Re-engineering redox-sensitive green fluorescent protein for improved response rate

Redox-sensitive variants of the green fluorescent protein (roGFPs) had previously been developed that allow "real-time" monitoring of the redox status of cellular compartments by fluorescence excitation ratiometry. However, the response time of these probes limits the study of certain rapid oxidative events, such as H2O2 bursts in cell signaling. The substitution of up to three positively charged amino acids adjacent to the introduced disulfide in roGFP1 (variants designated roGFP1-R1 through -R14) substantially improved the response rate. The pseudo first-order rate constants for oxidation by H2O2 and reduction by DTT and redox midpoint potentials were determined. The rate constants approximately doubled with each additional positively charged substitution, to nearly an order of magnitude total. The midpoint potentials are highly correlated with the rate increases, becoming more oxidizing with increasing numbers of positive substitutions. Crystal structures of two variants with opposite disulfide oxidation states have been determined: a 2.2 A resolution structure of oxidized "R7" containing two basic substitutions, and a 1.95 A resolution structure of reduced "R8" with one basic and one acidic substitution. Nonlinear Poisson-Boltzmann (PB) calculations are shown to accurately predict the effects of the substitutions on the rate constants. The effects of the substitutions on dimer formation, relative oxidative midpoint potentials, and oxidation and reduction rates are discussed. roGFPs are demonstrated to constitute an excellent model system for quantitative analysis of factors influencing thiol transfer reactions. roGFP1-R12 is most suitable for use in live cells, due to significantly increased reaction rate and increased pI.     

Quantification of DNA and protein adsorption by optical phase shift

A primary advantage of label-free detection methods over fluorescent measurements is its quantitative detection capability, since an absolute measure of adsorbed material facilitates kinetic characterization of biomolecular interactions. Interferometric techniques relate the optical phase to biomolecular layer density on the surface, but the conversion factor has not previously been accurately determined. We present a calibration method for phase shift measurements and apply it to surface-bound bovine serum albumin, immunoglobulin G, and single-stranded DNA.Biomolecules with known concentrations dissolved in salt-free water were spotted with precise volumes on the array surface and upon evaporation of the water, left a readily calculated mass. Using our label-free technique, the calculated mass of the biolayer was compared with the measured thickness, and we observed a linear dependence over 4 orders of magnitude. We determined that the widely accepted conversion of 1 nm of thickness corresponds to 1 ng/mm² surface density held reasonably well for these substances and through our experiments can now be further specified for different types of biomolecules. Through accurate calibration of the dependence of thickness on surface density, we have established a relation allowing precise determination of the absolute number of molecules for single-stranded DNA and two different proteins.     

植物组织切片谷氨酸传感器的研究

本文应用南瓜中果皮组织切片与BSA-戊二醛交联,然后与二氧化碳电极偶合组成了南瓜组织切片谷氨酸传感器.对电极的动力学响应特性,适宜的缓冲介质,不同pH及温度对电极响应的影响;电极的选择性及使用寿命作了研究。该电极在5.0×10~(-4)～1.0×10~(-2)mol/L谷氨酸浓度范围内,其动力学响应与谷氨酸浓度呈线性关系,相关系数为0.9586。计算得其Km值为7.13×10~(-3)mol/L,检测下限为3.0×10~(-4)mol/L,斜率为43mv/dec。该电极有良好的选择性。使用寿命可达一周以上。     

生物传感器法测定麦芽汁中赖氨酸含量的研究

本研究利用固定化赖氨酸氧化酶和生物传感器,建立了麦芽汁中赖氨酸含量的测定方法。研究了该方法的线性范围、检出限、最适pH值范围、稳定性及专一性。以大生产麦芽汁为样品,与其它检测方法进行了比较。结果表明,生物传感器法测定赖氨酸含量操作简单而准确,加标回收率为97．41％～103．23％,RSD为2．23％;线性范围为2～100 mg/100 mL,检出限为2 mg/100 mL;稳定性及专一性强,不受其他氨基酸的干扰,能够用于氨基酸种类复杂的麦汁赖氨酸的检测。本方法与氨基酸分析仪、DBL、HPLC等方法相比较,测定结果无显著差异（P＞0．05）。     

CRISPR-Cas生物传感器研究进展

成簇规律间隔短回文序列(clustered regularly interspaced short palindromic repeats,CRISPR)系统是广泛存在于细菌中的一种特有的免疫防御机制,与特殊的Cas蛋白结合后能够有效的对外源的核酸分子进行特异性片段化,并进一步促进其降解。CRISPR-Cas系统具有独特的靶向性,为开发针对于核酸为底物的生物传感器提供了新的概念。越来越多的研究人员根据不同Cas蛋白的性质,建立了独特的逻辑系统对靶标物质进行准确识别,基于CRISPR技术的生物传感器也开拓了该技术在基因编辑以外领域的应用。介绍了CRISPR-Cas系统的起源、作用机制和科学分类,根据生物传感器的作用方式以及识别底物进行了分类,并对基于CRISPR-Cas系统的高效生物传感器的应用前景进行了展望。     

Fiber optic monitoring of carbamate pesticides using porous glass with covalently bound chlorophenol red

An optical fiber biosensor for the determination of the pesticides propoxur (Baygon®) and carbaryl, two of the most commonly used carbamate insecticides in vegetable crops, is described. A pH indicator, chlorophenol red, is used as optical transducer of the inhibition of the enzyme acetylcholinesterase by the analytes. The biorecognition element is covalently immobilized onto controlled pore glass beads (CPG) and packed in a thermostatized bioreactor connected to a flow-through cell that contains CPG-immobilized chlorophenol red placed at the common end of a bifurcated fiber optic bundle. In the presence of a constant acetylcholine concentration, the colour of the pH sensitive layer changes and the measured reflectance signal can be related to the carbamate concentration in the sample solution. The performance of the biosensor has been optimized using a flow injection system. The linear dynamic range for the determination of carbaryl and propoxur spans from 0.8 to 3.0 mg l⁻¹ and from 0.03 to 0.50 mg l⁻¹, respectively. The detection limit (3 s) of the biosensor for propoxur (0.4 ng) is lower than that measured for carbaryl (25 ng). Reproducibility, stability and interference studies of the optical device are reported. The biosensor has been applied to the determination of propoxur in spiked vegetables (onion and lettuce) using ultrasound extraction, achieving recovery values between 93 and 95% for onion samples at the different concentration levels assayed.     

葡萄糖传感器在微囊化动物细胞灌注培养过程中的应用

本文提出了用葡萄糖传感器监测在灌注培养微囊化动物细胞时,培液中葡萄糖量的变化。实验过程中,葡萄糖传感器的线性检测范围为１０－５００ｍｇ／１０ｍｌ．将葡萄糖氧化酶膜贮存于４℃条件下的０．０５ｍｏｌ／Ｌ ＰＢＳ（ｐＨ７．０）中,它的活力在４０天之内保持不变,本方法操作简便,灵敏度高,耗时少,准确可靠。     

Biosensor technology: technology push versus market pull

Biosensor technology is based on a specific biological recognition element in combination with a transducer for signal processing. Since its inception, biosensors have been expected to play a significant analytical role in medicine, agriculture, food safety, homeland security, environmental and industrial monitoring. However, the commercialization of biosensor technology has significantly lagged behind the research output as reflected by a plethora of publications and patenting activities. The rationale behind the slow and limited technology transfer could be attributed to cost considerations and some key technical barriers. Analytical chemistry has changed considerably, driven by automation, miniaturization, and system integration with high throughput for multiple tasks. Such requirements pose a great challenge in biosensor technology which is often designed to detect one single or a few target analytes. Successful biosensors must be versatile to support interchangeable biorecognition elements, and in addition miniaturization must be feasible to allow automation for parallel sensing with ease of operation at a competitive cost. A significant upfront investment in research and development is a prerequisite in the commercialization of biosensors. The progress in such endeavors is incremental with limited success, thus, the market entry for a new venture is very difficult unless a niche product can be developed with a considerable market volume.     

压电石英晶体生物传感器及其应用

压电石英晶体生物传感器是利用压电石英晶体振荡频率对晶体表面质量负载和表面性状如密度、粘度、电导、介电常数等的高度敏感性与生物识别分子的高度特异性相结合发展起来的一种新型传感器。它具有灵敏度高、特异性好;操作简单,不需任何标记;检测速度快,成本低廉等特点,有望成为临床实验诊断大规模应用的方法。仪器体积小,重量轻,能实时检测,是获得在线信息强有力的手段,特别适于环境监测、食品卫生监督、工业生产实时监测等野外流动作业和在线检测。本就压电石英晶体生物传感器的原理、基本结构、特点及其应用等方面进行综述。     

Amperometric glucose biosensors based on layer-by-layer assembly of chitosan and glucose oxidase on the Prussian blue-modified gold electrode

A glucose biosensor based on layer-by-layer (LBL) self-assembling of chitosan and glucose oxidase (GOD) on a Prussian blue film was developed. First, Prussian blue was deposited on a cleaned gold electrode then chitosan and GOD were assembled alternately to construct a multilayer film. The resulting amperometric glucose biosensor exhibited a fast response time (within 10 s) and a linear calibration range from 6 μM to 1.6 mM with a detection limit of 3.1 μM glucose (s/n = 3). With the low operating potential, the biosensor showed little interference to the possible interferents, including ascorbic acid, acetaminophen and uric acid, indicating an excellent selectivity.