Development of a fluorogenic sensor for activated Cdc42

Cdc42, a member of the Rho GTPase family, is a fundamental regulator of the actin cytoskeleton during cell migration. To generate a sensor for Cdc42 activation, we employed a multi-pronged approach, utilizing cysteine labeling and expressed protein ligation, to incorporate the environment sensitive fluorophore 4-N,N-dimethylamino-1,8-naphthalimide (4-DMN) into the GTPase binding domain of the WASP protein. These constructs bind only the active, GTP-bound conformation of Cdc42 to produce a fluorescence signal. Studies with a panel of five sensor analogs revealed a derivative that exhibits a 32-fold increase in fluorescence intensity in the presence of activated Cdc42 compared to incubation with the inactive GDP-bound form of the protein. We demonstrate that this sensor can be exploited to monitor Cdc42 nucleotide exchange and GTPase activity in a continuous, fluorescence assay.     

Specific detection of cysteine and homocysteine in biological fluids by tuning the pH values of fluorosurfactant-stabilized gold colloidal solution

This study describes the use of 14 nm nonionic fluorosurfactant-capped gold nanoparticles (FSN-capped AuNPs) for the simultaneous detection of cysteine (Cys) and homocysteine (Hcy) using colorimetric method, requiring no use of separation techniques. It was found that the kinetics of Cys/Hcy-induced aggregation of the 14 nm FSN-capped AuNPs strongly depends on the pH value of gold colloidal solution. At a pH of 6.5, the Cys-induced aggregation kinetics of the FSN-capped AuNPs was almost identical to that induced by Hcy, facilitating simultaneous detection of total Cys and Hcy up to a concentration as low as 0.15 μM; while at pH 12.0, the kinetics of Cys-induced aggregation was much faster than that inducted by Hcy, leading to selective detection of Cys at concentration as low as 1.0 μM in the presence of Hcy. The applicability of the method was validated by spiking known amount of Cys and Hcy in human urine and plasma samples, obtaining a recovery of 95.4–105.5%. The present approach is simple, high selective and provides high reproducibility, and has a great potentiality in disease diagnosis.     

Interdigitated capacitive biosensor based on molecularly imprinted polymer for rapid detection of Hev b1 latex allergen

Allergen protein detection was performed by a surface imprinted layer combined with an interdigitated capacitance (IDC) transducer that allowed label-free measurements. The immobilized imprinted polymers are the probes that bind to rubber allergen proteins extracted from products such as rubber gloves. Copolymers made from methacrylic acid–vinylpyrrolidone–dihydroxyethylene-bisacrylamide (MAA–NVP–DHEBA) are soluble in aqueous solution and eliminate the denaturation of protein. When deposited as a coating onto an IDC microelectrode transduction system, such materials lead to sensors that produce capacitance responses that are clearly dependent on the concentration of the latex protein (10–900 ng ml⁻¹) in pH 7.4 buffer. The biosensor can detect Hev b1 within minutes and with a detection limit of 10 ng ml⁻¹. Different but related hevein allergenic proteins isolated from natural rubber latex from the rubber tree (Hev b1, Hev b2, and Hev b3) were distinguished by the imprinted material, depending on the dimension and conformation of these proteins with a selectivity factor of 4. They recognized Hevea latex proteins better than non-Hev b proteins, such as lysozyme, ovalbumin, and bovine serum albumin, by a factor of 2. Moreover, the sensor exhibited good operational stability of up to 180 days when used continuously at room temperature.     

Protein design for pathway engineering

Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds.     

荧光纳米生物传感器研究进展

荧光纳米生物传感器检测物质具有灵敏度高、响应迅速、抗干扰性强、无需参比电极等特点而被广泛地运用于生物传感技术领域。本文综述了荧光纳米生物传感器种类和特点,介绍了国内外近期在荧光纳米生物传感器及在生物检测方面的一些研究成果及进展,并作了分析比较。着重讨论了纳米粒子荧光生物传感器和光纤纳米荧光生物传感器的特性及其在生物分析中的应用。     

Nanoscopic modeling of a carbon nanotube force-measuring biosensor

In this paper we perform a theoretical study of a potential design of a carbon nanotube device able to transduce forces developed at the scale of basic cellular processes into electric current variations. The first part of this study consists of an assessment of the sensitivity of the device with forces in the tens of pico Newtons (pN), developed typically at the cellular scale. In the second stage, we focus on the transduction of the deflection of a cantilever into an electrical signal, employing methods borrowed from non-equilibrium Green's functions. Several issues related to the importance of thermal effects in the proper operation of the sensor are then discussed. Following a simple method we include non-zero temperature through molecular dynamics in quantum conductance calculations that results in the displacement-current characteristic found at the end of this paper.     

刺激响应型DNA水凝胶的性质及其应用 *

DNA水凝胶作为一种生物合成分子,既具有DNA分子的特异性,生物可降解性和分子识别等特性,又具有水凝胶的高亲水性等特征.刺激响应型DNA水凝胶主要是在环境因素的刺激下,利用常规DNA序列经Watson-Crick碱基互补配对形成的DNA分支结构或多种功能核酸的特殊DNA序列形成的i-motif结构;T-A·T三螺旋结构,C-G·C ⁺三螺旋结构及G-四链体结构等对环境的响应行为使水凝胶形成及应用.近年来,刺激响应型DNA水凝胶因其在温度,pH,光,金属离子,生物分子等单刺激因素,以及光热,金属离子,有机物,温度与pH等多刺激因素下的独特应答性质,在生物传感,生物成像,药物递送,生物材料等方面得到了广泛的应用.综述了刺激响应型DNA水凝胶的形成方法,分类及其核酸来源,形成后的表征手段以及在环境刺激下的响应行为与应用,概括了目前刺激响应型DNA水凝胶的研究热点,并就其未来发展趋势做出了预测.     

Direct Tests of Enzymatic Heme Degradation by the Malaria Parasite Plasmodium falciparum

Malaria parasites generate vast quantities of heme during blood stage infection via hemoglobin digestion and limited de novo biosynthesis, but it remains unclear if parasites metabolize heme for utilization or disposal. Recent in vitro experiments with a heme oxygenase (HO)-like protein from Plasmodium falciparum suggested that parasites may enzymatically degrade some heme to the canonical HO product, biliverdin (BV), or its downstream metabolite, bilirubin (BR). To directly test for BV and BR production by P. falciparum parasites, we DMSO-extracted equal numbers of infected and uninfected erythrocytes and developed a sensitive LC-MS/MS assay to quantify these tetrapyrroles. We found comparable low levels of BV and BR in both samples, suggesting the absence of HO activity in parasites. We further tested live parasites by targeted expression of a fluorescent BV-binding protein within the parasite cytosol, mitochondrion, and plant-like plastid. This probe could detect exogenously added BV but gave no signal indicative of endogenous BV production within parasites. Finally, we recombinantly expressed and tested the proposed heme degrading activity of the HO-like protein, PfHO. Although PfHO bound heme and protoporphyrin IX with modest affinity, it did not catalyze heme degradation in vivo within bacteria or in vitro in UV absorbance and HPLC assays. These observations are consistent with PfHO''s lack of a heme-coordinating His residue and suggest an alternative function within parasites. We conclude that P. falciparum parasites lack a canonical HO pathway for heme degradation and thus rely fully on alternative mechanisms for heme detoxification and iron acquisition during blood stage infection.     

FluRox based biosensors

A biosensor is an integrated device of biomaterials and electronic components which detects physiological change or physico-chemical response. The efforts towards the development of a supersensitive FluoRox biosensor are discussed in this paper. FluoRox principle is based on the novel concept of monitoring redox events in vitro and in vivo by fluorescence detection based on forster resonance energy transfer (FRET). Unlike conventional electrochemical biosensors fluorescence based sensors has the advantage of higher sensitivity which under suitable conditions can detect single molecules. Thus a highly sensitive and a miniaturized device is aimed at, which will enable the detection of trace amounts of pollutants and the detection of diseases at an early stage. Think of a biosensor and youwould conjure up with the question of sensitivity. Nusrat Sanghamitra, Fellow of EdRox network explains how a simple but yet novel concept of detecting redox reaction induced fluorescence change shoots up the sensitivity.     

A FRET-based biosensor for NO detection

In this paper we explore the use of fluorescently labeled cytochrome c peroxidase (CcP) from baker's yeast for monitoring nitric oxide (NO) down to the sub-micromolar level, by means of a FRET (Förster Resonance Energy Transfer) mechanism. The binding affinity constant (K_d) for the NO binding to CcP was determined to be 10 ± 1.5 µM. The rate of NO dissociation from the CcP (k_off) and the second order rate constant for the NO association (k_on) were found to be 0.22 ± 0.08 min^− 1 and 0.024 ± 0.002 µM^− 1 min^− 1 respectively. The immobilization of fluorescently labeled CcP into a polymeric matrix for use in a solid state NO sensing device was also explored. The results provide proof-of-principle that labeled CcP can be successfully implemented in a fast, simple, quantitative and sensitive NO sensing device.