Zhou Nie

Label-Free Colorimetric Assay for Methyltransferase Activity Based on a Novel Methylation-Responsive DNAzyme Strategy

DNA methylation catalyzed by methyltransferase (MTase) is a significant epigenetic process for modulating gene expression. Traditional methods to study MTase activity require a laborious and costly DNA labeling process. In this article, we report a simple, colorimetric, and label-free methylation-responsive DNAzyme (MR-DNAzyme) strategy for MTase activity analysis. This new strategy relies on horseradish peroxidase (HRP) mimicking DNAzyme and the methylation-responsive sequence (MRS) of DNA which can be methylated and cleaved by the MTase/endonuclease coupling reaction. Methylation-induced scission of MRS would activate the DNAzyme that can catalyze the generation of a color signal for the amplified detection of methylation events. Taking Dam MTase and DpnI endonuclease as examples, we have developed two colorimetric methods based on the MR-DNAzyme strategy. The first method is to utilize an engineered hairpin-DNAzyme hybrid probe for facile turn-on detection of Dam MTase activity, with a wide linear range (6-100 U/mL) and a low detection limit (6 U/mL). Furthermore, this method could be easily expanded to profile the activity and inhibition of restriction endonuclease. The second method involves a methylation-triggered DNAzyme-based DNA machine, which achieves the ultrahigh sensitive detection of Dam MTase activity (detection limit = 0.25 U/mL) by a two-step signal amplification cascade.

Impedimetric Aptasensor with Femtomolar Sensitivity Based on the Enlargement of Surface-Charged Gold Nanoparticles

A simple and ultrasensitive label-free electrochemical impedimetric aptasensor for thrombin based on the cascaded signal amplification was reported. The sandwich system of aptamer/thrombin/aptamer-functionalized Au nanoparticles (Apt-AuNPs) was fabricated as the sensing platform. The change of the interfacial feature of the electrode was characterized by electrochemical impedance analysis with the redox probe [Fe(CN)(6)](3-/4-). For improving detection sensitivity, the three-level cascaded impedimetric signal amplification was developed: (1) Apt-AuNPs as the first-level signal enhancer; (2) the steric-hindrance between the enlarged Apt-AuNPs as the second-level signal amplification; (3) the electrostatic-repulsion between sodium dodecylsulfate (SDS) stabilized Apt-AuNPs and the redox probe [Fe(CN)(6)](3-/4-) as the third-level signal amplification. Enlargement of Apt-AuNPs integrated with negatively charged surfactant (SDS) capping could not only improve the detection sensitivity of the impedimetric aptasensor for thrombin but also present a simple and general signal-amplification model for impedimetric sensor. The aptasensor based on the enlargement of negatively charged Apt-AuNPs showed an increased response of the electron-transfer resistance to the increase of thrombin concentration through a wide detection range from 100 fM to 100 nM. The linear detection range was 0.05-35 nM, and thrombin was easily detectable to a concentration of 100 fM. The aptasensor also has good selectivity and reproducibility.

A sensitive and stable biosensor based on the direct electrochemistry of glucose oxidase assembled layer-by-layer at the multiwall carbon nanotube-modified electrode

A novel strategy for fabricating the sensitive and stable biosensor was present by layer-by-layer (LBL) self-assembling glucose oxidase (GOD) on multiwall carbon nanotube (CNT)-modified glassy carbon (GC) electrode. GOD was immobilized on the negatively charged CNT surface by alternatively assembling a cationic poly(ethylenimine) (PEI) layer and a GOD layer. And the direct electrochemistry of GOD in the self-assembled {GOD/PEI}(n) film was investigated. CNT as an excellent nanomaterial greatly improved the direct electron transfer between GOD in {GOD/PEI}(n) film and the electrode. And the ultrathin {GOD/PEI}(n) film on the CNT surface provided a favorable microenvironment to keep the bioactivity of GOD. Moreover, PEI used as an out-layer was adsorbed on the top of the {GOD/PEI}(n) film to form the sandwich-like structure (PEI/{GOD/PEI}(n)), improving the stability of the enzyme electrode. On basis of these, the developed PEI/{GOD/PEI}(n)/CNT/GC biosensor has a high sensitivity of 106.57 μA mM(-1) cm(-2), and can measure as low as 0.05 mM glucose. In addition, the biosensor has excellent operational stability with no decrease in the activity of enzyme over a 1-week period. Therefore, the developed strategy making use of the advantages of CNT and LBL assembly is ideal for the direct electrochemistry of the redox enzymes and the construction of the sensitive and stable enzyme biosensor.

Sensitive Bifunctional Aptamer-Based Electrochemical Biosensor for Small Molecules and Protein

In this paper, a bifunctional electrochemical biosensor for highly sensitive detection of small molecule (adenosine) or protein (lysozyme) was developed. Two aptamer units for adenosine and lysozyme were immobilized on the gold electrode by the formation of DNA/DNA duplex. The detection of adenosine or lysozyme could be carried out by virtue of switching structures of aptamers from DNA/DNA duplex to DNA/target complex. The change of the interfacial feature of the electrode was characterized by cyclic voltammertic (CV) response of surface-bound [Ru(NH(3))(6)](3+). On the other hand, DNA functionalized Au nanoparticles (DNA-AuNPs) were used to enhance the sensitivity of the aptasensor because DNA-AuNPs modified interface could load more [Ru(NH(3))(6)](3+) cations. Thus, the assembly of two aptamer-contained DNA strands integrated with the DNA-AuNPs amplification not only improves the sensitivity of the electrochemical aptasensor but also presents a simple and general model for bifunctional aptasensor. The proposed aptasensor has low detection limit (0.02 nM for adenosine and 0.01 microg mL(-1) for lysozyme) and exhibits several advantages such as high sensitivity and bifunctional recognition.

Selective collection and detection of leukemia cells on a magnet-quartz crystal microbalance system using aptamer-conjugated magnetic beads

A novel method for selective collection and detection of human acute leukemia cells has been proposed using aptamer-conjugated magnetic beads (apt-MBs) and a magnet-quartz crystal microbalance (QCM) system. The sgc8c aptamer-conjugated MBs specifically binding to CCRF-CEM cells were used for target cell extraction from complex matrixes, and the magnet-QCM system was successfully applied for quantitative cell detection, requiring no further labeling of cells. The accumulation of MBs-conjugated CCRF-CEM cells on a quartz crystal gold electrode surface under a magnetic field resulted in decreased resonant frequency. A linear relationship between the frequency shift and cell concentration over the range of 1 x 10(4)-1.5 x 10(5)cells mL(-1) was obtained, with a detection limit of 8 x 10(3)cells mL(-1). The applicability of the method for target cell detection from cell mixture was satisfactory.

Graphene Oxide-Peptide Nanocomplex as a Versatile Fluorescence Probe of Protein Kinase Activity Based on Phosphorylation Protection against Carboxypeptidase Digestion

The research on complicated kinomics and kinase-target drug discovery requires the development of simple, cost-effective, and multiplex kinase assays. Herein, we propose a novel and versatile biosensing platform for the detection of protein kinase activity based on graphene oxide (GO)-peptide nanocomplex and phosphorylation-induced suppression of carboxypeptidase Y (CPY) cleavage. Kinase-catalyzed phosphorylation protects the fluorophore-labeled peptide probe against CPY digestion and induces the formation of a GO/peptide nanocomplex resulting in fluorescence quenching, while the nonphosphopeptide is degraded by CPY to release free fluorophore as well as restore fluorescence. This GO-based nanosensor has been successfully applied to sensitively detect two model kinases, casein kinase (CKII) and cAMP-dependent protein kinase (PKA) with low detection limits of 0.0833 mU/μL and 0.134 mU/μL, respectively. The feasibility of this GO-based sensor was further demonstrated by the assessment of kinase inhibition by staurosporine and H-89, in vitro kinase assay in cell lysates, and simultaneous detection of CKII and PKA activity. Moreover, the GO-based fluorescence anisotropy (FA) kinase assay has been also developed using GO as a FA signal amplifier. The proposed sensor is homogeneous, facile, universal, label-free, and applicable for multiplexed kinase assay, presenting a promising method for kinase-related biochemical fundamental research and inhibitor screening.

Non-Redox Modulated Fluorescence Strategy for Sensitive and Selective Ascorbic Acid Detection with Highly Photoluminescent Nitrogen-Doped Carbon Nanoparticles via Solid-State Synthesis

Highly photoluminescent nitrogen-doped carbon nanoparticles (N-CNPs) were prepared by a simple and green route employing sodium alginate as a carbon source and tryptophan as both a nitrogen source and a functional monomer. The as-synthesized N-CNPs exhibited excellent water solubility and biocompatibility with a fluorescence quantum yield of 47.9%. The fluorescence of the N-CNPs was intensively suppressed by the addition of ascorbic acid (AA). The mechanism of the fluorescence suppression of the N-CNPs was investigated, and the synergistic action of the inner filter effect (IFE) and the static quenching effect (SQE) contributed to the intensive fluorescence suppression, which was different from those reported for the traditional redox-based fluorescent probes. Owing to the spatial effect and hydrogen bond between the AA and the groups on the N-CNP surface, excellent sensitivity and selectivity for AA detecting was obtained in a wide linear relationship from 0.2 μM to 150 μM. The detection limit was as low as 50 nM (signal-to-noise ratio of 3). The proposed sensing systems also represented excellent sensitivity and selectivity for AA analysis in human biological fluids, providing a valuable platform for AA sensing in clinic diagnostic and drug screening.

A novel and simple strategy for selective and sensitive determination of dopamine based on the boron-doped carbon nanotubes modified electrode

The Boron-doped carbon nanotubes (BCNTs) modified glassy carbon (GC) electrode was obtained simply and used for highly selective and sensitive determination of dopamine (DA). Comparing with the bare GC and CNTs/GC electrodes, the BCNTs have higher catalytic activity toward the oxidation of DA and ascorbic acid (AA). Moreover, the voltammetric peaks of AA and DA were separated enough (ca. 238 mV) at the BCNTs/GC electrode, which is superior to that at the CNTs/GC electrode (ca. 122 mV). Thus, the selective determination of DA was carried out successfully in the presence of AA. A wide concentration range (2.0 x 10(-8)-7.5 x 10(-5)M) and low detection limit (1.4 nM, S/N=3) for the DA detection were obtained. The possibility of the BCNTs/GC electrode for the determination of DA in human blood serum has also been evaluated. The advantageous properties of this electrode for the DA determination lie in its excellent catalytic activity, selectivity and simplicity. The more edge plane sites presented on the BCNTs surface were partially responsible for its good analytical behavior.

Amino acid ionic liquids as chiral ligands in ligand-exchange chiral separations.

Recently, amino acid ionic liquids (AAILs) have attracted much research interest. In this paper, we present the first application of AAILs in chiral separation based on the chiral ligand exchange principle. By using 1-alkyl-3-methylimidazolium L-proline (L-Pro) as a chiral ligand coordinated with copper(II), four pairs of underivatized amino acid enantiomers-dl-phenylalanine (dl-Phe), dl-histidine (dl-His), dl-tryptophane (dl-Trp), and dl-tyrosine (dl-Tyr)-were successfully separated in two major chiral separation techniques, HPLC and capillary electrophoresis (CE), with higher enantioselectivity than conventionally used amino acid ligands (resolution (R(s))=3.26-10.81 for HPLC; R(s)=1.34-4.27 for CE). Interestingly, increasing the alkyl chain length of the AAIL cation remarkably enhanced the enantioselectivity. It was inferred that the alkylmethylimidazolium cations and L-Pro form ion pairs on the surface of the stationary phase or on the inner surface of the capillary. The ternary copper complexes with L-Pro are consequently attached to the support surface, thus inducing an ion-exchange type of retention for the dl-enantiomers. Therefore, the AAIL cation plays an essential role in the separation. This work demonstrates that AAILs are good alternatives to conventional amino acid ligands for ligand-exchange-based chiral separation. It also reveals the tremendous application potential of this new type of task-specific ILs.

A simple "clickable" biosensor for colorimetric detection of copper(II) ions based on unmodified gold nanoparticles.

A novel colorimetric copper(II) biosensor has been developed based on the high specificity of alkyne-azide click reaction to the catalysis of copper ions and unmodified gold nanoparticles (AuNPs) as the signal reporter. The clickable DNA probe consists of two parts: an azide group-modified double-stranded DNA (dsDNA) hybrid with an elongated tail and a short alkyne-modified single-stranded DNA (ssDNA). Because of low melting temperature of the short ssDNA, these two parts are separated in the absence of Cu(2+). Copper ion-induced azide-alkyne click ligation caused a structural change of probe from the separated form to entire dsDNA form. This structural change of probe can be monitored by the unmodified AuNPs via mediating their aggregation with a red-to-blue colorimetric read-out because of the differential ability of ssDNA and dsDNA to protect AuNPs against salt-induced aggregation. Under the optimum conditions, this biosensor can sensitively and specifically detect Cu(2+) with a low detection limit of 250 nM and a linear range of 0.5-10 μM. The method is simple and economic without dual-labeling DNA and AuNPs modification. It is also highly selective for Cu(2+) in the presence of high concentrations of other environmentally relevant metal ions because of the great specificity of the copper-caused alkyne-azide click reaction, which potentially meets the requirement of the detection in real samples.