葡萄糖氧化酶在水溶性CdTe量子点上的直接电化学研究

利用合成的Cd Te量子点(QDs)作修饰材料,将葡萄糖氧化酶(GOD)固定在水溶性Cd Te量子点表面,制备了葡萄糖氧化酶Cd Te量子点修饰碳糊电极(GOD/Cd Te/CPE),实现了GOD在电极表面的直接电化学。Cd Te QDs能有效地加速葡萄糖氧化酶(GOD)与电极表面的直接电子转移,电子传递效率比无QDs Cd Te存在时提高约8倍;电子转移速率常数(K)为0.14 s-1,传递系数(α)为0.60,GOD在GOD/Cd Te/CPE表面的平均覆盖量(Γ)为7.9×10-8mol/cm2。GOD/Cd Te/CPE电极作为第三代葡萄糖电化学生物传感器,成功应用于葡萄糖浓度的检测,其线性范围为0.050~0.32 mmol/L,检出限为0.020 mmol/L。GOD/Cd Te/CPE的制备方法简单,稳定性强,具有优良的选择性和重现性,且响应速度快。     

基于Dy_2(MoO_4)_3-AuNPs复合纳米材料的葡萄糖生物传感器

采用水热法合成了纳米材料钼酸镝[Dy_2(MoO_4)_3],并制备了Dy_2(MoO_4)_3-AuNPs复合材料,利用该复合材料固定葡萄糖氧化酶(GOD)构建了葡萄糖生物传感器.通过透射电子显微镜(TEM)、紫外-可见光谱(UV-Vis)和能谱分析(EDS)等手段对所制备的材料进行了表征,并利用电化学阻抗谱(EIS)和循环伏安(CV)曲线研究了该传感器的电化学性能.结果表明,Dy_2(MoO_4)_3-AuNPs复合材料具有较好的生物相容性,能增强固定化的GOD的生物活性,并促进GOD在电极表面的电子传递速率;该传感器在葡萄糖浓度为0.01~1.0 mmol/L范围内葡萄糖浓度与响应电流呈较好的线性关系,最低检出限为3.33μmol/L(S/N=3),该生物传感器还具有较好的稳定性和重现性.     

纳米铜修饰玻碳电极的制备及其对葡萄糖的催化氧化

在表面活性剂十六烷基三甲基溴化铵(CTMAB)的分散作用下,通过恒电位还原CuSO4在玻碳电极上沉积Cu,得到纳米Cu修饰玻碳电极(nano-Cu-GCE),该修饰电极对葡萄糖(Glu)的氧化具有明显的催化作用,利用该催化作用对Glu进行检测,通过研究沉积电位、沉积时间以及检测电位对电流信号的影响,优化了电极的制备条件和Glu的检测条件。沉积电位为-100mV,沉积时间8min。在检测电位400mV下,Glu在1.0×10-6~3.9×10-4mol/L范围内Glu电流与空白溶液电流值之差与其浓度呈线性关系,检出限为2.6×10-7mol/L(S/N=3),线性回归方程Δi(μA)=-1.02-125674.54C(mol/L),r=0.9981。抗坏血酸(AA)、对乙酰氨基酚(AP)和L-半胱氨酸(Cys)对Glu信号几乎无干扰。     

四磺基苯基铁卟啉催化反应研究及分析应用

用金属-有机配位化合物四磺基苯基铁卟啉(Iron-(4-sulphophenyl)porphyrin,FeTPPS4)作为辣根过氧化物酶(Horeseradish Peroxidase,HRP)模拟酶,催化活化H2O2氧化染料氢供体底物孔雀绿(MG),建立了FeTPPS4/H2O2/MG新的模拟酶催化反应体系。用该体系测定H2O2的表观摩尔吸光系数ε为5.54×103L.mol-1.cm-1,检出限为3.61×10-6mol/L。拟定的方法用于雨水中H2O2的测定,并与葡萄糖氧化酶(Glucose Oxidase,GOD)反应偶联测定葡萄糖(Glu)含量,结果令人满意。     

纸芯片显色法用于L-半胱氨酸的快速测定

该文以5,5'-二硫硝基苯甲酸(DTNB)为含巯基化合物的衍生化试剂,建立了其纸芯片快速检测新方法。显色剂DTNB和样品在显色区相遇并显色,用手机拍照记录,采用Photoshop软件分析显色强度,进行比色检测,并以L-半胱氨酸(L-Cys)为例,优化得到纸芯片检测L-Cys的最佳显色条件。结果显示,L-Cys浓度在0.01～0.1 mmol/L范围内与显色强度呈良好的线性关系(r=0.999 8),检出限达0.001 mmol/L。多种氨基酸和干扰离子对L-Cys在纸芯片上的测定干扰不超过±5%。采用该方法测得牛血清样品中L-Cys含量为0.013 mmol/L,加标回收率为99.1%～103%,该方法检测L-Cys具有操作简单、耗样量少、检测快速、重现性好等优点。     

超高效液相色谱–串联质谱法测定饮用水中微囊藻毒素

建立饮用水中微囊藻毒素(MC–RR,MC–LR)的超高效液相色谱–串联质谱检测方法。样品经PVDF针式过滤头过滤后直接进样,采用喷雾正离子源(ESI~+)和多重反应监测模式(MRM)测定。MC–RR的质量浓度在0.02~10.00μg/L范围内与色谱峰面积呈良好的线性,线性相关系数r~2=0.998 9,检出限为0.096μg/L,测定结果的相对标准偏差为6.6%~9.1%(n=7),加标回收率为99.0%~103.0%。MC–LR的质量浓度在0.1~20μg/L范围内与色谱峰面积呈良好的线性,线性相关系数r~2=0.999 2,检出限为0.188μg/L,测定结果的相对标准偏差为4.3%~10.0%(n=7),加标回收率为93.0%~114.0%。该方法灵敏度高、重现性好,可用于饮用水中微囊藻毒素的检测。     

PDMS芯片自由酶反应器检测葡萄糖

近年来,芯片酶反应器备受关注~([1]).根据芯片中酶的存在形式,可分为自由酶反应器和固定化酶反应器.固定化酶反应器能使酶的稳定性提高,减少了酶的消耗,不会对产物造成污染~([1]),但酶在芯片通道内的固定步骤比较复杂;相比而言,自由酶反应器具有更好的灵活性,制作方便,操作简单.葡萄糖(Glu)存在于人体的血浆和淋巴液中,是生命活动中不可缺少的物质,它在人体内直接参与新陈代谢过程.     

基于金属纳米粒子增敏的SPR生物传感器检测吲哚乙酸

本实验建立了表面等离子体共振(SPR)生物传感器检测3-吲哚乙酸(IAA)的方法。制备了两种SPR生物传感器检测IAA:传统模式的SPR生物传感器1和Au/Ag合金纳米粒子增敏的SPR生物传感器2。结果发现:传感器1在IAA浓度范围为175~350μg/L时,浓度与其波数位移值呈线性关系,检出限为25μg/L(S/N=3);传感器2在IAA浓度范围为17.5~250μg/L时,浓度与其波数位移值呈线性关系,检出限为2.2μg/L(S/N=3)。说明基于Au/Ag合金纳米粒子的传感器2比传感器1有较高的灵敏度和较低的检出限。加标回收实验测得加标回收率范围为96%~100.2%,平均值为98.4%。本实验制备的SPR生物传感器具有较好的精密度、稳定性、重现性和特异性。     

磁性纳米粒子固定葡萄糖氧化酶修饰电极电致化学发光葡萄糖传感器

通过交联剂将葡萄糖氧化酶(GOD)固定在Fe3O4磁性纳米粒子上,在磁力作用下将该磁性复合粒子修饰在石蜡碳糊电极(SPCE)表面,制成易更新酶电极.GOD催化氧化葡萄糖生成过氧化氢,并使鲁米诺产生电致化学发光(ECL),据此首次构建了易更新型电致化学发光葡萄糖传感器.其电致发光强度与葡萄糖浓度在1×10-5～1.0×10-2 mol/L范围内呈线性关系,线性回归方程I＝65.4374C+23.9017(r=0.9987); 检出限为1.0 μmol/L.此传感器响应快, 稳定性高, 表面易更新,已用于测定人血清中葡萄糖的含量.     

酶荧光毛细分析法测定葡萄糖

基于酶催化和荧光毛细分析法(FCA)开发了一种微量、快速测定葡萄糖的新方法(GE-FCA)。优选的实验条件为:反应时间15min;反应温度30℃;磷酸盐缓冲液(pH6.0),HRP浓度和GOD浓度分别是200和150U/L;线性范围0.1～4mg/L;检出限0.070mg/L。GE-FCA对血液中葡萄糖进行测定,其回收率在98.9%～103.8%之间。与其它方法相比,GE-FCA法操作简单,而且试样用量少(仅为18μL),节省了酶试剂用量,实验成本低,易于普及推广。     

Production of rapidly reversible antibody and its performance characterization as binder for continuous glucose monitoring

To effectively control diabetes, a method to reliably measure glucose fluctuations in the body over given time periods needs to be developed. Current glucose monitoring systems depend on the substrate decomposition by an enzyme to detect the product; however, the enzyme activity significantly decays over time, which complicates analysis. In this study, we investigated an alternative method of glucose analysis based on antigen-antibody binding, which may be active over an extended period of time. To produce monoclonal antibodies, mice were immunized with molecular weight (M(W)) 10K dextran chemically conjugated with keyhole limpet hemocyanin. Since dextran contains glucose molecules polymerized via a 1,6-linkage, the produced antibodies had a binding selectivity that could discriminate biological glucose compounds with a 1,4-linkage. Three antibody clones with different affinities were screened using the M(W) 1K dextran-bovine serum albumin conjugates as the capture ligand. Among the antibodies tested, the antibody clone Glu 26 had the lowest affinity (K(A) = 3.56 × 10(6) M(-1)) and the most rapid dissociation (k(d) = 1.17 × 10(-2) s(-1)) with the polysaccharide immobilized on the solid surfaces. When glucose was added to the medium, the sensor signal was inversely proportional to the glucose concentration in a range between 10 and 1000 mg dL(-1), which covered the clinical range. Under the optimal conditions, the response time was about 3 min for association and 8 min for dissociation based on a 95% recovery of the final equilibrium.     

Logic gates and elementary computing by enzymes

Different selected enzymes, glucose oxidase (GOx), catalase (Cat), glucose dehydrogenase (GDH), horseradish peroxidase (HRP), and formaldehyde dehydrogenase (FDH), are used alone or coupled to construct eight different logic gates. The added substrates for the respective enzymes, glucose and H(2)O(2), act as the gate inputs, while the biocatalytically generated gluconic acid or NADH are the output signals that follow the operation of the gates. Different enzyme-based gates are XOR, INHIBIT A, INHIBIT B, AND, OR, NOR, Identity and Inverter gates. By combining the AND and XOR or the XOR and INHIBIT A gates, the half-adder and half-subtractor are constructed, respectively, opening the way to elementary computing by the use of enzymes.     

The efficiency of immobilised glutamate oxidase decreases with surface enzyme loading: an electrostatic effect, and reversal by a polycation significantly enhances biosensor sensitivity

The apparent Michaelis constant, K(M), for glutamate oxidase (GluOx) immobilised on Pt electrodes increased systematically with enzyme loading. The effect was due, at least in part, to electrostatic repulsion between neighbouring oxidase molecules and the anionic substrate, glutamate (Glu). This understanding has allowed us to increase the Glu sensitivity of GluOx-based amperometric biosensors in the linear response region (100+/-11 nA cm(-2)microM(-1) at pH 7.4; SD, n=23) by incorporating a polycation (polyethyleneimine, PEI) to counterbalance the polyanionic protein. Differences in the behaviour of glucose biosensors of a similar configuration highlight a limitation of using glucose oxidase as a model enzyme in biosensor design.     

葡萄糖-BrO-3-Mn2+-H2SO4-丙酮体系的振荡反应

本文首次报道葡萄糖(以Glu表示)-丙酮(Act)-Mn~(2+)-BrO_3~--H_2SO_4体系的化学振荡反应,这种由糖类及丙酮作混和底物的均相B-Z振荡反应至今尚未见报道。实验部分1.试剂及药品:实验中各物质均采用分析纯,溶液在去离子水中配制。2.实验仪器及方法:实验在恒温30°±0.2℃下进行,以溴离子选择性电极和PXS-215型离子活度计测量电势(E)随时间(t)的变化来反映log[Br~-]的变化,以Hg|Hg_2SO_4|K_2SO_4为参比电极,振荡现象可通过XWT-台式自动平衡电位记录仪记录的E～t曲线观察,同时也可采用HP-8451A型紫外可见分光光度计,测定特定波长下吸光率的变化反映读物质浓度的变化,实验时溶液均匀搅拌。     

Density functional computational studies on the glucose and glycine Maillard reaction: Formation of the Amadori rearrangement products

Theoretical energy changes of various intermediates leading to the formation of the Amadori rearrangement products (ARPs) under different mechanistic assumptions have been calculated, by using open chain glucose (O‐Glu)/closed chain glucose (A‐Glu and B‐Glu) and glycine (Gly) as a model for the Maillard reaction. Density functional theory (DFT) computations have been applied on the proposed mechanisms under different pH conditions. Thus, the possibility of the formation of different compounds and electronic energy changes for different steps in the proposed mechanisms has been evaluated. B‐Glu has been found to be more efficient than A‐Glu, and A‐Glu has been found more efficient than O‐Glu in the reaction. The reaction under basic condition is the most favorable for the formation of ARPs. Other reaction pathways have been computed and discussed in this work. © 2007 Wiley Periodicals, Inc. Int J Quantum Chem, 2008     

Glycosylated Cell‐Penetrating Poly(disulfide)s: Multifunctional Cellular Uptake at High Solubility

The glycosylation of cell‐penetrating poly(disulfide)s (CPDs) is introduced to increase the solubility of classical CPDs and to achieve multifunctional cellular uptake. With the recently developed sidechain engineering, CPDs decorated with α‐d ‐glucose (Glu), β‐d ‐galactose (Gal), d ‐trehalose (Tre), and triethyleneglycol (TEG) were readily accessible. Confocal laser scanning microscopy images of HeLa Kyoto cells incubated with the new CPDs at 2.5 μm revealed efficient uptake into cytosol and nucleoli of all glycosylated CPDs, whereas the original CPDs and TEGylated CPDs showed much precipitation into fluorescent aggregates at these high concentrations. Flow cytometry analysis identified Glu‐CPDs as most active, closely followed by Gal‐CPDs and Tre‐CPDs, and all clearly more active than non‐glycosylated CPDs. In the MTT assay, all glyco‐CPDs were non‐toxic at concentrations as high as 2.5 μm . Consistent with thiol‐mediated uptake, glycosylated CPDs remained dependent on thiols on the cell surface for dynamic covalent exchange, their removal with Ellman's reagent DTNB efficiently inhibited uptake. Multifunctionality was demonstrated by inhibition of Glu‐CPDs with d ‐glucose (IC₅₀ ca. 20 mm ). Insensitivity toward l ‐glucose and d ‐galactose and insensitivity of conventional CPDs toward d ‐glucose supported that glucose‐mediated uptake of the multifunctional Glu‐CPDs involves selective recognition by glucose receptors at the cell surface. Weaker but significant sensitivity of Gal‐CPDs toward d ‐galactose but not d ‐glucose was noted (IC₅₀ ca. 110 mm ). Biotinylation of Glu‐CPDs resulted in the efficient delivery of streptavidin together with a fluorescent model substrate. Protein delivery with Glu‐CPDs was more efficient than with conventional CPDs and remained sensitive to DTNB and d ‐glucose, i.e., multifunctional.     

Ag@Au core/shell triangular nanoplates with dual enzyme-like properties for the colorimetric sensing of glucose

Due to the serious harm of diabetes to human health, development of sensitive assays for glucose level is of high significance for early prevention and treatment of diabetes. Currently, most conventional enzyme-based glucose sensors suffer from high cost and low stability due to the inherent defects of natural enzymes. Herein, we develop a pure nanozyme-based glucose detection method using Ag@Au core/shell triangular nanoplates (TNPs), which combines glucose oxidase (GOD)- and horseradish peroxidase (HRP)-like activities of the Au shell and inherent plasmonic properties of Ag TNPs. The sensing mechanism is based on the fact that the Au shell possessed GOD-like activity, enabling the oxidation of glucose to produce H₂O₂, which can further etch the silver core, leading to the decrease of absorbance at 800 nm and the color change from blue to colorless. Compared with the previous nanozymes-based glucose sensors, our method avoids the use of enzymes and organic chromogenic agent. Moreover, the stability of the Ag@Au core/shell TNPs is much better than that of Ag TNPs due to the protection by the coating of the Au shell. This method was successfully applied to the detection of urine samples from patients with diabetes, indicating its practical applicability for real sample analysis.     

Simultaneous determination of D-glucose and 3-hydroxybutyrate by chemiluminescence detection with immobilized enzymes in a flow injection system.

A chemiluminometric flow-through sensor for the simultaneous determination of glucose (Glu) and 3-hydroxybutyrate (HB) in a single sample has been developed. Coimmobilized 3-hydroxybutyrate dehydrogenase/NADH oxidase/peroxidase, a support material, and coimmobilized glucose dehydrogenase/NADH oxidase/peroxidase were packed sequentially in a transparent PTFE tube. The tube was then placed in front of a photomultiplier tube as a flow cell. A two-peak recording was obtained by one injection of the sample solution. The peak heights of the first and second peaks were dependent on the concentrations of HB and Glu, respectively. The calibration graphs for HB and Glu were linear at 0.05-10 and 0.1-30 microM, respectively. The maximum sample throughput was 30 h(-1). The sensor was stable for two weeks.     

A Microelectronic Sensor Device Powered by a Small Implantable Biofuel Cell

Biocatalytic buckypaper electrodes modified with pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase and bilirubin oxidase for glucose oxidation and oxygen reduction, respectively, were prepared for their use in a biofuel cell. A small (millimeter-scale; 2×3×2 mm³) enzyme-based biofuel cell was tested in a model glucose-containing aqueous solution, in human serum, and as an implanted device in a living gray garden slug (Deroceras reticulatum), producing electrical power in the range of 2–10 μW (depending on the glucose source). A microelectronic temperature-sensing device equipped with a rechargeable supercapacitor, internal data memory and wireless data downloading capability was specifically designed for activation by the biofuel cell. The power management circuit in the device allowed the optimized use of the power provided by the biofuel cell dependent on the sensor operation activity. The whole system (power-producing biofuel cell and power-consuming sensor) operated autonomously by extracting electrical energy from the available environmental source, as exemplified by extracting power from the glucose-containing hemolymph (blood substituting biofluid) in the slug to power the complete temperature sensor system and read out data wirelessly. Other sensor systems operating autonomously in remote locations based on the concept illustrated here are envisaged for monitoring different environmental conditions or can be specially designed for homeland security applications, particularly in detecting bioterrorism threats.     

pH gated glucose responsive biomimetic single nanochannels

The development of pH gated glucose (Glu) biosensor is of great significance to human health. Herein, we have designed a pH gated Glu responsive biomimetic nanochannel, modified with 3-aminobenzeneboronic acid. The Glu responsive property can be regulated by pH which can switch nanochannels from the "on" to "off" state.