基于电化学发光和阻抗技术研究DNA单碱基错配

单碱基错配的识别和稳定性差异在核酸多态性研究中至关重要。在同一电化学传感器平台上，采用电化学发光（ECL）和电化学阻抗（EIS）2种技术，协同研究DNA链中不同类型和不同位点的单碱基错配识别和稳定性差异。电极表面具有茎环构象的探针DNA与完全互补DNA、不同类型或不同位点单碱基错配DNA杂交前后的ECL和EIS信号强度变化有显著差异。信号强度变化可揭示单碱基错配识别的稳定性。结果表明，DNA链中心位点的C-A单碱基错配稳定性低于链两端的，靠近键合电极表面双链链端的C-A单碱基错配稳定性低于非键合电极表面双链链端的，同一中心位点C-X碱基对的稳定性顺序为C-G?C-T>C-A≥C-C。研究结果可为核酸多态性研究提供参考。     

A real-time,fluorescence-based assay for Rho-associated protein kinase activity

Inhibitors of Rho-associated protein kinase (ROCK) enzymatic activity have been shown to reduce the invasive phenotype observed in metastatic hepatocellular carcinoma (HCC). We describe the design, synthesis, and evaluation of a direct probe for ROCK activity utilizing a phosphorylation-sensitive sulfonamido-oxine fluorophore, termed Sox. The Sox fluorophore undergoes an increase in fluorescence upon phosphorylation of a proximal amino acid via chelation-enhanced fluorescence (CHEF, ex. = 360 nm and em. = 485 nm), allowing for the direct visualization of the rate of phosphate addition to a peptide substrate over time. Our optimal probe design, ROCK-S1, is capable of sensitively reporting ROCK activity with a limit of detection of 10 pM and a high degree of reproducibility (Z’-factor = 0.6 at 100 pM ROCK2). As a proof-of-principle for high-throughput screening (HTS) we demonstrate the ability to rapidly assess the efficacy of a 78 member, small molecule library against ROCK2 using a robotics platform. We identify two previously unreported ROCK2 inhibitor scaffolds, PHA665752 and IKK16, with IC₅₀ values of 3.6 μM and 247 nM respectively. Lastly, we define conditions for selectively monitoring ROCK activity in the presence of potential off-target enzymes (PKCα, PKA, and PAK) with similar substrate specificities.     

A Biosilification Fusion Protein for a ‘Self‐immobilising’ Sarcosine Oxidase Amperometric Enzyme Biosensor

Monomeric sarcosine oxidase (mSOx) fusion with the silaffin peptide, R5, designed previously for easy protein production in low resource areas, was used in a biosilification process to form an enzyme layer electrode biosensor. mSOx is a low activity enzyme (10–20 U/mg) requiring high amounts of enzyme to obtain an amperometric biosensor signal, in the clinically useful range <1 mM sarcosine, especially since the K_m is >10 mM. An amperometric biosensor model was fitted to experimental data to investigate dynamic range. mSOx constructs were designed with 6H (6×histidine) and R5 (silaffin) peptide tags and compared with native mSOx. Glutaraldehyde (GA) cross‐linked proteins retained ～5 % activity for mSOx and mSOx‐6H and only 0.5 % for mSOx‐R5. In contrast R5 catalysed biosilification on (3‐mercaptopropyl) trimethoxysilane (MPTMS) and tetramethyl orthosilicate (TMOS) particles created a ‘self‐immobilisation’ matrix retaining 40 % and 76 % activity respectively. The TMOS matrix produced a thick layer (>500 μm) on a glassy carbon electrode with a mediated current due to sarcosine in the clinical range for sarcosinemia (0–1 mM). The mSOx‐R5 fusion protein was also used to catalyse biosilification in the presence of creatinase and creatininase, entrapping all three enzymes. A mediated GC enzyme linked current was obtained with dynamic range available for creatinine determination of 0.1–2 mM for an enzyme layer ～800 nm.     

Catalytic amplification based on hole-transporting materials as efficient metal-free electrocatalysts for non-enzymatic glucose sensing

Hole-transporting materials with tunable structures and properties are mainly applied in organic light-emitting diodes as transport layer. But their catalytic properties as signal amplifiers in biological assays are seldom reported. In this paper, a starburst molecule, 4,4,4″-tri(N-carbazolyl)-triphenylamine (TCT), containing a triphenylamine as the central core and three carbazoles as the peripheral functional groups was designed and synthesized. Subsequently, the hole-transporting material based on the TCT polymer, poly(TCT) (PTCT), was achieved via a low-cost electrochemical method and exploited as an efficient metal-free electrocatalyst for non-enzymatic glucose detection. Here, this hole-transporting material served three purposes: electrochemical recognition (owing to hydrogen bonding interaction and the biomimetic microenvironment created by the polymer), electrocatalysis (owing to the hole-transporting capability of triphenylamine and the catalytic property of carbazole), and signal amplification (owing to energy migration along the conductive polymer backbone). The electrocatalytic and sensing performances of the sensor based on PTCT were evaluated in detail. Results revealed that the PTCT film could efficiently catalyze the oxidation of glucose at a less-positive potential (+0.20 V) in the absence of any enzymes. The response to glucose was linear in the concentration range of 1.0–6000 μM, and the detection limit was 0.20 μM. With good stability and selectivity, the proposed sensor could be feasibly applied to detect glucose in practical samples. The encouraging sensing performances suggest that the hole-transporting material is one of the promising biomimetic catalysts for electrocatalysis and relevant fields.     

Feasibility of application of conductometric biosensor based on acetylcholinesterase for the inhibitory analysis of toxic compounds of different nature

This study was aimed at the development of a conductometric biosensor based on acetylcholinesterase considering the feasibility of its application for the inhibitory analysis of various toxicants. In this paper, the optimum conditions for enzyme immobilization on the transducer surface are selected as well as the optimum concentration of substrate for inhibitory analysis. Sensitivity of the developed biosensor to different classes of toxic compounds (organophosphorus pesticides, heavy metal ions, surfactants, aflatoxin, glycoalkaloids) was tested. It is shown that the developed biosensor can be successfully used for the analysis of pesticides and mycotoxins, as well as for determination of total toxicity of the samples. A new method of biosensor analysis of toxic substances of different classes in complex multicomponent aqueous samples is proposed.     

Trends in the detection of pharmaceuticals and endocrine-disrupting compounds by Field-Effect Transistors (FETs)

Field-effect transistors (FETs) are one of the most widely-used electronic sensors for continuous monitoring and detection of contaminants such as pharmaceuticals and endocrine-disrupting compounds at low concentrations. FETs have been successfully utilized for the rapid analysis of these environmental pollutants due to their advantageous material properties like the disposability, rapid responses and simplicity. This paper presented an up-to-date overview of applied strategies with different bio-based materials in order to enhance the analytical performances of the designed sensors. Comparison and discussion were made between characteristics of recently engineered FET bio-sensors used for the detection of famous and selected pharmaceutical compounds in the literature. The recent progress in environmental research applications, comments on interesting trends, current challenge for future research in endocrine-disrupting chemicals’ (EDCs) detection using FETs biosensors were highlighted.     

Different PES nanofibrous membrane parameters effect on the efficacy of immunoassay performance

The contribution of electrospun nanofibrous membranes (e.NFMs) in the biosensing platforms opens up a new prospect for the invention of faster and more sensitive analytical devices. In this paper, we utilized e.NFM of polyethersulfone (PES) as a solid substrate for the protein immobilization through two different approaches: physical and covalent. Scanning electron microscopy (SEM) and Fourier‐transform‐infrared (FTIR) tests were performed to study the effect of plasma treatment on protein immobilization efficacy. Moreover, taking advantage of ELISA technique, the influence of different parameters, namely, nanofibers diameter, membrane thickness, plasma treatment time, an incubation time of ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide/N‐hydroxysuccinimide (EDC/NHS), and their ratio on antibody immobilization efficacy through two mentioned approaches, was also assessed.     

Electrochemical Signal Enhancer Fabricated Using Lysine‐rich Peptide for Ultrasensitive Electrochemical DNA Biosensor Analysis

Here we present a novel design of electrochemical signal enhancer to increase the detection sensitivity of electrochemical DNA biosensors. The key element of this enhancer is a lysine‐rich peptide (LRP). Its C‐terminal is conjugated with a planer molecule, being able to intercalate into the base pairs of probe‐target duplexes. The lysine residues of LRP are covalently linked with electrochemical signal indicators, acting as an assembly of electrochemical signal indicators. Experimental results proved the feasibility of the novel design. We have examined the effects of the numbers of lysine residues and the hybridization conditions on the detection sensitivity. The optimization procedures have led to significant sensitivity enhancement, and the LOD (limit of detection) has been determined to be 1.4 amol. This enhancer demonstrates advantages of easy operation, simple instrumentation, and high exemption from environmental influence.     

基于中空多孔金纳米/石墨烯复合纳米材料的葡萄糖氧化酶直接电化学及其生物传感研究

采用溶液相牺牲模板法制备中空多孔金纳米粒子（HPAuNPs）,并将该材料与还原氧化石墨烯（rGO）复合,用于葡萄糖氧化酶（GOx）在玻碳电极（GCE）表面的有效固定,构建GOx/HPAuNPs/rGO/GCE传感界面。利用扫描和透射电镜、X射线光电子能谱、X射线衍射谱、红外光谱及电化学等方法对材料的形貌与结构,GOx的固定化过程,以及传感器的直接电化学和电催化性能进行表征。结果表明,HPAuNPs和rGO的协同作用能有效促进GOx与电极之间的直接电子转移（DET）。基于GOx/HPAuNPs/rGO/GCE对葡萄糖的良好电催化性能,该方法有效实现了对葡萄糖的高灵敏度检测,其电流响应的线性范围为0.05～7.0 mmol/L,检出限（S/N=3）为16μmol/L。该传感器具有良好的选择性、重现性及稳定性,对实际样品血清中血糖的测定结果令人满意,回收率为98.0%～103%,相对标准偏差不大于5.0%。     

A Practical Approach for the Detection of Protein Tau with a Portable Potentiostat

Along with many factors, the change in protein tau isoforms, which has an obvious role in the function of microtubules, is an important biomarker of Alzheimer's disease. The aim of this study is to determine the protein Tau-441 with a portable potentiostat using a practical approach. For this purpose, screen printed electrodes (SPCEs) were first hydroxylated and then functional self-assembled monolayers were formed on the surface with 3-aminopropyltriethoxysilane (APTES). Evidence of anti-Tau being immobilized on to the surface was followed by techniques such as cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and Fourier transform infrared spectroscopy (FTIR). The constructed immunosensor showed a linear response within the concentration range of 0.0064–0.8 ng/mL for the target analyte Tau-441 and the limit of detection was found to be 0.0053 ng/mL. In addition, analytical behaviors such as reproducible measurements and storage life of the developed immunosensor with a portable potentiostat were also investigated. It has been demonstrated that Tau-441 can be captured with the help of portable device with sensitivity in CSF environment.