Qingwang Xue

Highly sensitive fluorescence assay of DNA methyltransferase activity by methylation-sensitive cleavage-based primer generation exponential isothermal amplification-induced G-quadruplex formation

Site-specific identification of DNA methylation and assay of MTase activity are imperative for determining specific cancer types, provide insights into the mechanism of gene repression, and develop novel drugs to treat methylation-related diseases. Herein, we developed a highly sensitive fluorescence assay of DNA methyltransferase by methylation-sensitive cleavage-based primer generation exponential isothermal amplification (PG-EXPA) coupled with supramolecular fluorescent Zinc(II)-protoporphyrin IX (ZnPPIX)/G-quadruplex. In the presence of DNA adenine methylation (Dam) MTase, the methylation-responsive sequence of hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease Dpn I. The cleaved hairpin probe then functions as a signal primer to initiate the exponential isothermal amplification reaction (EXPAR) by hybridizing with a unimolecular DNA containing three functional domains as the amplification template, producing a large number of G-quadruplex nanostructures by utilizing polymerases and nicking enzymes as mechanical activators. The G-quadruplex nanostructures act as host for ZnPPIX that lead to supramolecular complexes ZnPPIX/G-quadruplex, which provides optical labels for amplified fluorescence detection of Dam MTase. While in the absence of Dam MTase, neither methylation/cleavage nor PG-EXPA reaction can be initiated and no fluorescence signal is observed. The proposed method exhibits a wide dynamic range from 0.0002 to 20U/mL and an extremely low detection limit of 8.6×10(-5)U/mL, which is superior to most conventional approaches for the MTase assay. Owing to the specific site recognition of MTase toward its substrate, the proposed sensing system was able to readily discriminate Dam MTase from other MTase such as M.SssI and even detect the target in a complex biological matrix. Furthermore, the application of the proposed sensing strategy for screening Dam MTase inhibitors was also demonstrated with satisfactory results. This novel method not only provides a promising platform for monitoring activity and inhibition of DNA MTases, but also shows great potentials in biological process researches, drugs discovery and clinical diagnostics.

Sensitive detection of proteins using assembled cascade fluorescent DNA nanotags based on rolling circle amplification.

A novel cascade fluorescence signal amplification strategy based on the rolling circle amplification (RCA)-aided assembly of fluorescent DNA nanotags as fluorescent labels and multiplex binding of the biotin-streptavidin system was proposed for detection of protein target at ultralow concentration. In the strategy, fluorescent DNA nanotags are prepared relying on intercalating dye arrays assembled on nanostructured DNA templates by intercalation between base pairs. The RCA product containing tandem-repeat sequences could serve as an excellent template for periodic assembly of fluorescent DNA nanotags, which were presented per protein recognition event to numerous fluorescent DNA nanotags for assay readout. Both the RCA and the multiplex binding system showed remarkable amplification efficiency, very little nonspecific adsorption, and low background signal. Using human IgG as a model protein, the designed strategy was successfully demonstrated for the ultrasensitive detection of protein target. The results revealed that the strategy exhibited a dynamic response to human IgG over a three-decade concentration range from 1.0 pM to 1.0 fM with a limit of detection as low as 0.9 fM. By comparison with the assay of multiple labeling antibodies with the dye/DNA conjugate, the limit of detection was improved by 4 orders. The designed signal amplification strategy would hold great promise as a powerful tool to be applied for the ultrasensitive detection of target protein in immunoassay.

Quantitative Detection of Single Molecules Using Enhancement of Dye/DNA Conjugate-Labeled Nanoparticles

An ultrasensitive fluorescence immunoassay method for quantitative detection of single molecules is developed on the basis of counting single magnetic nanobeads (MNBs) with combined amplification of DNA and dye/DNA conjugate. Highly amplified fluorescence signal and low background signal are achieved by using mutilabel bioconjugates made by linking multiple dye/DNA conjugates to streptavidin-coated magnetic nanobeads (SA-MNBs) and magnetic separation. In this method, human IgG (Ag) is captured on the silanized glass substrate surface, followed by immunoreaction with biotinylated mouse antihuman antibody (BT-Ab). Then, SA-MNBs are attached to the BT-Ab through the biotin/streptavidin interaction at a ratio of 1:1. Subsequently, a 30 base pair double-stranded oligonucleotide terminated with biotin (BT-dsDNA) is conjugated to the SA-MNBs. The resultant Ag-BT-Ab-SA-MNBs/BT-dsDNA/SYBR Green I is achieved after a fluorescent DNA probe, SYBR Green I, is added to the substrate and bound to the oligonucleotide at high ratios. Finally, epifluorescence microscopy coupled with a high-sensitivity electron multiplying charge-coupled device is employed for human IgG fluorescence imaging and detection. The number of fluorescent spots corresponding to single protein molecules on the images is counted. It is found that the number of fluorescent spots resulting from the SA-MNBs/BT-dsDNA/SYBR Green I immuotargeted on the glass slides is correlated with the concentration of human IgG target antigen in the range 3.0-50 fM.

Te-template approach to fabricating ternary TeCuPt alloy nanowires with enhanced catalytic performance towards oxygen reduction reaction and methanol oxidation reaction

Fabricating ternary Pt-based alloys has emerged as a promising strategy to further enhance the catalytic performance of Pt catalysts in direct methanol fuel cells (DMFCs) for both the oxygen reduction reaction (ORR) and the methanol oxidation reaction (MOR). Herein, we reported for the first time the synthesis of ternary TeCuPt nanowires (NWs) by a Te-template-directed galvanic replacement reaction, in which Te NWs serve as both sacrificial templates and reducing agents. Compared with a binary TePt alloy and pure Pt catalysts, the ternary TeCuPt alloys exhibit a more positive half-wave potential and a higher specific area/mass activity for ORR, and also display a better CO tolerance ability and long-term stability for MOR. The enhanced catalytic performance for TeCuPt NWs is attributed to the electronic and geometric structure effects, originating from the Pt alloying with both Te and Cu components, which could weaken the binding strength between the Pt surface atoms and the intermediate species (e.g. OH*, CO*). Our studies have demonstrated a new alternative ternary Pt-based catalyst for both ORR and MOR, which could find application in DMFC.

Label-free, sensitivity detection of fibrillar fibrin using gold nanoparticle-based chemiluminescence system.

A novel, label-free, gold nanoparticles (AuNPs)-based chemiluminescence assay has been developed for the detection of fibrillar fibrin. The method relied on the interaction of fibrinogen (Fib) with AuNPs and the aggregated AuNPs induce a strong luminol-H2O2 chemiluminesecence (CL) signal. We prepared the 12-nm-diameter AuNPs which well dispersed in the solution. Fib was absorbed on the surface of AuNPs against the aggregation of AuNPs in 1.0M NaCl. Otherwise, Fib was catalyzed to form fibrillar fibrin in the presence of thrombin. The fibrin induced AuNPs aggregated in the presence of NaCl solution. The catalytic activity of aggregated AuNPs on the luminol-H2O2 CL reaction is greatly enhanced. This allows us to utilize the luminol-H2O2 CL system for quantitative analysis of thrombin, which was used to denote fibrosis degree of Fib. The assay showed a linear toward fibrillar fibrin concentration in the range of 2.7 × 10(-15)-2.7 × 10(-13)M with a correlation of 0.9920. The limit of detection for fibrin was experimentally determined to be 1 fM, based on a signal-to-noise ratio (S/N) of 3. Relative to conventional methods, this method offers the advantages of higher sensitivity and selectivity and lower cost, showing great potential for medical diagnosis.

Magnetic bead-liposome hybrids enable sensitive and portable detection of DNA methyltransferase activity using personal glucose meter.

DNA methyltransferase (MTase) plays a critical role in maintaining genome methylation patterns, which has a close relationship to cancer and bacterial diseases. This encouraged the need to develop highly sensitive, simple, and robust assays for DNA MTase detection and inhibitor screening. Herein, a simple, sensitive, and specific DNA MTase activity assay was developed based on magnetic beads-liposome hybrids combined with personal glucose meter (PGM) for quantitative detection of DNA MTase and inhibitor screening. First, a magnetic beads-liposome hybrid probe is designed by the hybridization of p1DNA-functionalized magnetic bead with p2DNA-functionalized glucoamylase-encapsulated liposome (GEL). It integrates target recognition, magnetic separation and signal amplification within one multifunctional design. Then, in the presence of Dam MTase, the hybrids probe was methylated, and cleaved by methylation-sensitive restriction endonuclease Dpn I, making liposome separated from magnetic bead by magnetic separation. Finally, the separated liposome was decomposed, liberating the encapsulated glucoamylase to catalyze the hydrolysis of the signal substrate amylose with multiple turnovers, producing a large amount of glucose for quantitative readout by the PGM. In the proposed assay, the magnetic beads-liposome hybrids offered excellent sensitivity due to primary amplification via releasing numerous glucoamylase from a liposome followed by a secondary enzymatic amplification. The use of portable quantitative device PGM bypasses the requirement of complicated instruments and sophisticated operations, making the method simple and feasible for on-site detection. Moreover, the proposed assay was successfully applied in complex biological matrix and screen suitable inhibitor drugs for DAM for disease(s) treatment. The results reveal that the approach provides a simple, sensitive, and robust platform for DNA MTases detection and screening potential drugs in medical research and early clinical diagnostics.

Target-responsive dumbbell probe-mediated rolling circle amplification strategy for highly sensitive Hg2+ detection

A novel label-free amplified fluorescent sensing scheme based on target-responsive dumbbell probe-mediated rolling circle amplification (D-RCA) has been developed for sensitive and selective detection of mercuric ions. In this strategy, we reported an ingeniously designed dumbbell-shaped DNA probe (D-DNA) that integrates target-binding, amplification and signaling within one multifunctional design. An Hg2+–primer DNA (Hg2+–p-DNA) was designed to be complementary to the region of D-DNA but with T–T mismatches. The mismatched Hg2+–primer cannot initiate the RCA reaction in the absence of Hg2+. Stable T–Hg2+–T can be formed in the presence of target Hg2+, thus it induces the elongation and amplification reaction by a RCA mechanism, resulting in numerous cascade dumbbell probes forming duplex G-rich quadruplex oligomeric structures . Upon addition of N-methyl mesoporphyrin IX (NMM), the signal reporter, a strong interaction between the G-quadruplex and NMM brings about a great fluorescence enhancement. In this way, we successfully converted each Hg2+-triggered D-RCA reaction event into detectable fluorescent signals, which were significantly amplified by RCA in an isothermal fashion. This approach can detect 80 fM of mercuric ions, much lower levels than previously reported biosensors, and exhibits high discrimination ability. More significantly, the dynamic range of D-RCA is extremely large, covering 5 orders of magnitude. We also demonstrate Hg2+ quantification with this highly sensitive and selective D-RCA strategy in real samples.

Sensitive fluorescent detection of fibrin based on the inner filter effect of gold nanoparticles

A simple, rapid and sensitive fluorescent assay for determination of fibrin has been developed based on the inner filter effect (IFE) of gold nanoparticles (AuNPs). When fibrinogen (Fib), as the precursor of fibrin, was added into the AuNPs solution, the fluorescence of fluorescein was very weak due to the intensive absorption of AuNPs. In the presence of thrombin, Fib was transformed to fibrin which interacted with AuNPs, thereby inducing the aggregation of AuNPs, which induced the recovery of fluorescence. As a result, the present IFE-based approach can detect fibrin ranging from 0.125–2.5 nM with a correlation of 0.9926. The limit of detection for fibrin was experimentally determined to be 40 pM, based on a signal-to-noise ratio (S/N) of 3. Notably, the present IFE-based approach had advantages of being simple, time-saving, and economical compared with conventional fluorescent assays. The method is successfully applied to the quantification of fibrin in human plasma samples.

A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification

Bisphenol A (BPA) detection in drinking water and food packaging materials has attracted much attention since the discovery that BPA can interfere with normal physiological processes and cause adverse health effects. Here, we constructed a label-free aptamer fluorescent assay for selective and sensitive detection of BPA based on the rolling circle amplification (RCA)/Exonuclease III (Exo III)-combined cascade amplification strategy. First, the duplex DNA probe (RP) with anti-BPA aptamer and trigger sequence was designed for BPA recognition and signal amplification. Next, under the action of BPA, the trigger probe was liberated from RP to initiate RCA reaction as primary amplification. Subsequently, the RCA products were used to trigger Exo III assisted secondary amplification with the help of hairpin probes, producing plenty of “G-quadruplex” in lantern-like structures. Finally, the continuously enriched “G-quadruplex lanterns” were lightened by zinc(II)-protoporphyrin IX (ZnPPIX) generating enhanced fluorescence signals. By integrating the primary RCA and secondary Exo III mediated cascade amplification strategy, this method displayed an excellent sensitivity with the detection limits of 5.4 × 10−17 M. In addition, the anti-BPA aptamer exhibits high recognition ability with BPA, guaranteeing the specificity of detection. The reporter signal probe (G-quadruplex with ZnPPIX) provides a label-free fluorescence signals readout without complicated labeling procedures, making the method simple in design and cost-effective in operation. Moreover, environmental samples analysis was also performed, suggesting that our strategy was reliable and had a great potential application in environmental monitoring.

Label-Free Fluorescent DNA Dendrimers for microRNA Detection Based On Nonlinear Hybridization Chain Reaction-Mediated Multiple G-Quadruplex with Low Background Signal

Various fluorescent sensing systems for miRNA detection have been developed, but they mostly contain enzymatic amplification reactions and label procedures. The strict reaction conditions of tool enzymes and the high cost of labeling limit their potential applications, especially in complex biological matrices. Here, we have addressed the difficult problems and report a strategy for label-free fluorescent DNA dendrimers based on enzyme-free nonlinear hybridization chain reaction (HCR)-mediated multiple G-quadruplex for simple, sensitive, and selective detection of miRNAs with low-background signal. In the strategy, a split G-quadruplex (3:1) sequence is ingeniously designed at both ends of two double-stranded DNAs, which is exploited as building blocks for nonlinear HCR assembly, thereby acquiring a low background signal. A hairpin switch probe (HSP) was employed as recognition and transduction element. Upon sensing the target miRNA, the nonlinear HCR assembly of two blocks (blocks-A and blocks-B) was initiated with the help of two single-stranded DNA assistants, resulting in chain-branching growth of DNA dendrimers with multiple G-quadruplex incorporation. With the zinc(II)-protoporphyrin IX (ZnPPIX) selectively intercalated into the multiple G-quadruplexes, fluorescent DNA dendrimers were obtained, leading to an exponential fluorescence intensity increase. Benefiting from excellent performances of nonlinear HCR and low background signal, this strategy possesses the characteristics of a simplified reaction operation process, as well as high sensitivity. Moreover, the proposed fluorescent sensing strategy also shows preferable selectivity, and can be implemented without modified DNA blocks. Importantly, the strategy has also been tested for miRNA quantification with high confidence in breast cancer cells. Thus, this proposed strategy for label-free fluorescent DNA dendrimers based on a nonlinear HCR-mediated multiple G-quadruplex will be turned into an alternative approach for simple, sensitive, and selective miRNA quantification.