Yuanfu Zhang

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.

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.

Aptamer-based fluorescent detection of ochratoxin A by quenching of gold nanoparticles

A simple, rapid, low cost and highly sensitive method for the detection of ochratoxin A (OTA) was developed based on the principle that dispersed AuNPs show a better fluorescence quenching effect than aggregated AuNPs. In the absence of OTA, the aptamer is adsorbed onto the surface of AuNPs, which helps to enhance the stability of AuNPs against salt-induced aggregation, and also enhances the fluorescence quenching of the fluorescein-labeled aptamer. With the addition of OTA, the conformation of the aptamer changed, which induced aggregation of AuNPs in the presence of high-salt conditions. The fluorescence intensity was clearly recovered. The assay showed a linear response toward OTA concentration in the range of 25 nM to 300 nM with a correlation coefficient of 0.9957. The limit of detection for OTA was experimentally determined to be 22.7 nM. This method has the advantages of simple operation, low-cost and high sensitivity compared to conventional methods and can be applied to the detection of real samples.

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 competitive fluorescence quenching-based immunoassay for bisphenol A employing functionalized silica nanoparticles and nanogold

We have developed a fast and sensitive immunoassay for the determination of bisphenol A (BPA), using the fluorescence quenching effect between gold nanoparticles and fluorescein isothiocyanate (FITC). Herein, carboxyl-modified single-strand DNA (COOH-ssDNAs) and anti-BPA antibodies were simultaneously conjugated with silica nanoparticles, and FITC-labeled single-strand DNA (FITC-ssDNA, the complement of COOH-ssDNA) was hybridized with COOH-ssDNA to form a signal platform, and the BPA coated antigen-functionalized nanogold was regarded as an acceptor. In the presence of BPA, a competitive immunoreaction takes place between BPA and BPA coated antigen-functionalized nanogold for the binding sites of the anti-BPA antibodies on the signal platform. Due to the fact that the photoluminescence of FITC was strongly quenched by the AuNPs, the fluorescence emission was decreased significantly. Under optimized conditions, the fluorescence intensity had a linear signal response range with a BPA concentration from 2.0 × 10−3 ng mL−1 to 1.0 ng mL−1 with a low limit of detection of 1.44 × 10−3 ng mL−1. Furthermore, this new signal platform was successfully applied to detect real samples for low levels of BPA.

A sensitive sandwich structure time-resolved fluorescence method for thrombin detection

We designed a sensitive and specific time-resolved fluorescence assay for detection of human thrombin. This design was based on the amplification of nanoparticles and the specificity of aptamers. The DC@AuNPs were prepared by the conjugation of AuNPs to BSA–Eu3+–DTPA and SH-Apt29. In the presence of thrombin, the formation of a quadruplex–thrombin complex could lead to the formation of a MNPs@Apt15–thrombin–DC@AuNP sandwich structure. In the presence of enhancement solution, Eu3+ interacted with the enhancement solution to produce strong fluorescence. Under the optimized conditions, the linear range of the method was from 1 pM to 100 pM of thrombin, and the limit of detection was 0.78 pM. Furthermore, the method could specifically recognize thrombin in the presence of other analogous proteins. The method is successfully used to determine thrombin in human plasma. This method has the advantages of high sensitivity and selectivity. It showed great potential for medical diagnosis.

A simple and sensitive fluorescence method for detection of telomerase activity using fusion protein bouquets

Telomerase is considered as a widely accepted cancer biomarker for early cancer diagnostics. Herein, we develop a simple, ultrahigh sensitivity method for detection of telomerase activity, which relied on that RecA-GFP fusion proteins wrapped around telomeric DNA to form fluorescence bouquets. RecA-GFP fusion protein was synthesized through fusion protein technology. In the presence of telomerase, telomerase elongation products are wrapped around by RecA-GFP fusion protein to form big fluorescent bouquets, which resulted in strong fluorescence. This method has the linear range from 50 to 1000 HeLa cells and the detection limit is 8 HeLa cells, based on a signal-to-noise ratio (S/N) of 3. Compared with conventional methods, this method has the advantages of low toxicity, outstanding sensitivity, and excellent selectivity. Hence, it provides a promising approach for the detection of telomerase activity and diagnosis of cancer.