Gold nanoparticles based colorimetric assay of protein poly(ADP-ribosyl)ation

A new method to assay protein poly(ADP-ribosyl)ation was proposed based on the interaction between the substrate of poly(ADP-ribosyl)ation nicotinamide adenine dinucleotide and gold nanoparticles, which needed no coupled enzymes or other modified catalytic substrate.     

Study of UVA irradiation on hemoglobin in the presence of NADH

Reduction of ferric methemoglobin (metHb) to its ferrous form is observed by short-time ultraviolet A (UVA) irradiation of metHb together with nicotinamide adenine dinucleotide (NADH). And, severely structural destruction of metHb occurs when long-time UVA irradiation is exerted. However, neither reduction nor destruction can be observed in the absence of NADH under otherwise the same experimental conditions. Accordingly, the O2-binding ability of the protein increases by short-time UVA irradiation of metHb together with NADH, which corresponds with the reduction of metHb, while it decreases by long-time UVA irradiation, which corresponds with the structural destruction. Besides, it is found that the reduction reaction and the conformational destruction proceed more rapidly with higher concentrations of NADH.     

Investigation of MTH1 activity via mismatch-based DNA chain elongation

Accumulation and misincorporation of oxidative damaged 8-oxo-7,8-dihydroguanine triphosphates (8-oxo-dGTP) in genomic DNA may cause serious cellular function disorders. MutT Homolog 1 (MTH1), a protein enzyme that can help to prevent 8-oxo-dGTP misincorporation, plays critical roles in oxidative stress neutralization, oncogene-associated tumor malignancy, and anticancer therapies. So, in this work, a simple and function-oriented method is developed for the assay of MTH1 activity. Specifically, a mismatch-based (“8-oxoG: A” mismatch) DNA chain elongation strategy (MB-DCE) is firstly proposed to reveal the misincorporation efficiency of 8-oxo-dGTP. Then, further coupled with the inherent activity of MTH1 to prevent 8-oxo-dGTP misincorporation, a relationship can be established to reveal the activity of MTH1 through MB-DCE. As the method is designed directly towards the cellular function of MTH1, activity of MTH1 in different breast cancer cell lines has been detected, implying the potential application of this assay method for biomedical research and clinical diagnose in the future.     

Colorimetric detection of proteins based on target-induced activation of aptazyme

The detection of protein is vital to fundamental research as well as practical applications. However, most detection methods depend on antibody-based assays which are faced with many shortcomings. Herein, we propose a colorimetric method for protein assays based on target-triggered activation of aptazyme, which may offer simple, rapid and cost-effective detection of the target protein. In this method, the conformation change of aptazyme induced by target protein is designed to be associated with aptazyme activation. Consequently, in the presence of the target protein, the designed DNA linkers will be cleaved into two fragments that fail to cross-link gold nanoparticles (GNPs), thus the color of GNP solution remains red, while the color will be changed in the absence of the target. Because of the advantages of aptazyme such as economic synthesis, stable, easy modification and its ability to accomplish signal recognition and signal amplification simultaneously, the method is thermostable, simple and cost-efficient. In this work, we have taken the detection of vascular endothelial growth factor (VEGF) as an example, which can present an analytical performance with as low as 0.1 nM detection limit, spanning a detection range of 3 orders of magnitude. What is more, the principle of this proposed new method can be extended as a universal assay method not only for the detection of analytes which have an aptamer but also for those analytes that have ligands.     

Electrochemical evaluation of self-disassociation of PKA upon activation by cAMP

The allosteric reaction of protein kinase A (PKA) upon binding of cyclic AMP (cAMP) is revealed with an electrochemical technique through the redox current change of an electrochemically active marker. The different effect of cAMP's regulation at a distinct concentration level is obtained in this system. The influence of structural analogues is also examined with respect to the affinity and special selectivity. This study presents an electrochemical approach to the rapid and sensitive investigation of the protein-ligand interaction in the signal transduction networks.     

A novel electrochemical method to detect mercury (II) ions

A novel and sensitive electrochemical method for determination of mercury (II) ions (Hg²⁺) based on the formation of thymine–Hg²⁺–thymine complexes and gold nanoparticle-mediated signal amplification is reported. Two 5′ end thiolated complementary oligonucleotides containing six strategically placed thymine–thymine mistakes were introduced in this work. One of the two oligonucleotides was immobilized on a gold electrode and the other one on gold nanoparticles (AuNPs). Due to six thymine–thymine mistakes the two oligonucleotides were not able to be hybridized, so AuNPs could not be immobilized onto the electrode surface after the electrode was immersed in the DNA–AuNPs solution. However, if Hg²⁺ existed, T–Hg²⁺–T complexes could be formed and AuNPs could be immobilized onto the electrode surface. Meanwhile, large numbers of [Ru(NH₃)₆]³⁺ molecules as electrochemical species could be localized onto the electrode surface. The Hg²⁺ detection limit of this assay could be as low as 10 nM, which is the US Environmental Protection Agency (EPA) limit of Hg²⁺ for drinkable water. This method is proven to be simple, convenient, high sensitive and selective.     

Third-generation biosensors based on the direct electron transfer of proteins.

Recent progress in third-generation electrochemical biosensors based on the direct electron transfer of proteins is reviewed. The development of three generations of electrochemical biosensors is also simply addressed. Special attention is paid to protein-film voltammetry, which is a powerful way to obtain the direct electron transfer of proteins. Research activities on various kinds of biosensors are discussed according to the proteins (enzymes) used in the specific work.     

聚苯胺修饰电极上抗坏血酸与多巴胺氧化峰的分离

本文介绍一种分离抗坏血酸(AA)和多巴胺(DA)氧化峰的新方法。聚苯胺(PA)对AA和DA有不同的催化性,因而在PA修饰电极上,AA和DA在不同的电位被氧化,从而解决了二者氧化峰不能分离的问题。实验表明,在PA膜电极上,AA和DA氧化峰可分开约140mV。     

聚苯胺修饰电极制备过程中几个问题的探讨

报道聚苯胺修饰电极制备中尚须引起人们重视的几个问题。主要包括：循环伏安法扫描电位上限对聚合的影响，恒电位法最佳的聚合电位，以及仅具２００ｍＶ和９００ｍＶ两峰聚苯胺膜的制备，聚合电量对不同制备方法影响的差异性等等。并对制备聚苯胺修饰电极的几种方法进行了比较，从而指出了最佳的聚合方法和条件。