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.     

A piezoelectric immunoassay based on self-assembled monolayers of cystamine and polystyrene sulfonate for determination of Schistosoma japonicum antibodies

A piezoelectric immunosensor based on an improved immobilization strategy combining self-assembled monolayers (SAM) of cystamine (Cys) and polystyrene sulfonate (PSS) has been developed for the determination of Schistosoma japonicum antibodies (SjAb) in rabbit serum. Cys SAM were first applied to the gold electrode surface of the crystal, serving as a positively-charged base. Schistosoma japonicum antigen (SjAg) was then electrostatically immobilized on the crystal by means of a negatively-charged PSS layer. When sealed by use of an appropriately selected blocking reagent for BSA and normal rabbit serum (NRS), non-specific adsorption could be substantially reduced.The immunosensor was used to determine SjAb in optimized buffer medium with addition of poly(ethylene glycol) (PEG), which served as an immunoreaction enhancer. It was shown experimentally that SjAg immobilized by the Cys-PSS adsorption procedure had higher immunological activity or binding efficiency than those immobilized by the glutaraldehyde (GLU) binding or direct attachment procedures. The immunosensor developed had satisfactory sensitivity and detection limit, and regeneration of the piezoelectric quartz-crystal was easy. Analytical results obtained with infected rabbit serum samples indicated that the proposed immunosensor is a promising alternative for qualitative and quantitative determination of SjAb in clinical diagnosis of infection with Schistosoma japonicum.     

基于等离子体聚合膜的日本血吸虫压电免疫传感器的研究

提出了一种测定日本血吸虫抗体的可逆压电免疫传感器。先在石英晶振上沉积正丁胺等离子体聚合膜,再自组装聚电解质,用以静电吸附固定日本血吸虫抗原。然后采用BSA和NRS作封闭剂,以封闭晶振上非特异必吸附位点,实现对日本血吸虫感染兔血清的测定。探讨了聚电解质(PSS和AASS)自组装、抗原包被和免疫反应等实难条件的影响;考察了该传感器的响应特性与再生性能,并与采用戊二醛共价键合固定法进行比较。发现该传感器具有灵敏度高、重现性好、非物异性吸附低、再生简便等优点。将它用于测定一系列不同感染程度的兔血清样本,结果表明,该传感系统是临床定性和定量诊断日本血吸虫病的一种有效工具。     

A novel biosensing interfacial design based on the assembled multilayers of the oppositely charged polyelectrolytes

A novel biosensing interfacial design strategy has been produced by the alternate adsorption of the oppositely charged polyelectrolytes. A quartz-crystal microbalance (QCM) as a model transducer was modified by use of mercaptoacetic acid (MAA) self-assembled monolayer (SAM) and the adsorption multilayers of the oppositely charged polyelectrolytes. MAA-SAM was first applied to the gold electrode surface of the crystal, and the positively charged chitosan was used as a double-sided linker to attach the negatively charged alginate-HSA antibodies to the negatively charged MAA-SAM layer. The assembly process and conditions were studied using the real-time output device and the surface topologies of the resulting crystals were characterized by atomic force microscopy (AFM) imaging. It is discovered that the optimal pH of immobilizing antibodies was 7.2 and the suited dilution ratio of antibodies was 10:30. The proposed immunosensor in optimal conditions has a linear detection range of 12.3-184.5 μg/mL for HSA detection. Comparing with the direct immobilization method of antibodies, the immunosensor with the proposed immobilization procedure shows some advantages, such as improved sensitivity due to the well-retained antibody activity and the significantly extended detection range. In particular, the regeneration of the developed immunosensor was simple and fast. Analytical results indicate that the developed immobilization procedure is a promising alternative for the immobilization of biorecognition element on the electrode surface.     

Application of a flow type quartz crystal microbalance immunosensor for real time determination of cattle bovine ephemeral fever virus in liquid

Bovine ephemeral fever (BEF) is a viral disease of cattle. A flow type quartz crystal microbalance (QCM) immunosensor was developed for the real time determination BEF virus (BEFV) that is suitable for clinical point-case diagnosis. Self-assembled monolayer (SAM) of thiols and sulphides by the cystamine–glutaraldehyde method was used for the immobilization of BEFV monoclonal antibody on the gold surface of a quartz crystal microbalance (QCM). A positive correlation was found between the virus concentration and frequency changes (R² = 0.9962) on this QCM system. The reproducible rates for the 50 and 10 μg/mL samples were 4 and 13.9%, respectively. There was no interference from non-specifically adsorbed phage. Using this flow type QCM immunosensor, BEFV could specifically be detected with sensitivity comparable to a conventional enzyme-linked immunosorbent assay (ELISA). The measurement could be obtained directly, within several minutes, rather than hours as required visualizing the results of ELISA. In addition, the observation of reproducible and constant changes after successive additions of BEFV suggests that a QCM immunosensor in a flow cell could be developed for automated or continuous real time operation.     

Enzymatic preparation of biotinylated naturally-occurring sialylglycan and its molecular recognition on a quartz-crystal microbalance

A biotinylated sialylglycan was prepared enzymatically by endo-M, and binding behavior of an SSA lectin was studied on a different coverage of a sialylglycan-immobilized 27 MHz quartz-crystal microbalance (QCM).     

Protein adsorption on surfaces: dynamic contact-angle (DCA) and quartz-crystal microbalance (QCM) measurements

Adsorption of the protein bovine serum albumin (BSA) on gold has been tested at various concentrations in aqueous solution by dynamic contact-angle analysis (DCA) and quartz-crystal microbalance (QCM) measurements. With the Wilhelmy plate technique advancing and receding contact angles and the corresponding hysteresis were measured and correlated with the hydrophilicity and the homogeneity of the surface. With electrical admittance measurements of a gold-coated piezoelectrical quartz crystal, layer mass and viscoelastic contributions to the resonator's frequency shift during adsorption could be separated. A correlation was found between the adsorbed mass and the homogeneity and hydrophilicity of the adsorbed film.     

In vitro selection and evaluation of RNA aptamers that recognize arginine-rich-motif model peptide on a quartz-crystal microbalance

To study RNA-peptide interactions, we performed an in vitro selection of RNA on a simple alpha-helical peptide-immobilized quartz-crystal microbalance (QCM) and evaluated the association constants (10(7) M-1) of the selected RNA to the model peptide on the same QCM plate.     

一种新的转铁蛋白压电免疫传感器的研究

报道了一种基于等离子体聚合膜、结合聚电解质设计的转铁蛋白压电免疫传感器.采用辉光放电等离子体聚合技术,在石英晶体上沉积一层正丁胺聚合膜,再在膜上自组装一层易再生的、带负电的聚电解质,调节抗体溶液的pH值使其带正电,经静电吸附包被抗体后用以测定抗原.探讨了自组装聚电解质的浓度和自组装时间,抗体的包被浓度、包被时间和pH值以及免疫反应的酸度、温度及响应频率与时间的关系等实验条件的影响.考察了传感器的灵敏度、选择性、重现性和再生性能.用传感器测定人血清中转铁蛋白的线性范围为0.10～12.65μg/mL.将其用于实际样品中转铁蛋白的测定,结果与酶联免疫法基本一致.     

基于邻苯二胺电聚合膜的抗体固定化方法研究

Si等 [1] 在压电石英晶体金电极表面先电聚合了一层聚苯胺膜 (PAn) ,再于 PAn膜上电聚合一层聚间苯二胺膜 (Pm PD) ,形成一双层膜 (Pm PD和 PAn) ,而后通过戊二醛共价键合固定化方法 ,实现对生物蛋白质分子的固定和对生物细胞的测定 .但在上述方法中 ,传感器难以再生且蛋白质分子的固定量较少 .参照文献 [2 ],本文提出了一种在电聚合邻苯二胺薄膜上进行可逆的抗体固定化的新方法 .通过控制溶液的 p H值 ,在带正电的电聚合邻苯二胺膜表面先自组装一层聚阴离子聚苯磺酸根 (PSS)层 ,使传感器得到一个带负电的载体表面 ,再通过静电吸附 ,…     

无标记型免疫传感器的原理及其应用

无标记型免疫传感器能够直接测定生物样品，测定过程中无需预先对被测物进行标记，适应直接、实时、原位、在线的痕量免疫分析，已经广泛应用于临床医学、环境、制药、食品等分析领域中。表面等离子共振(SPR)型免疫传感器、石英晶体微天平(QCM)型免疫传感器、电容型免疫传感器即是目前报道较多的3种无标记型免疫传感器。本文分别对这3种免疫传感器检测原理、传感器构建方式以及在生物分子检测中的应用进行了简要综述。     

Chiral discrimination of limonene by use of beta-cyclodextrin-coated quartz-crystal-microbalances (QCMs) and data evaluation by artificial neuronal networks

The enantiomeric composition of the chiral flavoring agent limonene was analyzed by means of a quartz-crystal microbalance (QCM) sensor. As chiral selectors three different modified beta-cyclodextrins were investigated. The selector molecules were applied as mixtures in different polysiloxane matrices. The chiral separation factors alpha for limonene obtained at 30 degrees C by gas chromatography and by use of the QCM sensor were comparable. Evaluation of sensor data was performed by use of an artificial neuronal network (ANN); this enabled prediction of the enantiomeric composition of the gas mixtures.     

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.     

A kinetic study of protein binding to ecabet sodium using quartz-crystal microbalance.

To define the mechanism of the protection by ecabet sodium of the gastric mucosa, the characteristics of protein binding of this drug were investigated using a quartz-crystal microbalance (QCM) method. The binding rate constants (kb) and the binding amounts (delta m) were obtained from time courses of the frequency decrease (mass increase) of the QCM. The binding constants to proteins of two ecabet analogues (G1, ecabet type and G2, non-ionic ecabet type) were dependent on the pH, leading to large kb values at the acidic region. Furthermore, the kb values of G1 with the addition of bovine serum albumin (BSA) and bovine serum fibrinogen (BSF) at the acid region were larger than those of G2. The difference in kb values between G1 and G2 for porcine gastric mucin (PGM) is hardly discernible. Ecabet seems to be more heavily distributed in the ulcerous areas than in the intact mucosa, judging from the large binding constants of this drug to BSA and BSF compared with those to PGM. It is suggested that ecabet is bound to proteins by hydrophobic interaction, moreover, the electrostatic interaction between this drug and proteins (BSA and BSF) occurs at acidic pH region. On account of these interactions, ecabet sodium seems to have a more protective effect on an ulcer at intraluminal acidity than sucralfate. Finally, QCM was found to be a useful technique for detecting quantitatively the time course of binding proteins with drug.     

Dynamic analyses on induced-fit gaseous guest binding to organic crystals with a quartz-crystal microbalance

The inclusion behavior of gaseous guest molecules in a solid apohost, an orthogonal anthracene-bis(resorcinol)tetraol (1), was investigated with a quartz-crystal microbalance (QCM). Compound 1 forms crystals composed of molecular sheets bound together by an extensive hydrogen-bonded network. An apohost of 1 was cast onto a QCM and the binding of gaseous guest molecules was followed as a function of time by observing the decrease in the oscillation frequency, which is directly related to the increase in mass. Ethyl acetate and methyl ethyl ketone were significantly included into the apohost, whereas benzene and cyclohexane were simply adsorbed onto the surface of the solid; all these guests have similar vapor pressures at 25 degrees C. On the other hand, a host analogue 2, a tetramethoxy derivative of 1, barely included these guest molecules. The inclusion amount and the rate of inclusion of ethyl acetate or methyl ethyl ketone showed a drastic increase above a threshold concentration of guests in the gas phase. Thus, the structure of the apohost changed cooperatively in order to bind guest molecules above the threshold guest concentration. This cooperativity of the binding behavior was kinetically analyzed.     

High sensitivity microgravimetric biosensor for qualitative and quantitative diagnostic detection of polychlorinated dibenzo-p-dioxins

A piezoelectric immunosensor system was developed for the rapid detection of polychlorinated dibenzo-p-dioxins (PCDDs). The system uses a competitive inhibition enzyme immunoassay (EIA) based on a mouse monoclonal antibody that is specific for 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and a conjugate of a dioxin-like competitor coupled to the enzyme horseradish peroxidase (HRP). The anti-dioxin antibody was deposited on a 10 MHz AT-cut quartz crystal resonator modified with a self-assembly monolayer of dithiobis-N-succinimidyl propionate. PCDDs at different concentrations in the range 0.001-10 ng mL-1 were mixed with a constant amount of HRP-conjugated competitor. The frequency responses due to the adsorption of the mixed samples on the biosensor surface were measured. The results show that 2,3,7,8-TCDD can be quantitatively detected with the developed immunosensor in the concentration range 0.01-1.3 ng mL-1. Cross-reactivities of the biosensor to various PCDD congeners were also investigated. The sensitivity and selectivity of the quartz crystal microbalance (QCM) biosensor is comparable to EIA and ELISA methods in the detection of polychlorinated dibenzo-p-dioxins. The developed QCM immunosensing system offers significant improvements in speed, sample throughout and cost for the qualitative and quantitative detection of PCDDs compared with GC-MS.     

Detection of hydrogen peroxide and glucose by enzyme product precipitation on sensor surface

Biocatalysed precipitation of an insoluble product accumulated on the enzyme-modified electrode surface was applied as the amplification path for low concentration sensing of hydrogen peroxide and glucose. Sensitive electrochemical and quartz-crystal microbalance (QCM) biosensors based on biocatalytic precipitation were developed. A horseradish peroxidase (HRP) monolayer-modified electrode was used to sense H₂O₂ via the oxidation of 4-chloro-1-naphthol (4CN) forming insoluble benzo-4-chlorocyclohexadienone. Additionally, the bienzyme system employed glucose oxidase (GOx) linked to HRP/4CN. The amount of the precipitate assembled on the sensing surface corresponded to the concentration of analytes and to the length of the incubation interval. The precipitated deposits were followed as a change of impedance using cyclic voltammetry (CV), mass change was determined continuously using a microgravimetric quartz-crystal microbalance, and optical microscopy enabled the visualisation of the precipitate. Regeneration of the enzyme-modified electrode was performed using cathodic reduction of the insoluble product. Thus, a simple biosensor for multiple analyses with low detection limits and of low cost can be developed.     

Use of polyclonal antibodies to ochratoxin A with a quartz–crystal microbalance for developing real-time mycotoxin piezoelectric immunosensors

A piezoelectric immunosensor was tested for ochratoxin A (OTA) mycotoxin detection through the immobilization of OTA–bovine serum albumin (OTA–BSA) conjugate on gold-coated quartz crystals (AT-cut/5 MHz). Immunoassays were performed in a flow-injection system through frequency decreases in a quartz–crystal microbalance (QCM) because of a mass increasing during immunoreaction with anti-OTA antibodies. Three immobilization procedures for OTA–BSA (direct adsorption and covalent attachment to two alkane thiol self-assembled monolayers) were characterized with QCM in real time. Covalent attachment of the OTA–BSA conjugates through gold nanoparticles was also tested for amplifying the signal. Binding of the excess of antibodies to the immobilized OTA in an indirect competitive analysis decreased linearly the resonant frequency in the range of the OTA concentration from 10 to 128 ng/mL, with a detection limit of 8 ng/mL (signal/noise ratio of 3). A pepsin 2 mg/mL (pH = 2.1) solution was used to release antigen–antibody complexes, regenerating the biorecognition surface.     

Development of an immunosensor for human ferritin, a nonspecific tumor marker, based on a quartz crystal microbalance

A new human ferritin immunosensor was developed using anti-human ferritin antibodies (Abs) immobilized on the gold disc of a quartz crystal microbalance (QCM). Two kinds of self-assembled monolayers (SAMs) prepared by cystamine-glutaraldehyde and cystamine method were applied to immobilize anti-ferritin monoclonal antibodies (MoAbs) and polyclonal antibodies (PoAbs) on the quartz, respectively. The reusabilities of quartz crystal adopting the SAMs were found to be better than those of the other immobilization methods used. The 10 cycles of measurements could be performed on the gold surface of the same crystal regenerated with a solution of glycine·HC1. This sensor system could be continuously performed for 15 days, the relative frequency shifts (the frequency shifts measured are relative to the response at the first day) were all found to be above 95%. A linear relationship existed between the frequency shifts (Hz) and the log values of human ferritin concentrations in the range from 0.1 to 100 ng/ml in buffer and mouse serum. This human ferritin immunosensor had some advantages: high sensitivity, high specificity, low sample requirement, high reusability, no label and no pretreatment etc.     

Immunosensor for okadaic acid using quartz crystal microbalance

An immunosensor for the determination of okadaic acid (OA) using a quartz crystal microbalance (QCM) was developed and optimised in standard solutions. Several coupling techniques, protein A, protein G and polyethylenimine (PEI) with glutaraldehyde (GA) cross-linking, were investigated for the determination of okadaic acid and a very good result was obtained with PEI coupling. With the PEI coupling method, the optimisation of incubation time for the activation of PEI on the crystal surface using GA, the effect of the dilution factor of OA-bovine serum albumin (BSA) conjugate and the amount of antibody on crystal frequency were studied. Different molar ratios (4:1, 14:1, 30:1) of OA to bovine serum albumin for the conjugation were examined and the results using ELISA and a QCM showed that a ratio of 14:1 was slightly better than the other two. The strong attachment of the cross-linked complex to the gold surface resulted in an excellent storage lifetime of 38 days. However, the detection limit (1.9 μg/ml) and the sensitivity of the sensor were not satisfactory. Significant improvement of the performance of the device was obtained by incorporating an antibody-BSA hydrogel. Initial results showed that the minimum amount of analyte detectable and the sensitivity of the device were improved by 524- and 80-fold, respectively.