Electronic Olfactory Sensor Based on A. mellifera Odorant‐Binding Protein 14 on a Reduced Graphene Oxide Field‐Effect Transistor

An olfactory biosensor based on a reduced graphene oxide (rGO) field‐effect transistor (FET), functionalized by the odorant‐binding protein 14 (OBP14) from the honey bee (Apis mellifera) has been designed for the in situ and real‐time monitoring of a broad spectrum of odorants in aqueous solutions known to be attractants for bees. The electrical measurements of the binding of all tested odorants are shown to follow the Langmuir model for ligand–receptor interactions. The results demonstrate that OBP14 is able to bind odorants even after immobilization on rGO and can discriminate between ligands binding within a range of dissociation constants from K_d=4 μM to K_d=3.3 mM . The strongest ligands, such as homovanillic acid, eugenol, and methyl vanillate all contain a hydroxy group which is apparently important for the strong interaction with the protein.     

聚脲多孔材料的简单制备及在酶固定和手性拆分中的应用

以甲苯二异氰酸酯(TDI)为单体,在水与丙酮混合溶剂中通过沉淀聚合一步法制备了富含胺基的聚脲多孔材料(PPU),通过扫描电镜和压汞法对其表面形貌和孔结构进行了表征.PPU经戊二醛(GA)活化后用于荧光假单胞菌脂肪酶(PFL)的固定,考察了GA活化过程中GA浓度对酶固定量及固定酶活性的影响.结果表明,PPU是一种粒子尺寸分布在30~50μm范围的形状不规则的多孔粒子,孔径在2 nm~100μm之间呈连续分布.在pH=8.0的缓冲溶液中用0.17 mol/L的GA对PPU进行改性,将改性后的PPU用于PFL的固定,当酶溶液浓度为2.56 mg/m L时,得到酶的最大固定量为95.2 mg/g,固定酶的活性为375 U/mg,相对活性为76%.将此固定酶作为催化剂,用于1-苯乙醇外消旋化合物的手性拆分,并与游离酶催化的结果相比较.结果表明,固定酶的反应活性和立体选择性都明显优于游离酶.通过沉淀聚合制备的聚脲多孔材料在酶固定及手性分子拆分方面具有应用前景.     

Potassium Acyltrifluoroborate (KAT) Ligations are Orthogonal to Thiol‐Michael and SPAAC Reactions: Covalent Dual Immobilization of Proteins onto Synthetic PEG Hydrogels

The covalent immobilization of peptides, proteins, and other biomolecules to hydrogels provides a biologically mimicking environment for cell and tissue growth. Bioorthogonal chemical reactions can serve as a tool for this, but the paucity of such reactions and mutual incompatibilities limits the number of distinct molecules that can be introduced. We now report that the potassium acyltrifluoroborate (KAT ) amide‐forming ligation is orthogonal to both thiol‐Michael and strain promoted azide alkyne cycloadditions (SPAAC ) and the requisite functional groups – KAT s and hydroxylamines – are stable and compatible to hydrogel formation, protein modification, and post‐assembly immobilization of biomolecules onto hydrogels. In combination these ligations enables stepwise covalent protein immobilization of multiple BSA ‐derivatives onto the hydrogel scaffold regardless of the order of addition.     

Evaluation of a Gelatin-Modified Poly(ε-Caprolactone) Film as a Scaffold for Lung Disease

Lung transplantation is a necessary step for the patients with the end stage of chronic obstructive pulmonary disease. The use of artificial lungs is a promising alternative to natural lung transplantation which is complicated and is restricted by low organ donations. For successful lung engineering, it is important to choose the correct combination of specific biological cells and a synthetic carrier polymer. The focus of this study was to investigate the interactions of human lung epithelial cell line NCl-H292 that is involved in lung tissue development with the biodegradable poly(ε-caprolactone) before and after its chemical modification to evaluate potential for use in artificial lung formation. Also, the effect of polymer chemical modification on its mechanical and surface properties has been investigated. The poly(ε-caprolactone) surface was modified using aminolysis followed by immobilization of gelatin. The unmodified and modified polymer surfaces were characterized for roughness, tensile strength, and NCl-H292 metabolic cell activity. The results showed for the first time the possibility for NCI-H292 cells to adhere on this polymeric material. The resazurin assay showed that the metabolic activity at 24?h postseeding of 80% in the presence of the unmodified and greater than 100% in the presence of the modified polymer was observed. The roughness of the poly(ε-caprolactone) increased from 4 to 26?nm and the film strength increased from 0.01 to 0.045 kN when the material was chemically modified. The results obtained to date show potential for using modified poly(ε-caprolactone) as a scaffold for lung tissue engineering.     

Polydopamine‐assisted immobilization of zeolitic imidazolate framework‐8 for open‐tubular capillary electrochromatography

In this work, we developed a capillary column modified with zeolitic imidazolate framework‐8 as a novel stationary phase for open‐tubular capillary electrochromatography. To immobilize zeolitic imidazolate framework‐8 onto the inner surface of silica capillary, a bio‐inspired polydopamine functionalization was used to functionalize the capillary surface with polydopamine. First, a polydopamine layer was assembled inside the capillary. Second, due to noncovalent adsorption and covalent reaction ability, polydopamine could attract and anchor zeolitic imidazolate framework‐8 onto the inner surface of capillary. It has been demonstrated that zeolitic imidazolate framework‐8 was successfully grafted on the inner wall of the capillary by scanning electron microscopy, and Fourier transform infrared spectroscopy. The electro‐osmotic flow characteristics of capillaries were also investigated by varying the pH value and acetonitrile content of mobile phase. The zeolitic imidazolate framework‐8 coating not only increased the phase ratio of open‐tubular column, but also improved the interactions between tested analytes and the stationary phase. Three groups of isomers including acidic, basic, and neutral compounds were well separated on the zeolitic imidazolate framework‐8 bonded column, with theoretic plate numbers up to 1.9 × 10⁵ N for catechol. The repeatability of the prepared columns was also studied, and the relative standard deviations for intra‐ and interday runs were less than 5%.     

Orthogonal Surface Tags for Whole‐Cell Biocatalysis

We herein describe the engineering of E. coli strains that display orthogonal tags for immobilization on their surface and overexpress a functional heterologous “protein content” in their cytosol at the same time. Using the outer membrane protein Lpp‐ompA, cell‐surface display of the streptavidin‐binding peptide, the SpyTag/SpyCatcher system, or a HaloTag variant allowed us to generate bacterial strains that can selectively bind to solid substrates, as demonstrated with magnetic microbeads. The simultaneous cytosolic expression of functional content was demonstrated for fluorescent proteins or stereoselective ketoreductase enzymes. The latter strains gave high selectivities for specific immobilization onto complementary surfaces and also in the whole‐cell stereospecific transformation of a prochiral C_S‐symmetric nitrodiketone.     

Immobilization of Eversa® Transform via CLEA Technology Converts It in a Suitable Biocatalyst for Biolubricant Production Using Waste Cooking Oil

The performance of the previously optimized magnetic cross-linked enzyme aggregate of Eversa (Eversa-mCLEA) in the enzymatic synthesis of biolubricants by transesterification of waste cooking oil (WCO) with different alcohols has been evaluated. Eversa-mCLEA showed good activities using these alcohols, reaching a transesterification activity with isoamyl alcohol around 10-fold higher than with methanol. Yields of isoamyl fatty acid ester synthesis were similar using WCO or refined oil, confirming that this biocatalyst could be utilized to transform this residue into a valuable product. The effects of WCO/isoamyl alcohol molar ratio and enzyme load on the synthesis of biolubricant were also investigated. A maximum yield of around 90 wt.% was reached after 72 h of reaction using an enzyme load of 12 esterification units/g oil and a WCO/alcohol molar ratio of 1:6 in a solvent-free system. At the same conditions, the liquid Eversa yielded a maximum ester yield of only 34%. This study demonstrated the great changes in the enzyme properties that can be derived from a proper immobilization system. Moreover, it also shows the potential of WCO as a feedstock for the production of isoamyl fatty acid esters, which are potential candidates as biolubricants.     

通过"双离子键"固载于季鏻盐聚合物的[Rh(CO)I3]2–物种在多相乙醇羰基化中的应用

贵金属物种(Rh或Ir络合物)在均相羰基化和氢甲酰化催化过程得到了广泛的应用,但始终存在分离繁琐等问题,其均相多相化可很大程度上简化分离操作,故一直广受重视.单位点催化剂因其具有可与均相相比拟的较高金属利用率和选择性而成为均相多相化的重要研究方向之一.研究发现,在碘物种存在的情况下用于固载金属物种的配位键容易断裂,进而导致金属物种的流失,而通过离子键固载的[Rh(CO)₂I₂]^–物种更加稳定,比如著名的甲醇羰基化“Acetica^TM”工艺中,[Rh(CO)₂I₂]^–负一价阴离子物种是以离子键的方式固定在带有阳离子骨架的甲基化聚乙烯吡啶树脂上.与甲醇羰基化过程类似的乙醇羰基化过程是生产重要化工中间体丙酸的主要途径之一,但该过程的均相多相化始终存在着稳定性差这一关键问题.为了解决这一问题,基于之前将固载于季鏻盐聚合物的[Rh(CO)I₃]^2–应用于甲醇羰基化的工作,我们将类似的季鏻盐聚合物固载Rh基催化剂Rh-TPISP用于多相乙醇羰基化过程,通过多种表征进一步证明了Rh物种和P物种结构,并提出了“双离子键”模型.P的K边XANES证明了聚合物TPISP的季鏻化阳离子骨架特征.HAADF-STEM测试表明Rh-TPISP中的Rh呈现单位点分散的状态.Rh的XPS和XANES结果证明了Rh-TPISP中Rh物种的价态介于0~+1.通过EXAFS的拟合解析给出了[Rh(CO)I₃]^2–活性中心结构.由于[Rh(CO)₂I₂]^–为经典的羰基化活性中心,为了进一步证明该结构的正确性,我们将Rh-TPISP的EXAFS和IR谱图与标样[PPh3Et]⁺[Rh(CO)₂I₂]^–对比发现:在EXAFS谱图中,Rh-TPISP中的Rh-C峰高低于[PPh3Et]+[Rh(CO)₂I₂]^–的Rh-C峰高,而Rh-TPISP中的Rh-I峰高高于[PPh3Et]+[Rh(CO)₂I₂]^–的Rh-I峰高,这就说明Rh-TPISP中Rh物种的Rh-C配位数小于2,而Rh-I配位数大于2;在IR谱图中,标样[PPh3Et]+[Rh(CO)₂I₂]^–中有两个羰基振动峰,与该物种的两个Rh-C配位键相符,而Rh-TPISP中的只有一个羰基振动峰,说明Rh-C配位数为1.因此,Rh-TPISP催化剂的季鏻盐骨架中的每个P物种带有一个正电荷,每个带有两个负电荷的[Rh(CO)I₃]^2–通过与两个[P]⁺的静电作用进行固载,形成“双离子键”结构.该催化剂在固定床乙醇羰基化过程中表现出优异的羰基化活性、选择性和稳定性.在3.5 MPa、195 oC反应近1000 h后,Rh-TPISP催化剂TOF保持在约350 h–1,丙酰基选择性为95%以上,高出所有文献报道的均相和多相乙醇羰基化活性.其较高的活性主要是因为[Rh(CO)I₃]^2–比传统Rh活性相[Rh(CO)₂I₂]^–具有更强的富电子性,而较高的稳定性主要是由于“双离子键”这种强静电作用比“AceticaTM”工艺中“单离子键”更有利于Rh物种的固载.故Rh-TPISP催化剂中的“双离子键”对其优异的催化性能具有极其重要的作用,对后续多相乙醇羰基化的发展具有重要意义.     

Activity of AC electrokinetically immobilized horseradish peroxidase

Dielectrophoresis (DEP) is an AC electrokinetic effect mainly used to manipulate cells. Smaller particles, like virions, antibodies, enzymes, and even dye molecules can be immobilized by DEP as well. In principle, it was shown that enzymes are active after immobilization by DEP, but no quantification of the retained activity was reported so far. In this study, the activity of the enzyme horseradish peroxidase (HRP) is quantified after immobilization by DEP. For this, HRP is immobilized on regular arrays of titanium nitride ring electrodes of 500 nm diameter and 20 nm widths. The activity of HRP on the electrode chip is measured with a limit of detection of 60 fg HRP by observing the enzymatic turnover of Amplex Red and H₂O₂ to fluorescent resorufin by fluorescence microscopy. The initial activity of the permanently immobilized HRP equals up to 45% of the activity that can be expected for an ideal monolayer of HRP molecules on all electrodes of the array. Localization of the immobilizate on the electrodes is accomplished by staining with the fluorescent product of the enzyme reaction. The high residual activity of enzymes after AC field induced immobilization shows the method's suitability for biosensing and research applications.     

过渡金属-联咪唑-Dawson型钨磷酸盐杂化化合物的酶固定化性能

利用水热法和直接沉淀法, 设计合成了5例由过渡金属(TM)-联咪唑配阳离子与Dawson型钨磷酸阴离子构成的多金属氧酸盐(POM)基有机-无机杂化化合物[Ni(H₂biim)₃]₄[Ni(H₂biim)₂(P₂W₁₈O₆₂)₂]·2H₂O(1), [Co^III(H₂biim)₃]₂[P₂W₁₈O₆₂]·8H₂O(2), [Cu(H₂biim)₂]₃[P₂W₁₈O₆₂]·4H₂O(3), [Co^II(H₂biim)₃]₂H₂[P₂W₁₈O₆₂]·9H₂O(4)和 [Ni(H₂biim)₃]₃[P₂W₁₈O₆₂]·2H₂O(5); 并利用X射线单晶衍射分析(SC-XRD)、 红外光谱(IR)和热重-差热分析 (TG-DTA)等对其进行了表征. 化合物1~5作为载体用于固定辣根过氧化物酶(HRP)时, 显示出了较高的酶固定化能力. 另外, 利用圆二色光谱(CD)和激光扫描共聚焦显微镜(LSCM)等方法评价了固定化酶HRP/1~HRP/5的重复使用性、 储存稳定性和检测过氧化氢(H₂O₂)的性能. 由于该类POMs与HRP间存在强的相互作用, 利用简单的物理吸附法即可实现POMs对HRP的固载. POMs对酶的固定不但提高了HRP对使用及储存环境的耐受性, 同时也拓展了POMs在酶固定化领域的应用.