Electrochemical detection of protein-protein interactions using a yeast two hybrid: 17-beta-estradiol as a model

In this work we present a modified yeast two-hybrid bioassay for the highly sensitive detection of protein-protein interactions, based on the electrochemical monitoring of beta-D-galactosidase reporter gene activity, using p-aminophenyl-beta-D-galactopyranoside (PAPG) as a synthetic substrate. In a model system, the sensitive detection of 17-beta-estradiol was achieved at concentrations as low as 10(-11)M (approx 2 pg/ml) by monitoring 17-beta-estradiol receptor dimerization after exposure to 17-beta-estradiol. The sensitivity of this system was higher than that of standard optical methods by three orders of magnitude.     

Simultaneous intra- and extracellular superoxide monitoring using an integrated optical and electrochemical sensor system

A new integrated optical and electrochemical sensor system for simultaneous monitoring of intra- and extracellular superoxide (O(2)(-)) was developed using an array-based cell chip. For in vitro assays, A172 human glioblastoma cells were transferred into the cell chip and stimulated by phorbol 12-myristate 13-acetate (PMA). Intracellular O(2)(-) generation was detected via fluorescence image analysis with a dye probe, dihydrorhodamine 123 (DHR 123). Extracellular O(2)(-) was detected using an amperometric sensor constructed by immobilisation of cytochrome c using a binder, 3,3'-dithiobis(sulphosuccinimidylpropionate), to attach the redox protein onto the surface of electrodeposited Au electrodes incorporated into the optically transparent cell chip. The simultaneous intra- and extracellular production of O(2)(-) was successfully observed from PMA-stimulated A172 cells and inhibited by superoxide dismutase (SOD). The quantification of O(2)(-) concentration based on a mathematical model study and possible applications using the sensor system developed were discussed. The results confirm that there was no detectable interference or crosstalk between the optical and electrochemical assays. Feasibility of the integration of the two methods, optical and electrochemical, and the neutralisation of the intra- and extracellular O(2)(-) levels by SOD have been demonstrated.     

Comparisons of optical pH and dissolved oxygen sensors with traditional electrochemical probes during mammalian cell culture

Small-scale upstream bioprocess development often occurs in flasks and multi-well plates. These culturing platforms are often not equipped to accurately monitor and control critical process parameters; thus they may not yield conditions representative of manufacturing. In response, we and others have developed optical sensors that enable small-scale process monitoring. Here we have compared two parameters critical to control in industrial cell culture, pH and dissolved oxygen (DO), measured with our optical sensors versus industrially accepted electrochemical probes. For both optical sensors, agreement with the corresponding electrochemical probe was excellent. The Pearson Correlations between the optical sensors and electrochemical probes were 98.7% and 99.7%, for DO and pH, respectively. Also, we have compared optical pH sensor performance in regular (320 mOsm/kg) and high-osmolality (450 mOsm/kg) cell culture media to simulate the increase in osmolality in pH-controlled cultures. Over a pH range of 6.38-7.98 the average difference in pH readings in the two media was 0.04 pH units. In summary, we have demonstrated that these optical sensors agree well with standard electrochemical probes. The accuracy of the optical probes demonstrates their ability to detect potential parameter drift that could have significant impact on growth, production kinetics, and protein product quality. We have also shown that an increase in osmolality that could result from controlling pH or operating the reactor in fed-batch mode has an insignificant impact on the functionality of the pH patches.     

Effectiveness of qPCR permutations, internal controls and dilution as means for minimizing the impact of inhibition while measuring Enterococcus in environmental waters

Aims: Draft criteria for the optional use of qPCR for recreational water quality monitoring have been published in the United States. One concern is that inhibition of the qPCR assay can lead to false‐negative results and potentially inadequate public health protection. We evaluate the effectiveness of strategies for minimizing the impact of inhibition. Methods and Results: Five qPCR method permutations for measuring Enterococcus were challenged with 133 potentially inhibitory fresh and marine water samples. Serial dilutions were conducted to assess Enterococcus target assay inhibition, to which inhibition identified using four internal controls (IC) was compared. The frequency and magnitude of inhibition varied considerably among qPCR methods, with the permutation using an environmental master mix performing substantially better. Fivefold dilution was also effective at reducing inhibition in most samples (>78%). ICs were variable and somewhat ineffective, with 54–85% agreement between ICs and serial dilution. Conclusions: The current IC methods appear to not accurately predict Enterococcus inhibition and should be used with caution; fivefold dilution and the use of reagents designed for environmental sample analysis (i.e. more robust qPCR chemistry) may be preferable. Significance and Impact of the Study: Suitable approaches for defining, detecting and reducing inhibition will improve implementation of qPCR for water monitoring.     

Carbohydrate analysis of bacterial polysaccharides by high-pH anion-exchange chromatography and online polarimetric determination of absolute configuration

A significant problem in structure determination of complex carbohydrates, especially for bacterial polysaccharides, is determination of the absolute configuration of the component monosaccharides. A number of analytical methods have been used for this purpose but, as a result of the wide variety of chemical properties of sugars found in complex polysaccharides, no single method is universally applicable. High-resolution gas chromatography of volatile derivatives with chiral reagents is the most widely used method. Optical activity, although direct and simple, lacks sensitivity generally requiring a large quantity of pure monosaccharide. We report a combination of high-performance anion-exchange chromatography (HPAEC) with combined electrochemical pulsed amperometric detection and in-line detection of optical rotation with an in-line laser polarimeter for analysis of a number of sugars found in complex polysaccharides. We show that application of the method for analysis of capsular polysaccharides of several gram-positive and gram-negative pathogenic bacteria provides useful information simultaneously on carbohydrate composition and the enantiomeric configuration of component sugars.     

Preliminary approach of real-time monitoring in vitro matrix mineralization based on surface plasmon resonance detection

Matrix mineralization is a terminal process in osteoblast differentiation, and several approaches have been introduced to characterize the process in tissues or cultured cells. However, an analytical technique that quantitates in vitro matrix mineralization of live cells without any labeling or complex treatments is still lacking. In this study, we investigate a simple and enhanced optical method based on surface plasmon resonance (SPR) detection that can monitor the surface-limited refractive index change in real-time. During monitoring MC3T3-E1 cells in vitro culture every 2 days for over 4 weeks, the SPR angle is shifted with a greater resonance change in cells cultured with osteogenic reagents than those without the reagents. In addition, the SPR results obtained have a close relevance with the tendency of conventional mineralization staining and an inductively coupled plasma-based calcium content measure. These results suggest a new approach of a real-time SPR monitoring in vitro matrix mineralization of cultured cells.     

Immunosensors: Antibody-based biosensors

The challenges in developing practical immunosensors lie in converting (without added reagents) the binding event into an electrical or optical signal, and in creating fully reversible systems capable of monitoring both increases and decreases in analyte concentration. Numerous approaches are under investigation, some of which should lead to commercial products within a couple of years; their strengths and limitations are reviewed.     

A regenerable flow-through affinity sensor for label-free detection of proteins and DNA

Label-free monitoring of biomolecular reactions in real-time is of great interest since it can provide valuable information about binding kinetics and equilibrium constants. In this report, a sensor based on White Light Reflectance Spectroscopy (WLRS) is presented that is capable of real-time monitoring of biomolecular reactions taking place on top of a polymer covered silicon dioxide reflective surface. The optical set-up consists of a visible–near infrared light source, a bifurcated optical fiber and a spectrometer. The outer part of the optical fiber guides the light vertically onto the surface where the biomolecular reactions occur, whereas the reflected light is driven from the central part of the fiber to the spectrometer. A microfluidic module in combination with a pump supplies the reagents at a constant rate. The biomolecular interactions are monitored as shifts of the wavelength of the interference minimum. The proposed methodology was applied for real-time and label-free monitoring mouse gamma-globulins binding onto immobilized anti-mouse IgG antibody. Mouse gamma-globulins at concentrations down to 150 pM were detected in reaction times of 1-min. Regeneration of immobilized antibody was accomplished up to seven times without loss of its activity. In addition, real-time monitoring of hybridization reaction between complementary oligonucleotides was accomplished. The proposed sensor provides a simple, fast, low cost approach for label-free monitoring of biomolecular interactions and therefore it should by suitable for a wide range of analytical applications.     

Advances in biomedical sensor technology: A review of the 1985 patent literature

The 1985 patent literature pertaining to biomedical sensor technology is reviewed and an assessment made of the types of devices that are most likely to reach commercialisation. Novel systems that are described comprise electrochemical, optical, thermal and other transducers for monitoring key components of clinical samples. These include blood gases and electrolytes, metabolites, enzymes, proteins, antibodies and antigens.     

光学透明技术在植物多尺度成像中的应用

光学透明技术是一种通过各种化学试剂,将原本不透明的生物样本实现透明化,并在光学显微镜下深度成像的技术。结合多种光学显微成像新技术,光学透明技术可对整个组织进行成像和三维重建,深度剖析生物体内部空间特征与形成机制。近年来,多种植物光学透明技术和多尺度成像技术被陆续研发,并取得了丰硕的研究成果。该文综述了生物体光学透明技术的基本原理和一些新技术,重点介绍基于光学透明技术开发的新型成像方法及其在植物成像与细胞生物学中的应用,为后续植物整体、组织或器官的透明、成像与三维重构及功能研究提供理论依据和技术支持。     

Biosensors

Biosensors are analytical devices that respond selectively to analytes in an appropriate sample and convert their concentration into an electrical signal via a combination of a biological recognition system and an electrochemical, optical or other transducer. Such devices will find application in medicine, agriculture, environmental monitoring and the bioprocessing industries. The last few years have seen great advances in the design of sensor architectures, the marriage of biological systems with monolithic silicon and optical technologies, the development of effective electron-transfer systems and the configuration of direct immunosensors. Recent progress in these areas has already led to the introduction of new-generation biosensors into the competitive diagnostics market place.     

Electrochemical optical waveguide lightmode spectroscopy (EC-OWLS): a pilot study using evanescent-field optical sensing under voltage control to monitor polycationic polymer adsorption onto indium tin oxide (ITO)-coated waveguide chips

A new technique has been developed that combines evanescent-field optical sensing with electrochemical control of surface adsorption processes. This new technique, termed "electrochemical optical waveguide lightmode spectroscopy" (EC-OWLS), proved efficient in monitoring molecular surface adsorption and layer thickness changes of an adsorbed polymer layer examined in situ as a function of potential applied to a waveguide in a pilot study. For optical sensing, a layer of indium tin oxide (ITO) served as both a high-refractive-index waveguide and a conductive electrode. In addition, an electrochemical flow-through fluid cell was provided, which incorporated working, reference, and counter electrodes, and was compatible with the constraints of optical sensing. Poly(L-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG) served as a model, polycation adsorbate. Adsorption of PLL-g-PEG from aqueous buffer solution increased from 125 to 475 ng/cm(2 )along a sigmoidal path as a function of increasing potential between 0 and 1.5 V versus the Ag reference electrode. Upon buffer rinse, adsorption was partially reversible when a potential of >/=0.93 V was maintained on the ITO waveguide. However, reducing the applied potential back to 0 V before rinsing resulted in irreversible polymer adsorption. PLL-g-PEG modified with biotin demonstrated similar adsorption characteristics, but subsequent streptavidin binding was independent of biotin concentration. Applying positive potentials resulted in increased adsorbed mass, presumably due to polymer chain extension and reorganization in the molecular adlayer.     

Immunosensors for detection of pesticide residues

Immunosensors are biosensors that use antibodies or antigens as the specific sensing element and provide concentration-dependent signals. There is great potential in the applications of immunosensing technologies for rapid detection of pesticide residues in food and environment. This paper presents an overview of various transduction systems, such as electrochemical, optical, piezoelectric, and nanomechanics methods, which have been reported in the literature in the design and fabrication of immunosensors for pesticide detection. Various immobilization protocols used for formation of a biorecognition interface are also discussed. In addition, techniques of regeneration, signal amplification, miniaturization, and antibodies are evaluated for the development and applications of these immunosensors. It can be concluded that despite some limitations of the immunosensing technologies, these immuosensors for pesticide monitoring are becoming more and more relevant in environmental and food analysis.     

基于微流控芯片的细胞-细菌相互作用光电检测技术

细胞/细菌及其相互作用研究对于生命科学、药物研发、医学诊疗等领域的研究具有重要意义。微流控芯片分析技术因微环境可控、生物相容性好、检测并行性、微型化等特性，正发展成为细胞/细菌及其相互作用研究的高效手段。本文在简要介绍基于微流控芯片分析技术的细胞-细菌分析方法和技术基础之上，对微流控芯片上细胞-细菌相互作用模型的建立进行了讨论，重点针对细胞-细菌及其相互作用过程的芯片检测进行了综述，尤其对芯片集成的光电检测技术及其测试效果进行总结和比较。通过芯片集成微流体控制、多种光电传感监测模块，使微流控芯片分析技术成为细胞/细菌及其相互作用过程分析和检测的支撑平台和优势手段。最后，对微流控光电检测技术在细胞-细菌相互作用检测中面临的挑战及发展趋势进行了讨论和展望。     

Doppler optical coherence tomography for measuring flow in engineered tissue

The engineering of human tissue represents a major paradigm shift in clinical medicine. Early embodiments of tissue engineering are currently being taken forward to the clinic by production methods that are essentially extensions of laboratory manual procedures. However, to achieve the status of routine large-scale clinical practice, automation and scale-out processes are required. This in turn will require the development of reliable on-line monitoring and control systems. This paper examines one demand of crucial importance, namely the real time in vitro monitoring of the flow characteristics through growing tissue since this has a complex interrelationship. Doppler optical coherence tomography (DOCT) is a recently developed imaging technique for studying the rheological properties of tissues in vivo. Capable of non-invasive imaging in real time with high resolution, it is potentially ideal for the continuous monitoring of engineered tissues in vitro. As a base line, the current status of DOCT in vivo is therefore reviewed. This paper also reports the first preliminary use of DOCT in tissue engineering. The application described involves the imaging of a fully developed laminar flow through a combined tissue fabrication/bioreactor with a tissue-engineered construct (substitute blood vessel) in situ.     

Recent development of advanced biotechnology for wastewater treatment

Abstract

The importance of highly efficient wastewater treatment is evident from aggravated water crises. With the development of green technology, wastewater treatment is required in an eco-friendly manner. Biotechnology is a promising solution to address this problem, including treatment and monitoring processes. The main directions and differences in biotreatment process are related to the surrounding environmental conditions, biological processes, and the type of microorganisms. It is significant to find suitable biotreatment methods to meet the specific requirements for practical situations. In this review, we first provide a comprehensive overview of optimized biotreatment processes for treating wastewater during different conditions. Both the advantages and disadvantages of these biotechnologies are discussed at length, along with their application scope. Then, we elaborated on recent developments of advanced biosensors (i.e. optical, electrochemical, and other biosensors) for monitoring processes. Finally, we discuss the limitations and perspectives of biological methods and biosensors applied in wastewater treatment. Overall, this review aims to project a rapid developmental path showing a broad vision of recent biotechnologies, applications, challenges, and opportunities for scholars in biotechnological fields for “green” wastewater treatment.     

A method of processing tissue sections for staining with cu-promeronic blue and other dyes, using CEC techniques, for light and electron microscopy

The application to connective tissues etc. of new histochemical and ultrastructural methods involving consecutive treatment with dye-salt baths, specific enzymes, antibodies and decalcifying fluids, is best carried out on sections on glass slides. This ensures uniform access of reagents to substrates and allows the accurate monitoring of the results, as well as exploiting the strengths of the newer reagents (e.g. Cupromeronic Blue) in that they are highly coloured as well as electron dense. However, very high losses of connective tissue sections usually occur in the various solutions, to the extent that many important investigations are rendered almost impossible because of the high cost of the reagents. We describe a technique which we use routinely, that gives very high recovery of sections of "difficult" tissues from multi-stage enzyme and stain treatments, for light microscopy followed by plastic embedding of the coloured sections for electron microscopy.     

Recent trends in antibody based sensors

This review details recent advances in the fields of immunosensors and closely related immunoassays in the past decade, together with a discussion of possible future trends. Immunosensors can be classified by the way in which they transduce the signal produced upon the formation of an antibody antigen complex. Recent advancements to these methods of detection and transduction are discussed in detail, with particular focus on electrochemical, optical, piezoelectric and magnetic based sensors. The varying applications of these sensors are also discussed. Some of the most significant advances include development of immunosensors for the continuous monitoring of analytes, point of care (PoC) devices, with lower unit costs, automation, reusability and ease of use. Immunosensor technology has advanced at a prolific rate since its conception and has grown into a diverse area of ongoing research.     

聚吡咯的生长及与神经组织相容性的研究

通过化学氧化和电化学氧化方法得到聚吡咯,用所得到的聚吡咯与神经组织联合培养,通过倒置光学显微镜和扫描电子显微镜观察其生物相容特性.得知聚吡咯与神经组织有较好的生物相容性.     

Current methods for detecting ethylene in plants

Background

In view of ethylene''s critical developmental and physiological roles the gaseous hormone remains an active research topic for plant biologists. Progress has been made to understand the ethylene biosynthesis pathway and the mechanisms of perception and action. Still numerous questions need to be answered and findings to be validated. Monitoring gas production will very often complete the picture of any ethylene research topic. Therefore the search for suitable ethylene measuring methods for various plant samples either in the field, greenhouses, laboratories or storage facilities is strongly motivated.

Scope

This review presents an update of the current methods for ethylene monitoring in plants. It focuses on the three most-used methods – gas chromatography detection, electrochemical sensing and optical detection – and compares them in terms of sensitivity, selectivity, time response and price. Guidelines are provided for proper selection and application of the described sensor methodologies and some specific applications are illustrated of laser-based detector for monitoring ethylene given off by Arabidopsis thaliana upon various nutritional treatments.

Conclusions

Each method has its advantages and limitations. The choice for the suitable ethylene sensor needs careful consideration and is driven by the requirements for a specific application.Key words: Ethylene, Arabidopsis thaliana, gas sampling, gas chromatography, electrochemical sensing, laser-based detector