化学修饰方法对聚乙二醇功能化碳纳米管的影响

通过酰氯化法与碳二亚胺缩合法(EDC/NHS)制备氨基化聚乙二醇(PEG1500N)修饰的多壁碳纳米管(MWNTs)并采用FTIR、Raman、TEM、原子力显微镜(AFM)、TGA-DTA-DSC、UV-Vis进行表征与分析。实验结果发现：两种方法PEG1500N都能很好地修饰MWNTs,但EDC/NHS缩合法采用更短的反应时间(反应1 d),达到了更好的接枝效果。EDC/NHS缩合法提高了碳管上羧基的利用率,接枝率大大提高。TGA-DTA分析表明缩合法接枝率为30%,而酰氯化法(反应4 d)为15%。UV-Vis分析表明EDC/NHS缩合法得到的产物溶解性也更好,溶解度由1.19 mg·mL^-1(酰氯化法得到的产物的溶解度)提高到2 mg·mL^-1以上。     

碳纳米管修饰金电极检测特定序列DNA

利用化学偶联法将末端修饰氨基的寡聚核苷酸固定在表面修饰有羧基化碳纳米管(CNTs-COOH)的金电极表面, 制备新型核酸探针, 可以特异性结合目标单链寡聚核苷酸. 以阿霉素作为嵌合指示剂, 利用示差脉冲法测定杂交的结果. 经过实验条件的优化, 测定DNA浓度在1.0×10^－6～1.0×10^－9 mol/L呈良好的线性关系. 检测限为: 2.54×10^－10 mol/L. 碳纳米管特有的纳米结构对检测结果的放大作用, 提高了该传感器的检测限和灵敏度.     

Click‐chemistry of polymersomes on nanoporous polymeric surfaces

A facile click chemistry method of immobilizing surface‐functionalized polymer vesicles on casted polymeric PAN substrates is described. Microporous PAN membranes were subjected to hydrochloric acid hydrolysis to obtain surface carboxylates. The carboxylic groups were activated with EDC/NHS‐solution and were then reacted with propargylamine to introduce alkyne groups for CuAAC reactions. The alkyne functionality of the modified membrane surface was verified by reaction with an azide functional click dye both before and after the immobilization of azide‐functionalized ABA vesicles. The efficient postfunctionalization of the membrane with alkyne allowed quantitative coverage of the membrane surface with a polymersome monolayer, as confirmed by immobilization of polymerzomes loaded with a fluorescent dye. Polymersome monolayers immobilized on alkyne functionalized PAN‐membranes were characterized by cryo‐SEM and monolayers were confirmed by atom force microscopy. These methods opens up new avenues for preparing membrane based filtration and sensor technologies. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 2032–2039     

Orthogonal Functionalisation of Upconverting NaYF4 Nanocrystals

A simple and straightforward method for the orthogonal functionalisation of upconverting NaYF₄ nanocrystals (UCNCs)—doped withYb³⁺ and Er³⁺—based on N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide/N‐hydroxysuccinimide (EDC/NHS) selective reactions between two dyes and two different reactive groups present at the periphery of the upconverting nanocrystals is reported. Organic‐soluble UCNCs of 10 and 50 nm in size are encapsulated efficiently in a 1:1 mixture of two commercial 3000 Da poly(ethylene glycol) derivatives with two different reactive groups (amino and carboxylic groups). The water‐dispersible UCNCs are non‐cytotoxic, stable in the physiological environment, and present free amine and carboxylic reactive groups on their periphery, allowing rapid, selective, and modular covalent conjugation to payloads through EDC/NHS reactions. PEG‐encapsulated UCNCs with and without covalent conjugation to payloads are characterised in vitro through spectroscopic, dynamic light scattering, and electron microscopy measurements. Living cell analyses coupled with TEM measurements confirm the uptake and low cytotoxicity of the coated UCNCs. They are linked covalently to two different dyes, internalised by living cells, and analysed by confocal microscopy. The related colocalisation measurements prove the reactivity of both amines and carboxylic acids on the periphery of the nanocrystals. This approach demonstrates that it is possible to produce water‐dispersible and cyto‐compatible dual‐functional UCNCs.     

Multilayer film of thiourea and gold nanoparticles as an effective platform for immobilization of activated non-labeled-DNA and construction of an ultrasensitive electrochemical DNA biosensor

In this work, self assembly of thiourea and gold nano-particle multilayer built up on a thiourea modified gold nanoparticles Au electrode, has been used as a platform for immobilization of activated ss-DNA. Two NH₂ group of thiourea on a multilayer surface can interact with an activated phosphate group of non-labeled ss-DNA. Activated non-labeled ss-DNA was prepared using N-(3 dimethylaminopropyl)-N-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxy-succinimide (NHS). The whole DNA biosensor fabrication process was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) methods with the use of ferricyanide as an electrochemical redox indicator. Methylene Blue (MB) was used as the electrochemical indicator for monitoring the hybridization reaction after hybridized with the target ssDNA and the reduction current of MB intercalation decreased with increasing the concentration of target DNA, ranging from 7.9 × 10^–13 to 1.2 × 10^–8 M with a very low detection limit of 3.8 × 10^–13 M (S/N = 3).     

Synthesis and characterization of new thermally stable poly(amide-imide)s based on bicyclo[2,2,2]oct-7-ene-2,3,5,6-tetracarboxylic diimide and aromatic diamines

N,N′-bis-(4-carboxyphenyl)bicyclo[2,2,2]oct-7-ene-2,3,5,6-tetracarboxylic diimide was prepared by reaction of bicyclo[2,2,2]oct-7-ene-2,3,5,6-tetra carboxylic dianhydride with p-aminobenzoic acid in a mixture of acetic acid and pyridine (3: 2). Polycondensation of N,N′-bis-(4-carboxyphenyl)bicyclo[2,2,2]oct-7-ene-2,3,5,6-tetracarboxylic diimide with six different aromatic diamines produced a series of new poly(amide-imide)s (PAIs) in high yields with inherent viscosities between 0.49–0.95 dl/g. All PAIs were characterized by means of elemental analysis, viscosity measurement, solubility test, FTIR spectroscopy and ¹H-NMR spectroscopy. Dynamic TGA of PAIs shows 10% weight loss temperatures from 410 to 435°C under nitrogen.     

Thiol Modified Chitosan Self-Assembled Monolayer Platform for Nucleic Acid Biosensor

A self-assembled monolayer (SAM) of thiol modified chitosan (SH-CHIT), with thioglycolic acid (TGA) as a modifier to bestow thiol groups, has been prepared onto gold (Au)-coated glass plates for fabrication of the nucleic acid biosensor. The chemical modification of CHIT via TGA has been evidenced by Fourier transform infrared spectroscopy (FT-IR) studies, and the biocompatibility studies reveal that CHIT retains its biocompatible nature after chemical modification. The electrochemical studies conducted onto SH-CHIT/Au electrode reveal that thiol modification in CHIT amino end enhances the electrochemical behavior indicating that it may be attributed to delocalization of electrons in CHIT skeleton that participates in the resonance process. The carboxyl group modified end of DNA probe has been immobilized onto SH-CHIT/Au electrode using N-ethyl-N′-(3-dimethylaminopropyl)carbodimide (EDC) and N-hydroxysuccinimide (NHS) chemistry for detection of complementary, one-base mismatch and non-complementary sequence using electrochemical and optical studies for Mycobacterium tuberculosis detection. It has been found that DNA-SH-CHIT/Au bioelectrode can specifically detect 0.01 μM of target DNA concentration with sensitivity of 1.69?×?10^?6 A μM^?1.     

Ferrocenylnaphthalene diimide-based electrochemical ribonuclease assay.

Messenger RNA (mRNA) poly(A)+RNA (from mouse kidney) was immobilized on a N-hydroxysuccinimide(NHS)-activated carboxylic acid modified electrode prepared by the treatment of a gold electrode with 3,3'-dithiodipropionic acid, followed by NHS and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). An electrochemical measurement using this mRNA electrode was carried out in an electrolyte containing ferrocenylnaphthalene diimide (1), and showed an electrochemical signal based on 1 concentrated on immobilized mRNA. After treating this electrode with water containing varied amounts of ribonuclease A (RNase A), the current peak based on 1 decreased with increasing in the amount of RNase A with a linear correlation in the range of 0.2-10 pg of RNase A.     

Bioconjugation of trypsin onto gold nanoparticles: Effect of surface chemistry on bioactivity

The systematic study of activity, long-time stability and auto-digestion of trypsin immobilized onto gold nanoparticles (GNPs) is described in this paper and compared to trypsin in-solution. Thereby, the influence of GNP's size and immobilization chemistry by various linkers differing in lipophilicity/hydrophilicity and spacer lengths was investigated with regard to the bioactivity of the conjugated enzyme. GNPs with different sizes were prepared by reduction and simultaneous stabilization with trisodium citrate and characterized by UV/vis spectra, dynamic light scattering (DLS), ζ-potential measurements and transmission electron microscopy (TEM). GNPs were derivatized by self-assembling of bifunctional thiol reagents on the nanoparticle (NP) surface via dative thiol-gold bond yielding a carboxylic acid functionalized surface. Trypsin was either attached directly via hydrophobic and ionic interactions onto the citrate stabilized GNPs or immobilized via EDC/NHS bioconjugation onto the carboxylic functionalized GNPs, respectively. The amount of bound trypsin was quantified by measuring the absorbance at 280 nm. The activity of bound enzyme and its Michaelis Menten kinetic parameter K_m and v_max were measured by the standard chromogenic substrate N_α-Benzoyl-DL-arginine 4-nitroanilide hydrochloride (BApNA). Finally, digestion of a standard protein mixture with the trypsin-conjugated NPs followed by analysis with LC–ESI-MS and successful MASCOT search demonstrated the applicability of the new heterogenous nano-structured biocatalyst. It could be shown that the amount of immobilized trypsin and its activity can be increased by a factor of 6 using a long hydrophilic spacer with simultaneous reduced auto-digestion and reduced digestion time. The applicability of the new trypsin bioreactor was proven by digestion of casein and identification of α- as well as κ-casein by subsequent MASCOT search.     

Electrochemical detection of hybridization using peptide nucleic acids and methylene blue on self-assembled alkanethiol monolayer modified gold electrodes

An electrochemical hybridization biosensor based on peptide nucleic acid (PNA) probes is presented. PNA probes were attached covalently through a competition of free amines on the guanine bases and also at the 5^′ end of the probe, using N-(3-dimethylamino)propyl)-N^′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) onto a carboxylate terminated alkanethiol self-assembled monolayer (SAM) preformed on a gold electrode (AuE). The covalently immobilized probe could selectively hybridize with the target DNA to form a hybrid on the surface despite the bases being attached to the SAM. The changes in the peak currents of methylene blue (3,7-bis(dimethylamino)phenothiazin-5-ium chloride, MB), an electroactive label, were observed upon hybridization of probe with the target. Effective discrimination against point mutation was also obtained. Performance characteristics of the sensor are described, along with future prospects.     

Traceless solid-phase synthesis of 1,4-disubstituted-6-nitro-3,4-dihydro-1H-quinoline-2-ones

A traceless solid-phase route to 1,4-disubstituted-6-nitro-3,4-dihydro-1H-quinazolin-2-ones is described. N-Alloc-3-amino-3-(2-fluoro-5-nitrophenyl)propionic acid was tethered to Rink resin via its carboxylic group. The protected amine was coupled with an organic acid after Alloc-deprotection and the arylfluorine was displaced with a primary amine to generate a resin-bound aniline with two diversity points. The aniline was released via cleavage to produce the desired products in high yield and purity.     

Facile preparation of N-acylsulfonamides by using sulfonyl isocyanate

N-Acylsulfonamide is widely used as a carboxylic acid bioisostere. A rapid and mild acyl sulfonamide preparation methodology from carboxylic acid and arylsulfonyl isocyanate in the presence of amine is described. The preparation of biologically active acylsulfonamide by our methodology is also presented.     

Synthesis,X-Ray Crystallography,and Reactions of N-Acyl and N-Carbamoyl Succinimides

A collection of N-acyl and N-carbamoyl succinimides were prepared by acylation of succinimide with acyl chlorides or by ethylene dichloride (EDC) coupling of carboxylic acids. The x-ray crystal structures of N-benzoyl and N-p-nitrobenzoyl succinimides were determined. The N-acyl succinimides were effective in acylating primary amines, a secondary amine, and an aromatic amine.

Supplemental materials are available for this article. Go to the publisher's online edition of Synthetic Communications® to view the free supplemental file.     

Covalent VEGF protein immobilization on resorbable polymeric surfaces

Vascular endothelial growth factor type protein (VEGF), a potent angiogenic effector molecule, was successfully covalently immobilized onto the surfaces of the resorbable polymers poly(L‐lactic acid) (PLLA) and poly(ε‐caprolactone) (PCL) through a three‐step strategy. The surfaces were first covalently grafted with poly(acrylic acid) using non‐destructive and solvent free vapor‐phase grafting. A diamine spacer was coupled to the carboxylic acid pendant groups on the graft chains using EDC/NHS chemistry and VEGF was finally covalently attached to the amine linkers. The chemistry and topography of the modified substrates were quantitatively and qualitatively verified with XPS, ATR‐FTIR, UV–VIS, SEM, and ELISA. Copyright © 2010 John Wiley & Sons, Ltd.     

Solid-phase synthesis of quinoxaline, thiazine, and oxazine analogs through a benzyne intermediate

A solid-phase synthetic route to quinoxaline, thiazine, and oxazine analogs is described. N-Alloc-3-amino-3-(2,4-difluoro-5-nitrophenyl)propanoic acid was tethered to Rink resin via its carboxylic acid group. The 4-arylfluorine was displaced with a primary amine, alcohol, or thiol to create, respectively, a resin bound aniline, phenol, or thiophenol derivative with one diversity element and one single atom (e.g., N, S, or O) diversity point. A fused heterocyclic system was subsequently created via a benzyne heterocyclization initiated by dehydrofluorination with strong base. Acid treatment released the desired products in high yield and moderate purity.     

Room-temperature polycondensation of β-amino acid derivatives VI. Synthesis of various N-(hydroxyethyl) nylons

Various N-(hydroxyethyl)amino acid esters having a methyl substituent or phenyl group between amine and ester groups have been synthesized and their polycondensation behavior was investigated. These substituted amino acid esters gave amorphous polyamides which were soluble in alcohol. A model reaction between N-(hydroxyethyl)-amine and carboxylic acid ester was carried out in order to elucidate the role of hydroxyethyl group on the polycondensation. It was found that the amidation reaction took place rapidly at room temperature when the alkyl group of the carboxylic acid was small. N-(Hydroxyethyl) polyamides were obtained from N,N′-(bishydroxyethyl)-dicamines and dicarboxylic acid esters. The reaction mechanism of the room-temperature polycondensation reaction is discussed.     

Synthesis of multifunctional nanogels using a protected macromonomer approach

Nanoparticles possessing multiple functionalities provide synthetic handles for varied surface chemistries, making them useful for a range of applications such as biotargeting and drug delivery. However, the combination of interfering functionalities on the same particle is often challenging. We have employed a synthetic scheme involving chemical protection/deprotection to combine interfering functional groups on the same hydrogel nanoparticle. The synthesis of amine-containing poly(N-isopropylacrylamide) nanogels was carried out via free radical precipitation polymerization by incorporating a Fmoc-protected amine poly(ethylene glycol) (PEG) macromonomer. The Fmoc group was then removed to obtain free amines, which were shown to be available for conjugation. We further explored pNIPAm-co-acrylic acid nanogels with a protected amine-PEG, yielding zwitterionic particles. With careful attention to the order of the chemoligation and deprotection steps, these interfering functional groups can be forced to behave in a pseudo-orthogonal fashion, allowing for multiple chemoligation steps that employ both the amine and carboxylic acid groups.     

α,α-Dialkylglycines obtained by solid phase Ugi reaction performed over isocyanide functionalized resins

The multicomponent Ugi reaction is a straightforward method that can be used for the synthesis of highly hindered C-tetrasubstituted amino acids by reacting an amine, a ketone or aldehyde, a carboxylic acid and an isocyanide. In the present work, the synthesis of several α,α-dialkylglycines (α,α-diethylglycine, Deg; α,α-dipropylglycine, Dpg; 1-amino-1-cyclohexanecarboxylic acid, Ac₆c) was achieved by solid phase Ugi reaction using resins functionalized with the isocyanide group. Since no resins with these features were available commercially, the functionalization of an aminomethylated resin started by the use of glycine (Gly), β-alanine (β-Ala) and γ-aminobutyric acid (GABA) as spacers. After spacer N-formylation, followed by dehydration, isocyanide functionalised resins were obtained. The resins were then used in solid phase Ugi reaction, using phenylacetic acid as the acid component, 4-methoxybenzylamine as the amine component and different ketones, to afford the desired N-acylated α,α-dialkylglycines in good overall yields (60–80%), after acidolytic cleavage from the resin, thus proving the feasibility of this approach.     

Electrochemical DNA biosensor for the detection of Listeria Monocytogenes using toluidine blue as a hybridization indicator

An electrochemical DNA biosensor was established for the determination of actin-assembly inducing protein (actA) gene sequences from Listeria monocytogenes and its polymerase chain reaction (PCR) product. The actA gene probe sequences were covalently immobilized on the surface of the mercaptoacetic acid self-assembled gold electrode with the help of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), which was further used to hybridize with the target sequence. Toluidine blue (TB) was used as an effective electrochemical indicator for the discrimination of the hybridization reaction on the electrode surface, which had stronger interaction with double-stranded DNA (dsDNA) than single-stranded DNA (ssDNA). The electrochemical parameters of TB on DNA modified electrodes were carefully calculated. Based on the different electrochemical responses of TB on DNA modified electrodes, the actA gene sequences can be detected in the concentration range from 1.0 × 10^-7 to 8.0 × 10^-5 M. The PCR product of Listeria monocytogenes was successfully detected by the proposed electrochemical biosensor.     

漆酶在纳米多孔金上的固定化及其酶学性质研究

利用纳米材料为载体对酶等生物大分子进行固定化近年来引起人们的浓厚兴趣. 以Au/Ag合金为原料, 通过控制浓硝酸的腐蚀时间再辅以退火处理得到了不同孔径的纳米多孔金(NPG), 利用扫描电镜(SEM)和N2气体吸附仪对孔性质进行了表征. 以NPG为载体, 用α-硫辛酸和N-乙基-N’-(3-二甲基氨基丙基)碳酰二亚胺/N-羟基琥珀酰亚胺(EDC/NHS)对金表面进行活化, 通过化学共价偶联的方法对产自Trametes versicolor的漆酶进行了固定化. 比较了孔径大小对酶固定化量及比活力的影响. 发现小孔径更有利于对该漆酶的固定化. 与游离酶相比, 固定化酶的最适pH没有改变, 但最适温度却从原来的40 ℃升到了60 ℃. 固定化后, 漆酶的pH和热稳定性都明显提高了. 重复使用8次仍能保持初始活力的65%, 且在4 ℃下保存1个月几乎观察不到酶活力的下降. 此外, 失活的固定化酶经浓硝酸处理后, NPG载体可重复利用. 本结果初步显示出了NPG在生物技术领域中的应用潜力.