•Dam MTase-induced methylation of dsDNA probes inactivates endonuclease activity.•The protected dsDNAs inhibit the formation of supersandwich DNAzyme structures.•Label-free and amplified ECL detection of Dam MTase activity is developed.•The method shows high sensitivity and the capability to screen anti-cancer drugs.Sensitive detection of DNA methyltransferase (MTase) activity plays important roles for the diagnosis of many types of genetic disorder diseases and human malignancies. In this work, we describe the development of a label-free, sensitive and signal-on electrochemiluminescence (ECL) method for detecting MTase activity based on a restriction enzyme inactivation strategy. The sequence-specific double stranded DNA (dsDNA) probes on the sensing electrode can be cleaved by the restriction enzyme to initiate hybridization chain reaction (HCR) formation of the supersandwich DNAzyme structures, which catalyze the reduction and depletion of the dissolved oxygen to significantly quench the ECL of the oxygen/persulfate (O2/S2O82−) system. The methylation of the dsDNA probes by DNA adenine methyltransferase (Dam MTase) can inactivate the restriction enzyme activity and inhibit subsequent HCR formation of the DNAzymes, leading to recovery of the ECL signal for signal-on detection of Dam MTase in a dynamic range from 0.01 U mL−1 to 50 U mL−1 with the detection limit of 6.4 × 10−3 U mL−1. The inhibition of Dam MTase activity by different drugs is also evaluated, offering this method new opportunity for highly sensitive detection of Dam MTase activity and for screening potential drugs for cancer therapy as well.Download high-res image (96KB)Download full-size image

Because of their irreversible toxicological impacts on the environment and human body, the development of reliable and sensitive Hg2+ detection methods with high selectivity is of great significance. On the basis of the substantial signal amplification by metallo-toehold-triggered, catalytic hairpin assembly (CHA) formation of three-way DNAzyme junctions, we have constructed a highly selective and sensitive fluorescent sensing system for the determination of Hg2+ in different environmental water samples. The presence of the target Hg2+ ions can lead to the generation of T-Hg2+-T base mismatched metallo-toeholds, which trigger the catalytic assembly of three split-DNAzyme containing hairpins to form many Mg2+-dependent DNAzyme junction structures upon binding to the fluorescently quenched substrate sequences. The Mg2+ ions then cyclically cleave the fluorescently quenched substrate sequences of the Mg2+-dependent DNAzymes to generate drastically enhanced fluorescent signals for sensitively detecting Hg2+ at the low 4.5 pM level. The developed sensing method offers high selectivity toward the target Hg2+ over other possible competing metal ions due to the specific T-Hg2+-T bridge structure chemistry in the metallo-toehold domain, and reliable detection of spiked Hg2+ in environmentally relevant water samples with this method is also verified. Considering the nucleic acid nature of the trigger and assembly sequences, the developed approach thus holds great potentials for designing new enzyme-free signal amplification strategies to achieve highly sensitive determination of different DNA and RNA targets.Keywords: catalytic hairpin assembly; DNAzyme; fluorescence; mercury(II); strand displacement;

The detection of protein/small molecule interactions plays important roles in drug discovery and protein/metabolite interactions in biology. In this work, by coupling the terminal protection of small molecule-linked ssDNA strategy with the unmodified and positively charged gold nanoparticle ((+)AuNP) nanoprobes, we have developed a sensitive and simple colorimetric sensor for the detection of folate receptor, a highly expressed protein in many kinds of malignant tumors. The target folate receptor binds the folate moieties of the folate-linked ssDNA through high affinity interactions and protects the protein-bound ssDNA from digestion by exonuclease I. The protected ssDNA thus adsorbs the ((+)AuNP) through electrostatic interactions, leading to a red-to-blue color change of the sensing solution for sensitive colorimetric detection of folate receptor at the sub-nanomolar level. Besides, this colorimetric sensor shows high selectivity toward folate receptor against other control proteins. The developed sensor avoids the modification/conjugation of the AuNP nanoprobes and the involvement of any expensive instruments for signal transduction in protein detection. Featured with these obvious advantages, the colorimetric sensor strategy demonstrated herein can be easily expanded for sensitive and convenient detection of various protein/small molecule interactions.

The development of electronic sensors with minimized usage of reagents and washing steps in the sensing protocols will significantly facilitate the detection of biomolecules. In this work, by using a new pseudoknot design of the aptamer probes, the construction of an electronic sensor for reagentless and single-step detection of immunoglobulin E (IgE) in human serum is described. The pseudoknot aptamer probes are self-assembled on the disposable electrode surface. The association of IgE with the aptamer probes leads to conformational changes of the pseudoknot aptamer structures and brings the redox-tags in close proximity to the electrode, resulting in amplified current response for monitoring IgE. The effects of the pseudoknot structure and the immobilization concentration of the aptamer probes on the sensor performance are evaluated. Under optimal conditions, the detection limit for IgE is estimated to be 60 pM. The sensor is also selective and can be employed to detect IgE in human serum samples. The developed sensor can achieve reagentless, washing-free and low-cost (with the disposable electrode) electrochemical detection of proteins, making this device a convenient sensing platform for the monitoring of different biomarkers when coupled with the appropriate aptamer probes.

•Multiplexed electrochemical detection of DNA/protein bindings is achieved.•The DNA/protein complexes protect the DNA probes from digesting by exonuclease.•The electrochemical labels exhibit distinct signals for multiplexed detection.•The proposed sensor is sensitive and selective to the target proteins.A simple, sensitive and multiplexed electrochemical sensor for the detection of DNA–protein binding based on the exonuclease protection strategy is described. Two electroactive species, methylene blue (MB)- and ferrocene (Fc)-labeled dsDNA probes are self-assembled on a gold electrode to prepare the sensor surface. The target proteins, vascular endothelial growth factor (VEGF) and estrogen receptor (ERα), bind to the dsDNA probes and protect the probes from digesting by exonuclease III due to the steric hindrance of the bound proteins. These protein-protected, MB/Fc-labeled sequences remaining on the sensor surface display two distinct voltammetric peaks, whose peak potentials (MB: −0.27 V; Fc: +0.27 V) and intensities reflect the identities and amounts of the corresponding target proteins, for simultaneous and multiplexed detection of DNA–protein bindings. The proposed sensor is also selective to the target proteins against other interference molecules. By using labels with distinct voltammetric peaks, the developed method can be easily expanded for simultaneous detection of multiple DNA–protein bindings.

•A label-free approach for fluorescent detection of folate receptor is established.•The detection mechanism is based on terminal protection of the folate-linked ssDNA.•SYBR Gold fluorescent dye is used as the signal indicator.•Selective and sensitive detection of 30 pM folate receptor is achieved.In this work, based on terminal protection of folate-linked ssDNA (FA-ssDNA) and the SYBR Gold fluorescent dye, we describe the development of a label-free fluorescent strategy for the detection of folate receptors (FRs). The binding between the target FR and the FA moiety of the FA-ssDNA protects the FR bound FA-ssDNA from digesting by Exo I. The binding of SYBR Gold to the terminal protected, un-digested FA-ssDNA leads to enhanced fluorescent emission for the monitoring of FR with a detection limit of 30 pM. Besides, the developed method also shows high selectivity toward FR against other control proteins. Moreover, our approach avoids the labeling of the probes with fluorescent tags and achieves label-free detection of FR. With these advantages, the proposed method thus holds promising potential for the development of simple and convenient strategies for the detection of other proteins by using different small molecule receptor/protein ligand pairs.

•A highly sensitive aptasensor for the detection of Ochratoxin A is developed.•Ochratoxin A leads to the inhibition of the electron transfer of the CdTe QDs.•The inhibition is significantly enhanced by Ochratoxin A recycling amplification.•The method is successfully employed to detect Ochratoxin A in red wine samples.Based on the recovery of the quantum dot (QD) electrochemiluminescence (ECL) and exonuclease-catalyzed target recycling amplification, the development of a highly sensitive aptasensor for Ochratoxin A (OTA) detection is described. The duplex DNA probes containing the biotin-modified aptamer are immobilized on a CdTe QD composite film-coated electrode. The presence of the OTA target leads to effective removal of the biotin–aptamers from the electrode surface via exonuclease-catalyzed recycling and reuse of OTA, which prevents the attachment of streptavidin–alkaline phosphatase (STV–ALP) through biotin–STV interaction. The electron transfer (ET) from the excited state CdTe QD ([CdTe]⁎) to the electro-oxidized species of the enzymatic product of ALP during the potential scan is thus inhibited and the QD ECL emission is restored for quantitative OTA detection. Due to the exonuclease-catalyzed target recycling amplification, the inhibition effect of ET is significantly enhanced to achieve sensitive detection of OTA down to 0.64 pg mL−1. The proposed method is selective for OTA and can be used to monitor OTA in real red wine samples. Our developed ECL recovery-based aptasensor thus offers great potential for the development of new ECL sensing platforms for various target analytes.

In this work, we describe the development of a sensitive and label-free aptasensor for thrombin detection based on background noise reduction by exonuclease I (Exo I) and signal amplification by direct electron transfer (DET) of hemin. The thrombin binding aptamers (TBAs) are self-assembled on a sensing electrode. In the absence of the target thrombin, the TBAs are digested by Exo I, which avoids the association of hemin and significantly minimizes the background current noise. The presence of thrombin stabilizes the folded TBA G-quadruplex and prevents it from degrading by Exo I. The G-quadruplex bound hemin thus generates amplified signal output. In our sensing approach, the introduction of Exo I significantly enhances the signal to noise ratio of the sensor response and achieves sensitive detection of thrombin. Our new method is also coupled with good selectivity against other non-target proteins and thus holds great potential for the development of robust aptasensors for the detection of different types of targets.Highlights► The use of exonuclease I leads to significant background current reduction. ► Direct electron transfer of hemin is used for quantitative thrombin detection. ► The signal to noise ratio of the sensor is enhanced. ► Sub nanomolar thrombin can be detected selectively in a label-free fashion.

The hybridizations between the HIV target DNA and the capture probes as well as the signal probes conjugated to the multi-invertase/nanoparticle composites lead to the conversion of sucrose to glucose, which is monitored by the personal glucometer and provides quantitative digital readings for point-of-care diagnosis of HIV DNA fragments.

•A sensitive fluorescent method for detecting Dam MTase activity is developed.•Signal amplification is achieved by the integration of HCR with DNAzyme recycling.•The method is convenient and shows high sensitivity.•The application of this method for anti-cancer drug screening is also demonstrated.Aberrant DNA methylation originated from changes in DNA methyltransferase activity can lead to many genetic diseases and tumor types, and the monitoring of methyltransferase activity is thus of great importance in disease diagnosis and drug screening. In this work, by combing hybridization chain reaction (HCR) and metal ion-dependent DNAzyme recycling, we have developed a convenient enzyme-free signal amplification strategy for highly sensitive detection of DNA adenine methyltransferase (Dam MTase) activity and its inhibitors. The Dam MTase-induced methylation and subsequent cleavage of the methylated hairpin DNA probes by DpnI endonuclease lead to the release of ssDNA triggers for HCR formation of many Mg2+-dependent DNAzymes, in which the fluorescently quenched substrate sequences are catalytically and cyclically cleaved by Mg2+ to generate remarkably amplified fluorescent signals for highly sensitive detection of Dam MTase at 7.23 × 10−4 U/mL. In addition, the inhibition of different drugs to Dam MTase activity can also be evaluated with the developed method. With the advantages of simplicity and significant signal amplification over other common methods, the demonstrated biosensing approach thus offers great potential for highly sensitive detection of various methyltransferases and provides a convenient platform for drug screening for therapeutic applications.Figure optionsDownload full-size imageDownload as PowerPoint slide

The hybridizations between the HIV target DNA and the capture probes as well as the signal probes conjugated to the multi-invertase/nanoparticle composites lead to the conversion of sucrose to glucose, which is monitored by the personal glucometer and provides quantitative digital readings for point-of-care diagnosis of HIV DNA fragments.