Fangzhen Li

Aptamer pseudoknot-functionalized electronic sensor for reagentless and single-step detection of immunoglobulin E in human serum.

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.

The synthesis and activities of novel mononuclear or dinuclear cyclen complexes bearing azole pendants as antibacterial and antifungal agents

A series of novel compounds containing 1,4,7,10-tetraazacyclododecane and azoles were synthesized and characterized by (1)H NMR, MS and elemental analysis. Bioactive assay manifested that some target compounds, such as 11a, 11b and 11d, displayed good and broad spectrum antimicrobial activities with relative low MIC values against most of tested strains. These dinuclear complexes gave comparable or even better antimicrobial efficiencies than the reference drugs Fluconazole and Chloromycin. The result showed that the metal ions were the key factors to enhance the antimicrobial activities for mononuclear or dinuclear complexed in varying degrees. The interaction evaluation of compound 11b with bovine serum albumin (BSA) as an example was tested by fluorescence method. The thermodynamic parameters indicated that the hydrogen bonds and van der waals forces played the major roles in the strong association between dinuclear compound and BSA. The CCK-8 tests also confirmed the safeties of these dinuclear compounds in vitro.

Copper and zinc complexes of a diaza-crown ether as artificial nucleases for the efficient hydrolytic cleavage of DNA

Artificial nucleases are a kind of new non-enzymatic breakage tool, which mimic the function of a restricted enzyme and can catalyze nucleic acid cleavage highly efficiently and selectively. Some metal complexes are highly efficient cleavage agents for DNA. In this report, a diaza-crown ether (L) was synthesized and its copper and zinc complexes (CuL and ZnL) were prepared to serve as artificial nucleases. The compositions of the two metal complexes were confirmed by LC-MS analysis, and the nuclease activity of the complexes towards pUC19 DNA cleavage was studied using agarose gel electrophoresis. The results indicate that the two metal complexes can accelerate the breakage of DNA from the supercoiled (form I) to the nicked form (form II) under appropriate conditions; the optimal pH value for the DNA catalytic cleavage is 8.01 for both CuL and ZnL; the complex ZnL was a better catalyst in the DNA cleavage process than the complex CuL at low concentrations; and unreactive μ-hydroxo dimers were formed hampering the DNA cleavage process at high concentrations. In the presence of free radical scavengers, the supercoiled plasmid DNA cleavage catalyzed by these complexes has been performed and the catalytic mechanism has been investigated.

Development of the aza-crown ether metal complexes as artificial hydrolase

Hydrolases play a crucial role in the biochemical process, which can catalyze the hydrolysis of various compounds like carboxylic esters, phosphoesters, amides, nucleic acids, peptides, and so on. The design of artificial hydrolases has attracted extensive attention due to their scientific significance and potential applications in the field of gene medicine and molecular biology. Numerous macrocyclic metal complexes have been used as artificial hydrolase in the catalytic hydrolysis of the organic substrate. Aza-crown ether for this comment is a special class of the macrocyclic ligand containing both the nitrogen atoms and oxygen atoms in the ring. The studies showed that the aza-crown complexes exhibited high activity of hydrolytic enzyme. However, the aza-crown ether metal complex as artificial hydrolase is still very limited because of its difficulty in synthesis. This review summarizes the development of the aza-crown ether metal complexes as the artificial hydrolase, including the synthesis and catalysis of the transition metal complexes and lanthanide metal complexes of aza-crown ethers. The purpose of this review is to highlight: (1) the relationship between the structure and hydrolytic activity of synthetic hydrolase; (2) the synergistic effect of metal sites and ligands in the course of organic compound hydrolysis; and (3) the design strategies of the aza-crown ethers as hydrolase.

Multiplexed electrochemical coding of DNA–protein bindings

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.

DNA binding and cleavage properties of the Ce(III) complex of a diaza-crown ether

The hydrolytic cleavage of pUC19 DNA as promoted by the trivalent cerium complex (CeL) of diaza-crown ether ligand L (1,4,10,13-tetraoxa-7,16-diazacyclooctadecane) was examined in detail. The interaction of CeL with calf thymus DNA was investigated by UV-Vis spectroscopy. Furthermore, studies on the effects of pH, reaction time, and the concentration of CeL on cleavage of pUC19 DNA were carried out by gel electrophoresis. The results indicate that CeL can bind to DNA electrostatically with a binding constant of 2.18x104 M−1; CeL can promote the cleavage of plasmid pUC19 DNA from supercoiled to the nicked form under the appropriate conditions, and the optimal pH value is 7.54. The lack of effect of radical scavengers indicates that the cleavage involves hydrolytic cleavage.

Nucleic acid and phosphoester hydrolytic cleavage catalysed by aza-crown ether metal complexes as synthetic nucleases

Aza-crown ethers are ligands in which the oxygen atoms are replaced by nitrogen atoms in the crown ether ring systems. This type of ligand possesses specific complexation with metal ions, such as those of transition-metals, rare earths, alkali metals and alkaline earths, which form metal complexes whose structures are similar to those of some biological enzymes. In recent decades, research on aza-crown ethers and their metal complexes as mimics of nucleases in hydrolysing nucleic acids has attracted increasing attention. These studies illuminate the mechanism of nucleic acid hydrolytic cleavage as catalysed by natural nucleases. In order to assist the design and synthesis of highly active, selective and stable mimic nucleases, this paper reviews recent progress in the investigation of aza-crown ether metal complexes as mimic nucleases, including: the relationship between the structures and activities of synthetic metallonucleases; multicentre synergistic catalysis of metal ions in multinuclear complexes; bifunctional cooperative catalysis of the branches and ions in the complexes; and especially, the structural characteristic and catalytic mechanism of aza-crown ether metal complexes as mimic nucleases.

The Metallomicelle of Lanthanide Metal (Ce, La) Aza-Macrocyclic Complexes with a Carboxyl Branch: The Catalytic Activity and Mechanism in the Hydrolysis of a Phosphate Diester

An aza-macrocyclic ligand, 5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane-N/- acetic acid (L), was synthesized and characterized. The chemical composition of the complexes (CeL and LaL) was determined by a UV–Vis spectrophotometric method and kinetic Job plot. Two metallomicellar systems made of an lanthanide metal complex and n-lauroylsarcosine (LSS) micelle were used as catalyst in the hydrolysis of bis(4-nitrophenyl) phosphate ester (BNPP), and their catalytic activity was studied by the comparative method. The interaction between the complex and BNPP in the metallomicelle was studied by its fluorescent spectroscopy. The catalytic rate of BNPP hydrolysis was measured kinetically by UV–Vis spectrophotometry. The results indicate that CeL systems (aqueous solution and metallomicelle) exhibit higher catalytic activity than those of the LaL systems in BNPP hydrolysis, and the micelle provides an effective catalytic environment in the catalytic reaction. A reaction mechanism was also proposed on the basis of the results.

Unmodified and positively charged gold nanoparticles for sensitive colorimetric detection of folate receptor via terminal protection of small molecule-linked ssDNA

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.

DNA cleavage activity: comparison of two lanthanum complexes based on aza-crown ethers with different numbers of nitrogen atoms

Based on the unique characteristics of lanthanum ion and aza-crown ethers, the lanthanum complexes of two aza-crown ether (L1: 1,10-Dioxa-4,7,13,16-tetraazacyclo-octadecane and L2: 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane) were designed and synthesised. The interaction between these two complexes and DNA was measured by UV-Vis spectroscopy and gel electrophoresis. Moreover, a series of experiments of cleavage of pUC19 DNA were conducted to illustrate the acidity, time and concentration effects. The results indicated that the two metal complexes can accelerate the breakage of DNA from its supercoiled form (form I) to the nicked form (form II) at near-physiological conditions, and the optimum acidity of DNA catalytic cleavage is pH=6.5 and pH=7.0 for LaL1 and LaL2, respectively. Furthermore, the LaL1 exhibited better cleavage activity than LaL2 under the same conditions, thus supercoiled DNA was thoroughly cleaved to the nicked form under the appropriate conditions. The hydrolytic mechanism was verified by applying several oxygen-scavengers to the DNA catalytic cleavage.