基于巯基自组装单层膜的日本血吸虫石英晶体微天平免疫传感器

应用自组装膜技术在压电石英晶振金电极表面自组装一带羧基的巯基丙酸单层膜,通过盐酸1-乙基-3-(3-二甲基氨基丙基)碳二亚胺及N-羟基琥珀酰亚胺共价固定32KD的日本血吸虫分子抗原(SjAg32),设计了石英晶振微天平免疫传感器,用于测定日本血吸虫抗体.比较了巯基自组装单层膜与HEMA-MMA共聚物涂层修饰的石英晶振在溶液中的振荡行为,发现巯基自组装单层膜修饰的石英晶振稳定快,且稳定性好.在优化条件下,测得IRS(49-2000)的滴度为1:1500.此外,对不同程度血吸虫感染的兔血清进行了测试,结果表明,该传感器能较好地定量区别血吸虫感染程度.     

Biofouling of dextran-derivative layers investigated by quartz crystal microbalance

This study reports the fouling of carboxymethyl dextran (CMD) layers in cell culture medium, fibronectin, and serum solutions. CMD layers were covalently immobilized onto amine groups available either on an n-heptylamine plasma polymer (HApp) layer or onto a polyethylenimine (PEI) coating grafted to an acetaldehyde plasma polymer (AApp) layer. The successful immobilization of the graft layers was demonstrated by X-ray photoelectron spectroscopy (XPS). Primary amines on HApp and AApp + PEI surfaces were quantified using a colorimetric assay. Quartz crystal microbalance (QCM) was used to investigate in real-time the fouling of the graft layers upon incubation in cell culture medium (RPMI), fibronectin, and foetal bovine serum (FBS) solutions. HApp, AApp and AApp + PEI layers exhibited large fouling in fibronectin and FBS solutions, while fouling in RPMI cell culture medium was not significant. Protein repellent properties of CMD layers in FBS and fibronectin have been demonstrated compared to the other tested surfaces. QCM has shown that both CMD layers were fouled to a small extent in RPMI medium.     

Quartz-crystal microbalance immunosensor for Schistsoma-japonicum-infected rabbit serum.

A quartz-crystal microbalance immunosensor (QCM) has been developed for the direct determination of Schistosoma-japonicum-infected rabbit serum. A self-assembled monolayer with carboxyl groups was first coated on a gold electrode of a quartz-crystal resonator by the spontaneous adsorption of 3-mercaptopropionic acid. Schistosoma-japonicum molecular antigen of 32 kD molecular weight was then covalently attached to the crystal surface. The QCM immunosensor was used to detect infected rabbit serum (IRS49-2000); a maximum titer of 1:800 was achieved.     

Adsorption of bovine serum albumin onto poly(methyl methacrylate) stereocomplex films with a molecularly regulated nanostructure

Stereoregular poly(methyl methacrylate)s (PMMAs) were stepwise assembled on a quartz crystal microbalance (QCM) substrate after the immersion of the QCM into alternating acetonitrile solutions at ambient temperature. A quantitative QCM analysis at each step showed stereocomplex formation on the substrate surface. The adsorption of bovine serum albumin (BSA) onto stereocomplex films with a molecularly regulated nanostructure was analyzed quantitatively. The adsorption constant and the maximum adsorption amount, calculated by the assumption of Langmuir‐type adsorption, showed that BSA adsorbed with a relatively weak interaction onto the stereocomplex films. The BSA adsorption onto the stereocomplex films occurred in an end‐on manner, with a smaller adsorption constant than for that onto individual spin‐coated films. The amount of BSA adsorbed was significantly affected by the molecular weight of syndiotactic PMMA. Attenuated total reflection spectra indicated that BSA adsorbed onto the films with or without denaturing. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 1807–1812, 2003     

Influence of Poly(ethylenimine) on the Monolayer of Oleic Acid at the Air/Water Interface

The effect of poly(ethylenimine) (PEI) dissolved in water on the surface pressure-area isotherm of oleic acid (OA) at the air/water interface was investigated. On a concentrated PEI solution, the isotherm of the OA monolayers exhibited a noticeable difference as a function of subphase pH. PEI caused the collapse pressure of the OA monolayer to increase up to 45 mN/m, due to a stronger acid-base-type interaction occurring between the amine group of the PEI and the carboxyl group of OA; on a pure water subphase, the collapse pressure was;28 mN/m. On the other hand, owing to a stronger OA-PEI interaction, the OA monolayers favored a liquid-expanded state more on the PEI-containing water subphase than on the pure water. From the QCM measurement, each OA molecule appeared to interact, on average, with 4.3-5.8 ethylenimine repeating units at basic pHs. We also found that OA multilayers could be assembled on a hydrophilic substrate by a Z-type Langmuir-Blodgett (LB) deposition in a PEI-containing subphase at basic pHs. The ATR-IR spectral data revealed that, in a Z-type LB film, the headgroup of OA was mostly present as carboxylate, interacting in an ionic state with the protonated amine groups of PEI. In acidic conditions, neither a Y-type nor a Z-type deposition was really accomplished. Nonetheless, the ATR-IR spectral data suggested that OA molecules should exist in a monomeric state in a LB film assembled at acidic pHs without PEI while they would form intermolecular hydrogen bridges and/or dimers in the presence of PEI. Copyright 2000 Academic Press.     

牛血清白蛋白在超薄纳米二氧化钛膜表面的印迹与吸附

基于溶胶凝胶分子印迹方法,以溶胶二氧化钛TiO2为基质印迹了牛血清白蛋白分子。用1%的NaOH溶液可有效地除去纳米TiO2印迹膜中的模板分子。采用石英晶体微天平现场技术,研究了牛血清白蛋白在超薄纳米TiO2膜表面的吸附行为。研究表明,牛血清白蛋白在印迹膜和非印迹膜上的吸附量都随溶液浓度增加而增大,印迹膜具有吸附的特异性和可再生性,其吸附量是非印迹膜的3~5倍;在非印迹膜上的吸附符合Langmuir吸附模型,而在印迹膜上的吸附符合allosteric吸附模型;牛血清白蛋白在非印迹膜上的吸附量先随pH升高而增大,当pH为5左右时达到最大值,随后吸附量又随pH的增大而减小;而在印迹膜上其吸附量仅随pH增大而增大。     

Electrochemical decomposition of layer-by-layer thin films composed of TEMPO-modified poly(acrylic acid) and poly(ethyleneimine)

The decomposition of layer-by-layer (LbL) thin films composed of 2,2,6,6-tetramethylpiperidine-1-oxyl free radical-appended poly(acrylic acid) (TEMPO-PAA) and poly(ethylenimine) (PEI) was studied by using a quartz crystal microbalance (QCM) and cyclic voltammetry. The electrode potential of the (PEI/TEMPO-PAA)₄/PEI film-coated Au resonator was scanned from +0.2 to +0.8 V vs Ag/AgCl. The CV showed that the oxidation peak current decreased as the number of scans increased. The change in the resonance frequency of the QCM increased after electrolysis, indicating that the film was decomposed by electrolysis. The positive charges originating from the oxoammonium ions probably destabilized the (PEI/TEMPO-PAA)₄/PEI film. Furthermore, the release of 5,10,15,20-tetraphenyl-21H,23H-porphine tetrasulfonic acid (TPPS) from TPPS-loaded (PEI/TEMPO-PAA)₄/PEI-coated ITO electrodes was investigated. TPPS was released at electrode potentials greater than +0.6 V by the decomposition of the film. The results suggest that TEMPO-PAA/PEI LbL films are suitable for electrochemically controlled drug delivery systems.     

不同表面性质聚电解质多层膜的制备及蛋白质吸附和血液相容性能

利用层层自组装方法制备了聚烯丙基铵盐酸盐(PAH)/聚苯乙烯磺酸钠(PSS)多层膜. 通过吸附或共价偶联, 在多层膜表面修饰了聚乙二醇(PEG)、牛血清白蛋白(BSA)或肝素, 通过石英晶体微天平(QCM)、椭圆偏振光谱和原子力显微镜(AFM)研究了多层膜的表面形貌及修饰方法对各种蛋白的吸附性能. 经修饰后的多层膜较基底膜的厚度均有所增大; 最外层经修饰后的多层膜吸附的BSA、纤维蛋白原及血浆蛋白的量较未修饰多层膜均有所减少. 采用SEM观察了血小板在多层膜上的黏附情况和形态变化, 计算了血小板的黏附率. 比较各多层膜的凝血酶原时间(PT), 发现修饰后的多层膜的凝血酶原时间均有所延长, 但各组间无显著性差异.     

Quartz crystal microbalance studies on bilirubin adsorption on self-assembled phospholipid bilayers

Bilirubin adsorption on self-assembled phospholipid bilayers was studied using quartz crystal microbalance, and factors influencing its adsorption such as pH, temperature, and solution ionic strength were discussed in detail. The results show the amount of adsorbed bilirubin on self-assembled phospholipid bilayers is small at higher temperature and large at higher pH and solution ionic strength, and the adsorption kinetic parameter estimated from the in situ frequency measurement is (1.8+/-0.27)x10(6) M(-1) (mean +/- S.D.). With the present method, the desorption of adsorbed bilirubin caused by human serum albumin and the photoinduced decomposition of adsorbed bilirubin under light illumination were also examined. QCM measurement provides a useful method for monitoring the adsorption/desorption process of bilirubin on self-assembled phospholipid bilayers.     

液相色谱-串联质谱法同时测定贝类中大田软海绵酸、鳍藻毒素、蛤毒素和虾夷扇贝毒素

建立了同时测定贝类中大田软海绵酸(okadaic acid, OA)及其衍生物鳍藻毒素(dinophysistoxin-1, DTX-1)、蛤毒素(pectenotoxin-2, PTX-2)和虾夷扇贝毒素(yessotoxin, YTX)的液相色谱-串联质谱分析方法。样品经甲醇提取,固相萃取柱净化,C18色谱柱分离,经含甲酸和甲酸铵的乙腈-水溶液为流动相梯度洗脱,选择反应监测(SRM)模式检测,正、负离子切换扫描,基质标准校正,外标法定量。结果表明,OA、DTX-1和YTX的线性范围为2.0～200.0 μg/L,定量限(以信噪比(S/N)≥10计)为1.0 μg/kg; PTX-2的线性范围为1.0～100.0 μg/L,定量限为0.5 μg/kg;几种化合物的添加平均回收率为83.1%～105.7%,相对标准偏差(RSD)为3.16%～9.29%。成功应用本法对黄海灵山湾海域采集的贝类样品进行了分析,发现部分样品中含有大田软海绵酸、鳍藻毒素、蛤毒素和虾夷扇贝毒素。     

Prussian blue-glutamate oxidase modified glassy carbon electrode: A sensitive l-glutamate and β-N-oxalyl-α,β-diaminopropionic acid (β-ODAP) sensor

A sensitive biosensor has been developed for the neurotoxin β-N-oxalyl-α,β-diaminopropionic acid (β-ODAP) contained in the seeds of grass pea (Lathyrus sativus) and for l-glutamate based on glutamate oxidase (GlOx) and a Prussian blue (PB) modified glassy carbon (GC) electrode. The configuration of the system is so as to detect the hydrogen peroxide released during the enzymatic cycle at a low applied potential, −50 mV versus Ag|AgCl, in the flow injection mode. For this purpose GlOx was coupled to PB electrodeposited onto a glassy carbon electrode and stabilised by treatment with tetrabutylammonium toluene-4-sulfonate (TTS) during one of the steps in the electrodeposition. GlOx was cross-linked with glutaraldehyde (GA), bovine serum albumin (BSA) and Tween-20 on the surface of the PB modified GC electrodes. Addition of 0.01% and 0.001% polyethyleneimine (PEI) to the immobilisation mixture resulted in an enhancement of the response signal with about 35% and 62% for glutamate and β-ODAP, respectively, when using 0.01% PEI and with 164% and 200% for glutamate and β-ODAP, respectively, when using 0.001% PEI. The linear response range for β-ODAP was extended from 0.05-0.5 mM to 0.01-1 mM, when 0.001% PEI was used. However, a higher concentration of PEI, 0.1%, caused a decrease in the sensitivity of the biosensor.     

胶体金免疫层析法快速检测腹泻性贝毒软海绵酸的研究

腹泻性贝毒是一类分布较广的赤潮毒素,严重威胁到人类的健康和安全。本文用胶体金标记利用细胞融合技术制备的抗软海绵酸单克隆抗体,使用卵清蛋白合成高偶联比的包被抗原,以硝酸纤维素膜为载体,利用免疫层析技术原理,建立了快速检测软海绵酸的免疫层析试纸条方法。方法检出限12 ng/mL(0.96纳克/条)。     

预富集装置在QCM传感器检测芥子气中的运用

在化学战剂的诸种检测方法中, 质量型微传感器以其响应快速、使用简便等优点成为一种理想的检测手段. 但是这种传感器的使用往往受到检出限的限制, 对于低浓度的毒剂不能及时报警. 预富集技术的运用可以提高微传感器的检测限. 本文研制了一种预富集装置并对其进行了初步测试. 芥子气通过此预富集装置之后在QCM(石英晶体微天平)传感器上的检出限可以达到0.1 mg/m3.     

Assay of Glomalin Using a Quartz Crystal Microbalance Biosensor

Glomalin is a soil protein abundantly occurring in the soil. In the current time, knowledge about glomalin is limited and there are also missing simple test for the determination of glomalin in the environment. This work is devoted to construction of a biosensor which is expected to be a simple device for the determination of glomalin in extracts from soil samples. The biosensor was constructed using an antibody against glomalin and piezoelectric quartz crystal microbalance (QCM) sensor platform allowing label free assay. Electrodes of QCM were activated using cysteamine and glutaraldehyde and finally, an antibody against glomalin was immobilized. Glomalin was acquired from various soil samples by extraction in an autoclave and its content was determined by a standard spectrophotometric test. Time necessary to bind sufficient amount of glomalin was discovered for the biosensor and four hours incubation interval corresponded with maximal efficacy. Limit of detection for the biosensor based assay was found to be equal to 3.40 μg/g which is enough to cover all the tested soil samples containing glomalin in a concentration from 291 μg/g to 3.47 mg/g. The assay also fully correlated with the standard tests. In a conclusion, the piezoelectric biosensor seems to be a suitable platform for the determination of glomalin in samples of environment origin. The method represents an improvement of the current analytical platforms that are based on measurement of total protein content in soil extract.     

Characterization of fatty acid esters of okadaic acid and related toxins in blue mussels (Mytilus edulis) from Norway

Marine algal toxins of the okadaic acid group can occur as fatty acid esters in blue mussels, and are commonly determined indirectly by transformation to their parent toxins by alkaline hydrolysis. Some data are available regarding the identity of the fatty acid esters, mainly of palmitic acid (16:0) derivatives of okadaic acid (OA), dinophysistoxin-1 (DTX1) and dinophysistoxin-2 (DTX2). Other fatty acid derivatives have been described, but with limited mass spectral data. In this paper, the mass spectral characterization of the [M-H](-) and [M+Na](+) ions of 16 fatty acid derivatives of each of OA, DTX1 and DTX2 is presented. The characteristic fragmentation of [M+Na](+) ions of OA analogues provided a useful tool for identifying these, and has not been described previously. In addition, a set of negative ion multiple reaction monitoring (MRM) methods was developed for direct determination of 16 fatty acid esters of OA, 16 fatty acid esters of DTX1 and 16 fatty acid esters of DTX2 in shellfish extracts. The MRM methods were employed to study the profiles of fatty acid esters of OA analogues in blue mussels and to compare these with fatty acid ester profiles reported for other groups of marine algal toxins.     

GC‐FID determination and pharmacokinetic studies of oleanolic acid in human serum

Analytical interest of OA determination in human serum has increased owing to the increasing interest in pharmaceutical research by pharmaceutical properties. A simple, specific, precise and accurate GC method with flame ionization detector (FID) developed and validated for the determination of oleanolic acid (OA) in human serum (HS). To an aliquot of HS, internal standard was added and a combination of liquid–liquid extraction with a mixture of diethyl ether‐isopropyl alcohol, filtration and consecutive GC resulted in separation and quantification of OA. The organic phase was analyzed using a GC system equipped with a 30 × 0.25 mm i.d. Rtx‐65TG capillary column and FID detection. Total chromatographic time was 10 min and no interfering peaks from endogenous components in blank serum were observed. The OA/internal standard peak area ratio was linearly fitted to the OA concentration (r = 0.992) over the range 10–1500 ng/mL. The mean serum extraction recovery of OA was 96.7 ± 1.0% and the lower limit of quantification based on 5 mL of serum was 10.7 ng/mL. The intra‐day coefficient of variation ranged from 1.3 to 3.6% and inter‐day varied from 1.4 to 4.5%. The developed method was used to study the pharmacokinetics of OA after oral administration in humans. The assay was simple, sensitive, precise and accurate for the use in the study of the mechanisms of absorption and distribution of OA in humans. Copyright © 2015 John Wiley & Sons, Ltd.     

New nanostructured electrochemical biosensors based on three-dimensional (3-mercaptopropyl)-trimethoxysilane network

The design and characterization of a new nanostructured organic-inorganic hybrid material and its application to L-lactic acid determination are described. This material is based on the integration of the enzyme lactate oxidase (LOx) and gold nanoparticles (AuNPs) into a sol-gel 3D polymeric network derived from (3-mercaptopropyl)-trimethoxysilane (MPTS) previously formed onto a gold surface. MPTS presents the advantage of forming a 3D polymeric network containing a large number of thiol tail groups distributed throughout its structure that enable both its anchoring onto gold surfaces and the AuNPs incorporation. Moreover, this matrix provides a biocompatible environment that preserves the catalytic activity of LOx after its immobilization and allows the incorporation of a high amount of enzyme, which is expected to improve the sensitivity of the final biosensing device. Characterization of the designed biosensing platform was performed using quartz crystal microbalance (QCM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) techniques. From the conjunction of these techniques, information about (i) the kinetic of LOx adsorption process in real time, (ii) the amount of LOx incorporated into the network, and (iii) the morphological characteristics at the nanometre level of the designed biosensing material was obtained. This information is very useful on the development of successful biosensing devices. Finally, the response of the biosensor to L-lactic acid was evaluated. The biosensor responds linearly to L-lactic acid in the range of 50 μM to 0.25 mM, with a sensitivity of 3.4 μA mM(-1) and a detection limit of 4.0 μM.     

Quartz crystal microbalance immunosensor for highly sensitive 2,3,7,8-tetrachlorodibenzo-p-dioxin detection in fly ash from municipal solid waste incinerators

A quartz crystal microbalance (QCM) immunosensor was developed for the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxins (TCDD) in environmental pollutants. An anti-TCDD antibody was immobilized on the gold surface of the QCM via chemical coupling, and its immunologic activity was then maintained by treatment with an artificial stabilizing reagent such as poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate). A competitive immunoreaction with TCDD conjugated ovalbumin (TCDD-ovalbumin) was used to detect TCDD. A calibration curve was obtained through the competitive immunoreaction, and linearity was shown from 100 ng mL(-1) to 0.1 ng mL(-1). Also, the cross-reactivities of the anti-TCDD monoclonal antibody were thoroughly evaluated with several TCDD derivatives. The relationships between GC-MS, ELISA, and QCM were compared using fly ash samples from a municipal solid waste, which were prepared using an accelerated solvent extractor. For 23 samples, the experimental relationship between the TCDD concentration by QCM vs. the TCDD concentration by ELISA was y= 1.07x + 2.70, r= 0.99, and the TCDD concentration by QCM vs. the toxic equivalent quantity (TEQ) value by GC-MS was y= 2.46x - 14.98, r= 0.89.     

Immunosensor for the Determination of Okadaic Acid Based on Screen-Printed Electrode

A disposable immunosensor for okadaic acid (OA), using a screen-printed electrode (SPE), was developed and characterised. Detection of the product, p-aminophenol, resulting from the reaction catalysed by alkaline phosphatase (AP), was carried out using an amperometric three-electrode system poised at a voltage of + 300 mV versus Ag/AgCl. Alkaline phosphatase was used as a label for the antigen, OA, and two kinds of alkaline phosphatase preparation were studied for the conjugation of okadaic acid. The calibration curve for okadaic acid obtained from the conjugate created from low-activity AP, 969 units/mg, was unsatisfactory in terms of sensitivity, but a high-activity conjugate delivered the required sensitivity and limit of detection. Studies on the stability of the sensor with α-OA antibody and OA-AP conjugate showed that the current response decreased drastically after one day. Stabilisation strategies have been formulated to overcome this problem. The calibration curve obtained with the high activity conjugate was linear up to 40 ng/ml of okadaic acid with a minimum concentration of analyte detected of 5 ng/ml and a detection limit of 2 ng/ml.     

pH effect on protein G orientation on gold surfaces and characterization of adsorption thermodynamics

The pH effect on adsorbed antibody-binding protein (protein G) orientation on gold (Au) and its adsorption thermodynamic characteristics were investigated using quartz crystal microbalance (QCM) and X-ray photoelectron spectroscopy (XPS). The adsorbed protein G orientation was measured by binding response of two antibody-antigen systems: the model bovine serum albumin (BSA) and the foodborne pathogen E. coli O157:H7. Surface coverage was not significantly affected by pH, but its orientation was. The most properly oriented protein G for antibody binding was achieved at near-neutral pH. Adsorption was verified by XPS measurements using nitrogen (N) 1s, oxygen (O) 1s, and Au 4p peak heights. Adsorption energetics were determined by van't Hoff and Langmuir kinetic analyses of adsorption data obtained at 296, 303, and 308 K. Large characteristic entropy change of protein adsorption was observed (ΔS° = 0.52 ± 0.01 kcal/mol·K). The adsorption process was not classical physisorption but exhibited chemisorption characteristics based on significant enthalpy change (ΔH° = -25 ± 6 kcal/mol).