Real‐Time Monitoring of Mass‐Transport‐Related Enzymatic Reaction Kinetics in a Nanochannel‐Array Reactor

To understand the fundamentals of enzymatic reactions confined in micro‐/nanosystems, the construction of a small enzyme reactor coupled with an integrated real‐time detection system for monitoring the kinetic information is a significant challenge. Nano‐enzyme array reactors were fabricated by covalently linking enzymes to the inner channels of a porous anodic alumina (PAA) membrane. The mechanical stability of this nanodevice enables us to integrate an electrochemical detector for the real‐time monitoring of the formation of the enzyme reaction product by sputtering a thin Pt film on one side of the PAA membrane. Because the enzymatic reaction is confined in a limited nanospace, the mass transport of the substrate would influence the reaction kinetics considerably. Therefore, the oxidation of glucose by dissolved oxygen catalyzed by immobilized glucose oxidase was used as a model to investigate the mass‐transport‐related enzymatic reaction kinetics in confined nanospaces. The activity and stability of the enzyme immobilized in the nanochannels was enhanced. In this nano‐enzyme reactor, the enzymatic reaction was controlled by mass transport if the flux was low. With an increase in the flux (e.g., >50 μL min^?1), the enzymatic reaction kinetics became the rate‐determining step. This change resulted in the decrease in the conversion efficiency of the nano‐enzyme reactor and the apparent Michaelis–Menten constant with an increase in substrate flux. This nanodevice integrated with an electrochemical detector could help to understand the fundamentals of enzymatic reactions confined in nanospaces and provide a platform for the design of highly efficient enzyme reactors. In addition, we believe that such nanodevices will find widespread applications in biosensing, drug screening, and biochemical synthesis.     

Beyond labels: A review of the application of quantum dots as integrated components of assays, bioprobes, and biosensors utilizing optical transduction

A comprehensive review of the development of assays, bioprobes, and biosensors using quantum dots (QDs) as integrated components is presented. In contrast to a QD that is selectively introduced as a label, an integrated QD is one that is present in a system throughout a bioanalysis, and simultaneously has a role in transduction and as a scaffold for biorecognition. Through a diverse array of coatings and bioconjugation strategies, it is possible to use QDs as a scaffold for biorecognition events. The modulation of QD luminescence provides the opportunity for the transduction of these events via fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), charge transfer quenching, and electrochemiluminescence (ECL). An overview of the basic concepts and principles underlying the use of QDs with each of these transduction methods is provided, along with many examples of their application in biological sensing. The latter include: the detection of small molecules using enzyme-linked methods, or using aptamers as affinity probes; the detection of proteins via immunoassays or aptamers; nucleic acid hybridization assays; and assays for protease or nuclease activity. Strategies for multiplexed detection are highlighted among these examples. Although the majority of developments to date have been in vitro, QD-based methods for ex vivo biological sensing are emerging. Some special attention is given to the development of solid-phase assays, which offer certain advantages over their solution-phase counterparts.     

血糖近红外光谱分析的Savitzky-Golay平滑模式与偏最小二乘法因子数的联合优选

利用偏最小二乘法(PLS)和光谱Savitzky-Golay(SG)平滑方法,建立血清葡萄糖近红外光谱分析的优化模型。基于最优单波数模型的预测效果,提出划分校正集和验证集的一种新方法。采用10000～5300cm-1和4920～4160cm-1的组合波段,光谱经过SG平滑处理,利用PLS方法建立定标预测模型。将平滑点数扩充为5,7,…,87(奇数),多项式次数扩充为n=2,3,4,5,6,得到包含582个平滑模式的14个平滑系数表。对所有平滑模式和PLS因子数(1～40)分别建立PLS模型。按照预测效果进行优选,得到最优SG平滑模式为1阶导数平滑,3、4次多项式类型,SG平滑点数为53,最优PLS因子数为7,最优RMSEP达到0.376mmol/L。所采用的划分校正集和验证集的方法、SG平滑模式的扩充、SG平滑模式和PLS因子数的联合大范围筛选能够有效地应用于近红外光谱分析的模型优化。     

Fructose-selective calorimetric biosensor in flow injection analysis

A highly selective, interference free biosensor for the measurement of fructose in real syrup samples was developed. The assay is based on the phosphorylation of d(−)fructose to fructose-6-phosphate by hexokinase and subsequent conversion of fructose-6-phosphate to fructose-1,6-biphosphate by fructose-6-phosphate-kinase. The heat liberated in the second reaction is monitored using an enzyme thermistor. The major advantages of this biosensor are rapid and selective measurement of fructose without the need to eliminate glucose and inexpensive FIA-based, mediator-free calorimetric measurement suitable for regular fructose analysis. This method was optimised for parameters, such as pH, ionic strength, interference, operational stability and shelf life. Good and reproducible linearity (0.5-6.0 mM) with a detection limit of 0.12 mM was obtained. Fructose determination in commercial syrup samples and spiked samples confirmed the reliability of this set-up and technique. The biosensor gave reproducible results with good overall stability for continuous measurements over a period of three months besides a useful shelf life of six months. The method could be used for routine fructose monitoring in food samples.     

含葡萄糖的混浊介质的Mueller矩阵模拟与分析

利用矢量Monte Carlo算法结合葡萄糖的旋光特性模拟了含糖混浊介质的背散射Mueller矩阵.数值模拟表明:含糖混浊介质的背散射Mueller矩阵元素中除Mll、M14、M41、M44外的其它元素花样均发生龙卷风状的不同程度的扭曲,其中M24、M34、M42、M43的扭曲程度更为显著.研究发现M24、M34、M4...     

N‐glycosylation approach to glucose‐functionalized diamine and its use for protection‐free synthesis of polyurea bearing glucoside pendant

A glucose‐functionalized diamine was prepared and used as a new monomer for polyurea synthesis. The diamine was prepared by N‐glycosylation of 1,6‐hexamethylenediamine with D ‐glucose. Upon adding diisocyanates to the diamine, isocyanate reacted selectively with the amino groups, not with the hydroxyl groups of the glucose‐derived structure, to give the corresponding polyureas. The polyureas exhibited highly hydrophilic nature due to the presence of the glucose‐derived side chain. A ternary system consisting of the glucose‐functionalized diamine, piperazine, and diisocyanate gave the corresponding polyureas, where content of the glucose‐derived moiety was tunable by feed ratio between the diamine and piperazine. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013     

A sensitive, rapid ferricyanide-mediated toxicity bioassay developed using Escherichia coli

A need for rapid toxicity techniques has seen recent research into developing new microbiological assays and characterising their toxicity responses using a range of substances. A microbiological bioassay that determines changes in ferricyanide-mediated respiration for toxicity measurement (FM-TOX) shows particular promise. The development and optimisation of an improved FM-TOX method, incorporating novel features, is described using Escherichia coli as the biocatalyst. Omission of an exogenous carbon source, used in previously described FM-TOX assays, substantially improves the assay sensitivity. In addition, the development of a two-step procedure (toxicant exposure followed by determination of microbial respiratory activity) was found to enhance the inhibition of E. coli by 3,5-dichlorophenol and four other toxicants, compared to single-step procedures. Other assay parameters, such as the ferricyanide concentration, exposure times and optical density of the biocatalyst were also optimised, sometimes based on practical aspects. Toxicity tests were carried out using the adopted technique on both organic and inorganic toxicants, with classic sigmoid-shaped dose-response curves observed, as well as some non-standard responses. IC₅₀ data is presented for a number of common toxicants. The optimised assay provides a good foundation for further toxicity testing using E. coli, as well as the potential for expanding the technique to utilise other bacteria with complementary toxicity responses, thereby allowing use of the assay in a range of applications.     

Instrumental Requirements for Non-Invasive Blood Glucose Measurement Using NIR Spectroscopy

The magnitude of spectral change in blood glucose measurements with diffuse reflectance spectroscopy is investigated. Spectral change is estimated by simulation of light propagation in skin tissue and measurements of absorbance spectra of aqueous glucose solution. Required sensitivity of spectrophotometers for monitoring change in the blood glucose concentration as small as 10 mg/dL has been obtained using the estimated change in the absorbance spectrum and mean pathlength of light in tissue.