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.     

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.     

Rapid Detection of Staphylococcal Enterotoxin B by Two-Dimensional Molecularly Imprinted Film-Coated Quartz Crystal Microbalance

We fabricated a two-dimensional (2D) molecularly imprinted sol-gel thin film-coated quartz crystal microbalance (QCM) for the rapid detection of staphylococcal enterotoxin B (SEB) by combining organosilanes and the template protein SEB on the surface of piezoelectric quartz crystal (PQC) Au-electrode by in-situ immobilization. The detection process was monitored by the QCM's frequency shift (Δf). The working range of this method was 1.0 × 10^?1–1.0 × 10³ µg/mL. The detection limit was 6.1 ng/mL, which was lower than that of the PQC immunosensor, and the detection period was within 0.5 h. The reproducibility of the imprinted film-coated QCM was satisfactory due to no significant statistical difference (P > 0.05) in the rapid detection of SEB between intra- and inter-batch. The selectivity of the imprinted sol-gel film showed that it could discriminate the template molecule from its analogues and other guest molecules. Compared with immunochip, the imprinted film-coated QCM is more advantageous in terms of simplicity, rapidity, low cost, and sensitivity. Moreover, in real sample analysis, the recoveries of this method were 89.4–106.63%, which can be considered a favorable and applicable method for the rapid determination of SEB in real samples.     

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 ,…     

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。     

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.     

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.     

Highly reproducible hybridization assay of zeptomole DNA based on adsorption of nanoparticle-bioconjugate

A nanoparticle-bioconjugate was formed by homogeneous hybridization of one polynucleotide target with two oligonucleotide probes labelled by thiol and a nanoparticle, respectively. Deposition of the nanoparticle-bioconjugate on a gold surface by thiol-gold reaction was monitored in situ by quartz crystal microbalance (QCM) and applied for flow analysis of zeptomole amounts of polynucleotide. The formation in solution and adsorption of thiolated conjugates on gold could be fast, uniform and effective, and has been successfully exploited to construct a highly reproducible and sensitive platform for detection of target sequences. Being more rapid, reproducible, sensitive and amenable to automation than previously reported microgravimetric hybridization assays, this technology has great promise for practical applications in molecular diagnostics.     

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.     

QCM‐Based Immunosensor for the Determination of Ochratoxin A

Abstract

A piezoelectric immunosensor based on a competitive format was developed for determination of ochratoxin A (OTA) concentration. Surface modifications via two self‐assembled monolayers (SAMs) were investigated respectively and a better result was obtained with the SAM of 16‐mercaptohexadecanoic acid (16‐MHDA). The quartz crystal microbalance (QCM)‐based immunosensor was fabricated by immobilizing anti‐OTA antibodies onto the surface of the 16‐MHDA‐modified electrode, and allowing competition between free OTA and that conjugated with BSA to occur. The assay exhibited a working range of 50–1000 ng/mL and a detection limit of 16.1 ng/mL. Studies of interference and matrix effects were performed to evaluate the feasibility of the developed immunosensor for the direct analysis of OTA in real samples. Recoveries were conducted at 50, 200, and 1000 ng/g and were determined to be in the range of 142%–76%. The OTA assay is specific. No cross‐reactivates were observed with citrinin.     

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.     

A novel piezoelectric biosensor for the detection of phytohormone beta-indole acetic acid.

A novel piezoelectric immunosensor has been developed for the determination of beta-indole acetic acid (IAA) in dilute solutions. The detection is based on competitive immunoreaction between a hapten (IAA) and an antigen (IAA-BSA, hapten-protein conjugation) bound to an anti-IAA antibody, immobilized on a quartz crystal microbalance (QCM). The frequency change (y) of the sensor caused by antigen is linearly related to the logarithm of the concentration of IAA (x) in the range of 0.5 ng/ml - 5 microg/ml with a regression equation of the form y = -23x + 151 (r = 0.9937).     

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.     

QCM-DNA biosensor based on plasma modified PT/TiO2 nanocomposites

A quartz crystal microbalance DNA biosensor based on plasma prepared polythiophene /titanium dioxide (PT/TiO₂) nanocomposite was developed for the detection of genetically modified organisms (GMOs). DNA hybridization was studied by quartz crystal microbalance (QCM) and cyclic voltammetry (CV) measurements. Single stranded DNA probes were immobilized on the PT/TiO₂ coated quartz crystal electrode and the hybridization between the immobilized probe and the target complementary sequence in solution was monitored. The developed QCM-DNA biosensor represented promising results for a real-time, label-free, direct detection of DNA samples for the screening of genetically modified organisms.     

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.     

A review on novel developments and applications of immunosensors in food analysis

The present review deals with novel developments in immunosensors destined for final application in food analysis. In this perspective particular emphasis will be given to the most important approaches which recently have been used for immunosensor construction and assembling. For this reason, electrochemical, surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) techniques will be explored in detail and recent and practical examples on food matrices will be reviewed. Objective of this survey is to give a general overview of the possible application of immunosensors to the food analysis field.     

QCM/HCC as a platform for detecting the binding of warfarin to an immobilized film of human serum albumin

Quartz crystal microbalance/heat conduction calorimetry (QCM/HCC) is a new measurement technology that has been used to monitor simultaneously the mass and motional resistance of a thin film in conjunction with the heat flow produced by a chemical change in the film initiated by reaction with a gas. In this work we examine the applicability of the QCM/HCC in detecting chemical changes at the solution/thin film interface. Human serum albumin (HSA) was bound to the gold electrode of a 5 MHz AT-cut quartz resonator using three types of linkers and then exposed to buffered solutions of the anticoagulant drug warfarin. Changes in resonator frequency and motional resistance as well as changes in heat flow produced by warfarin binding to HSA were monitored as a function of the warfarin concentration. Differences in frequency and motional resistance changes depend upon the linker and vary both in magnitude and sign, whereas the integrated heat signal is proportional to the concentration of warfarin and independent of the linker chemistry. Quartz crystal microbalance/heat conduction calorimetry can thus be a useful tool for studying protein-ligand interactions at the solution-surface interface, even though the quartz resonator does not behave as a microbalance.     

Quartz crystal microbalance immunoassay for carcinoma antigen 125 based on gold nanowire-functionalized biomimetic interface

Gold nanowires with of designed length on a solid substrate have been proven as an efficiently immobilized affinity support for the detection of carcinoma antigen 125 (CA 125) in this study. The presence of gold nanowires provides a well-defined three-dimensional structure, and greatly amplifies the coverage of the anti-CA 125 protein on the probe surface. Moreover, the amount of anti-CA 125 varied with the change of the morphology of the probe, and achieved an optimal quartz crystal microbalance (QCM) response towards anti-CA 125 adsorption at the number of gold nanolayers of 5. The formed immune-probe exhibits good QCM responses for the detection of CA 125, and allows the detection of CA 125 at concentrations as low as 0.5 U ml(-1). The QCM immunosensor exhibited good precision, high sensitivity, acceptable stability, accuracy and reproducibility. The as-prepared immunosensors were used to analyze CA 125 in human serum specimens. Analytical results suggest that the developed immunoassay method is a promising alternative approach for detecting CA 125 in the clinical diagnosis. Compared with conventional ELISA, the proposed immunoassay system was simple and rapid without multiple labeling and separation steps. Importantly, the route provides an alternative approach to incorporate multiple gold nanolayers onto the solid matrix for biosensing applications.     

Development of molecularly ımprınted polymer based quartz crystal mıcrobalance nanosensor for the determınatıon of tryptophan

ABSTRACT

In this study, a quartz crystal microbalance (QCM) nanosensor was prepared to detect tryptophan. QCM nanosensor was prepared through the formation of tryptophan memories on the gold surface of QCM electrode using Methacryloylamidohistidine-Cu(II)-tryptophan ([MAH-Cu(II)]-tryptophan) pre-organised monomer system. The designed pre-organised monomer system was characterised by use of Fourier Transform Infrared (FTIR) and Atomic Force Microscope (AFM) was used to characterise the QCM nanosensors. After the characterisation studies, imprinted and non-imprinted sensors were connected to QCM system to determine the binding of the target molecule, selectivity and the detection of the amount of target molecule in real samples. The results showed that the imprinted QCM nanosensor had high selectivity for tryptophan.     

Electrochemically oriented immobilization of antibody on poly-(2-cyano-ethylpyrrole)-coated gold electrode using a cyclic voltammetry

Using quartz crystal microbalance (QCM) as an immunosensor, this work investigates the contribution of a cyclic voltammetry (CV) on the proper immobilization of antibodies with the aim of enhancing its target recognition and binding ability. Primarily, CV in the range of −0.1 to 0.9 V was applied to form a layer of poly-(2-cyano-ethylpyrrole) (PCEPy) on gold quartz crystal electrode. Then the efficiencies of antibodies (anti-IgG, AIgG) immobilized electrochemically with CV applied in 0-0.65 V were compared to those immobilized via physical adsorption, by observing relative affinity towards AIgG-Fab and AIgG-Fc fragments. The results showed antibody-AIgG-Fab interaction could be enhanced about 4 times when CV is applied (11.2 ± 1.3 vs 41.6 ± 3.4 relative fluorescence unit). On the contrary, physisorbed antibodies showed a higher degree of affinity towards AIgG-Fc indicating inappropriate orientations of physisorbed antibodies. AIgG immobilized PCEPy-gold QC electrode was characterized further for its sensitivity towards a new target bovine albumin with both a QCM and fluorescence measurement. Such electrode exhibited a good sensitivity as well as a large linear dynamic range, from 0.4 μg/ml to 1.0 μg/ml and from 0.5 μg/ml to 10.0 μg/ml, at QCM and fluorescence measurement, respectively.