with sensitivity down to aM concentration. Further, the same cascade detected VEGF protein with a sensitivity of 20nM showing that this universal method for amplifying signal may be linked to the detection of different types of analytes in an isothermal format.