Real-time QCM-D immunoassay through oriented antibody immobilization using cross-linked hydrogel biointerfaces

This report presents the development of pre-cross-linked and in situ cross-linked polyethyleneimine-carboxymethylcellulose antibody immobilization platforms for real-time QCM-D immunoassay of sepsis-related biomarkers. These platforms differ significantly from recent trends in QCM-based assays, a rapidly expanding field given the affordability and sensitivity of the transduction system, by providing ultrafast biointerface deposition through cross-linking of polysaccharides. Using rhIL-1ra (17 kDa), a known sepsis biomarker, for development, various immunoassay modifications to increase sensitivity were investigated, including the use of Protein A, Protein G, and anti-IgG Fc specific antibody capture ligands for oriented antibody immobilization, higher-frequency QCM-D crystals, and amplification using secondary antibodies. The optimized assay employs Protein A oriented immobilization on pre-cross-linked polymer and secondary antibodies to achieve a detection limit of 25 ng/mL on 5 MHz crystals. Assay repeatability using the optimized chemistry is robust, with no loss in 100 ng/mL antigen detection over 20 cycles of the 10 min sandwich assay. Nonspecific adsorption of human serum albumin, as characterized by ToF-SIMS, is minimal and negligible for the pre-cross-linked and in situ cross-linked compositions, respectively.     

Stabilization of phospholipid polymer surface with three-dimensional nanometer-scaled structure for highly sensitive immunoassay

A phospholipid polymer platform and an antibody as a bioaffinity ligand were used to construct a biointerface for a highly sensitive immunoassay. The platform had a nanometer-scaled particle deposition surface and it was constructed with poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl poly(ethylene glycol) methacrylate (MEONP)] (PMBN) by an electrospray deposition (ESD) method. The PMBN surface could immobilize specific antibodies through covalent chemical bonding by the reaction between MEONP units and amino groups in the antibody. In addition, the PMBN could prevent nonspecific protein adsorption from an analyte. However, the nanometer-scaled structure of the PMBN lost its shape after immersion in an aqueous medium. To stabilize the nanometer-scaled structure in an aqueous medium, the PMBN was cross-linked with 1,4-butylenediamine and then heat-treated. These treatments effectively improved the stability of the nanometer-scaled structure, that is, the structure had a high porosity even after immersing in an aqueous medium. The stabilization affected the specific signal in the enzyme-linked immunosorbent assay (ELISA), that is, the specific signal in ELISA was enhanced.     

A Direct Competitive Homogeneous Immunoassay for Progesterone – the Redox Quenching Immunoassay

A direct competitive amperometric immunoassay format for the detection of haptens and proteins was developed. The method is based on the quenching of electroactivity of ferrocenium, which is coupled to the antigen and used as the primary reporter, upon binding to a monoclonal anti‐ferrocenium antibody, which is coupled to the detection antibody and used as a secondary reporter. A separation‐free progesterone immunoassay with a lower detection limit of 1 ng mL^?1 (3.18 nmol L^?1) in 1 : 2 diluted blood serum was realised by combining two bifunctional conjugates, a ferrocenium‐PEG‐progesterone tracer and a bioconjugate of one anti‐progesterone and one anti‐ferrocenium antibody. The immune complex is formed within 30 s upon addition of progesterone, resulting in a total analysis time of 1.5 min.     

纳米金自组装膜的IgM压电免疫传感器的研究

利用等离子体聚合膜沉积技术和纳米金亚单层自组装技术设计传感器界面，用 于固定羊抗人M抗体，研制了一种新的M压电免疫传感器．先在石英品振上沉积正丁 胺等离子体聚合膜，通过戊二醛交联结合一半肮胺单层膜，利用膜上流基与纳米金 键合组装纳米金亚单层，得到可用于固定18kI抗体的界面，再以牛血清白蛋白 (BSA)和聚乙二醇(PEG)封闭晶振上的非特异性吸附位点．实验探讨了影响纳米金自 组装和抗体包被等主要实验参数和条件；考察了采用此固定化方法传感器的响应性 能，与戊二醛共价交联固定法和金电极表面直接吸附固定法进行了比较．结果表明 ，以纳米金单层作界面固定抗体时，具有传感界面不需活化、固定抗体的活性高、 检测时的非特异性吸附小、传感器能反复再生等优点．将传感器用于实际样品的检 测，结果令人满意．     

Surface modification for enhancing antibody binding on polymer-based microfluidic device for enzyme-linked immunosorbent assay

A novel surface treatment method using poly(ethyleneimine) (PEI), an amine-bearing polymer, was developed to enhance antibody binding on the poly(methyl methacrylate) (PMMA) microfluidic immunoassay device. By treating the PMMA surface of the microchannel on the microfluidic device with PEI, 10 times more active antibodies can be bound to the microchannel surface as compared to those without treatment or treated with the small amine-bearing molecule, hexamethylenediamine (HMD). Consequently, PEI surface modification greatly improved the immunoassay performance of the microfluidic device, making it more sensitive and reliable in the detection of IgG. The improvement can be attributed to the spacer effect as well as the functional amine groups provided by the polymeric PEI molecules. Due to the smaller dimensions (140x125 microm) of the microchannel, the time required for antibody diffusion and adsorption onto the microchannel surface was reduced to only several minutes, which was 10 times faster than the similar process carried out in 96-well plates. The microchip also had a wider detection dynamic range, from 5 to 1000 ng/mL, as compared to that of the microtiter plate (from 2 to 100 ng/mL). With the PEI surface modification, PMMA-based microchips can be effectively used for enzyme linked immunosorbent assays (ELISA) with a similar detection limit, but much less reagent consumption and shorter assay time as compared to the conventional 96-well plate.     

基于AuNPs/PDDA-GO纳米复合物的电化学免疫传感器的构建及对SirT1蛋白的检测

基于AuNPs/PDDA-GO纳米复合物制备了一种新型电化学免疫传感器, 并将其用于SirT1的检测. 首先, 在电极表面修饰复合材料AuNPs/PDDA-GO, 然后将目标蛋白SirT1固定到修饰了AuNPs/PDDA-GO的电极表面, 再通过特异性免疫反应结合一抗(Ab₁)和辣根过氧化酶标记的二抗分子(HRP-Ab₂), 最后用示差脉冲伏安法检测电流信号, 实现了对SirT1蛋白水平的测定. 在优化的实验条件下, SirT1蛋白的浓度在0.1~100 ng/mL范围内与响应电流呈良好线性关系, 检出限为0.029 ng/mL.     

A novel piezoelectric immunosensor for detection of carcinoembryonic antigen

A simple, rapid, and highly sensitive immunosensor for the direct determination of carcinoembryonic antigen (CEA) in human serum using a piezoelectric crystal has been developed and optimized. In order to improve sensitivity of the immunosensor, a protein A-based orientation-controlled immobilization method for antibodies was adopted together with an immunoreactive accelerant of polyethyleneglycol (PEG) used to amplify the signal response of frequency. Human normal serum was utilized as a reference background. The linear range for CEA concentration obtained by the end-point method was 66.7-466.7 ng/mL. Clinical samples from cancer patients were analyzed by the proposed piezoelectric immunoassay, and the analytical results were reasonably comparable with those obtained by the chemiluminescence immunoassay (CLIA). The proposed immunosensor provides a new promising method for the highly sensitive immunoassay of CEA in clinical laboratory.     

Surface plasmon resonance aided electrochemical immunosensor for CK-MB determination in undiluted serum samples

This article presents a simple chronoamperometric immunosensor for the quantitative assessment of creatine kinase MB (CK-MB) in 50 μL undiluted serum samples. The immunosensor consists of gold working and counter electrodes patterned onto a glass chip by thin-film photolithography and an external Ag|AgCl reference electrode. The detection limit (DL) of the chronoamperometric method is 13 ng mL⁻¹ (DL = 2×RMSD/S, where RMSD is the residual mean standard deviation of the measured points around a calibration curve with a slope of S). In spiked serum samples, the response was linear up to 300 ng mL⁻¹ of CK-MB. A surface plasmon resonance (SPR) system with simultaneous electrochemical detection (EC-SPR) aided the development of the sandwich immunoassay. Real-time monitoring of the SPR signal was used to optimize the capture antibody immobilization, CK-MB and detection antibody binding, as well as to minimize the nonspecific adsorption of serum proteins to the sensor surface. The detection antibody has been labeled with alkaline phosphatase (ALP) enzyme for sensitive electrochemical detection. ALP catalyzes the hydrolysis of ascorbic acid phosphate and generates ascorbic acid, which is measured chronoamperometrically. The electrochemical immunoassay for CK-MB was less sensitive to nonspecific adsorption related interferences, had a better detection limit, and required a lower volume of sample than the SPR method.     

具有内皮细胞选择性的细胞膜仿生支架材料的研究

利用AIBN引发自由基反应,由单体2-(甲基丙烯酰氧基)乙基-2-(三甲基氨基)乙基磷酸酯(MPC)、甲基丙烯酸十八酯(SMA)、对硝基苯氧羰基聚乙二醇甲基丙烯酸酯(MEONP)合成了一种新型类细胞膜仿生涂层材料.MPC可以阻抗非特异性吸附;MEONP可以结合抗体或多肽促进特异性识别.通过表面固定的方法引入多肽序列Arg-Glu-Asp-Val(REDV),使涂层具有内皮细胞选择性.核磁、紫外吸收、红外光谱表征证实聚合物的组成以及REDV多肽在表面的固定;并通过血浆复钙化实验表征涂层的血液相容性.细胞黏附与增殖实验反映REDV多肽构建的涂层表面具备良好的特异性识别并结合内皮细胞的能力.     

Magnetic beads-based electrochemical immunosensor for detection of pseudorabies virus antibody in swine serum

A novel magnetic electrochemical immunosensor has been developed for the detection of pseudorabies virus antibody in swine serum. The magnetic glass carbon electrode was fabricated to manipulate magnetic beads for the direct sensing applications. Magnetic beads were employed as the platforms for the immobilization and immunoreaction process, and gold nanoparticles were chosen as electroactive labels for the electrochemical detection. The parameters concerning the assay strategy were carefully investigated. Under the optimal conditions, the linear response range of pseudorabies virus antibody dilution ratio (standard positive serum) was 1:250 to 1:1000 with a detection limit of 1:1000. Finally, this developed immunoassay method was successfully applied in the detection of pseudorabies virus antibody in swine serum, and had a good diagnostic accordance in comparison with ELISA.     

Determination of immunoglobulin G in bovine colostrum and milk by direct biosensor SPR-immunoassay

An automated biosensor surface-plasmon resonance-based assay was developed for the determination of immunoglobulin G (IgG) in bovine milk and colostrum with either goat or rabbit antibovine IgG or protein G used as detecting molecule. The method is configured as a direct and nonlabeled immunoassay, with quantitation against an authentic IgG calibrant. Whole colostrum or milk is prepared for analysis by dilution into buffer. Analysis conditions, including ligand immobilization, flowrate, contact time, and regeneration, were optimized, and nonspecific binding was evaluated. Performance parameters included working range of 15-10 000 ng/mL, method detection limit of 0.08 mg/mL, overall instrument response reproducibility relative standard deviation (RSD(R)) of 0.47%, mean between-run RSD(R) of 10.5% for colostrum, and surface stability over 200 analyses. The proposed method was compared with independent alternative methods. The technique was applied to the measurement of IgG content during early lactation transition from colostrum to milk, as well as in consumer milk, colostrum, and hyperimmune milk powders.     

A review on tough and sticky hydrogels

In this review, we survey recent literature (2009–2013) on hydrogels that are mechanically tough and adhesive. The impact of published work and trends in the field are examined. We focus on design concepts, new materials, structures related to mechanical performance and adhesion properties. Besides hydrogels made of individual polymers, concepts developed to toughen hydrogels include interpenetrating and double networks, slide ring polymer gels, topological hydrogels, ionically cross-linked copolymer gels, nanocomposite polymer hydrogels, self-assembled microcomposite hydrogels, and combinations thereof. Hydrogels that are adhesive in addition to tough are also discussed. Adhesive properties, especially wet adhesion of hydrogels, are rare but needed for a variety of general technologies. Some of the most promising industrial applications are found in the areas of sensor and actuator technology, microfluidics, drug delivery and biomedical devices. The most recent accomplishments and creative approaches to making tough and sticky hydrogels are highlighted. This review concludes with perspectives for future directions, challenges and opportunities in a continuously changing world.     

Preparation and structural evolution of SiO(2)-TiO(2) pillared layered manganese oxide nanocomposite upon intercalating reaction

Poly(ethylene glycol) possessing pentaethylenehexamine at one end (N6-PEG) was prepared via a reductive amination reaction of aldehyde-ended PEG with pentaethylenehexamine. Using N6-PEG, an antibody/PEG co-immobilized surface was constructed on magnetic particles via an active ester reaction method. After immobilization of the antibody on the active ester surface, N6-PEG was reacted on the magnetic beads. A sandwich enzyme-linked immunosorbent assay (ELISA) system was newly constructed using PEG/antibody co-immobilized magnetic beads combined with an alkaline phosphatase (ALP)-assisted fluorescent detection system using alpha-fetoprotein (AFP) as a model antigen. The co-immobilization of both antibody and PEG on the magnetic bead surfaces reduced the nonspecific adsorption of proteins from cell lysates. Especially, when the magnetic particle surface was modified by N6-PEG mixtures with different molecular weights of 6000 and 2500 (6 kDa:2.5 kDa=9:1 w/w), the nonspecific adsorption of proteins was strongly suppressed. It is rather surprising for us that the sensitivity of the antibody on the surface was enhanced significantly when the PEG tethered chain was constructed in between the surface antibodies. Consequently, the mixed N6-PEG treatment showed a much higher S/N ratio than for the corresponding beads treated with bovine serum albumin (BSA), a conventional blocking reagent. Actually, when alpha-fetoprotein was analyzed by the magnetic bead-assisted ELISA thus constructed, the S/N ratio was about 20-fold higher for the mixed coating with PEG (6 kDa):PEG (2.5 kDa)=9:1, compared to the conventional BSA.     

基于网状混合自组装膜的压电免疫传感器及其应用

结合纳米金及混合自组装技术, 制备了一种新型网状混合膜, 提出了一种新的生物分子固定化方法, 研制了一种用于检测人血清抗精子抗体的压电免疫传感器. 首先, 将纳米金溶胶、巯基丙酸和1,6-二巯基己烷按一定的比例混合制得网状混合自组装膜, 然后将此膜组装到压电石英晶振的金电极表面, 经EDC/NHS活化后, 再将抗原固定到电极上, 实现对抗精子抗体的检测. 结果表明, 该方法能明显提高抗体抗原结合效率, 从而提高传感器的灵敏度, 并降低传感界面的非特异性吸附. 将此传感器应用于人血清抗精子抗体的检测, 线性范围为10~800 mU/mL, 检出限为7 mU/mL. 此传感器为抗精子抗体的临床检测提供了新平台.     

Fibrous crystalline hydrogels formed from polymers possessing a linear poly(ethyleneimine) backbone

Novel thermoreversible physical hydrogels formed from polymers with linear and star architectures possessing a linear poly(ethyleneimine) (PEI) backbone have been investigated. The hydrogelation occurred simply upon natural cooling of hot aqueous solutions of PEIs to room temperature. The X-ray diffraction and differential scanning calorimetry measurements for the resultant hydrogels unambiguously indicated that the hydrogelation originated from the formation of dihydrate crystalline structures of PEI. These crystalline hydrogels are structurally unique and hierarchical. Microscopic images revealed that the morphologies of the crystalline hydrogels depend on their molecular architectures. The linear PEI resulted in branched fibrous bundles organized by unit crystalline nanofibers with a width of ca. 5-7 nm. The six-armed star with benzene ring core produced fanlike fibrous bundles while the four-armed star with porphyrin core assembled into asterlike aggregates. The critical concentration of gelation (C(G)) was low (about 0.2 approximately 0.3%) and the thermoreversible gel-sol transition temperatures (T(G)) were controllable from approximately 43 to approximately 79 degrees C. The hydrogels formed in the presence of the various aqueous additives including organic solvents, hydrophilic polymers, physical cross-linker, chemical cross-linker, and base enabling modification and functionalization during synthesis. The mechanical properties of the hydrogels could be improved by chemical cross-linking of preformed hydrogels by glutaraldehyde. Physically and physical/chemical cross-linked hydrogels served as excellent template roles in biomimetic silicification, which produced silica-PEI hybrid powder or monolith constructed by nanofibers.     

基于胶体金标记的阳极溶出伏安免疫分析用于免疫球蛋白G的测定

提出了一种基于胶体金标记的阳极溶出伏安免疫分析方法。免疫反应在聚苯乙烯微孔板中以夹心分析模式进行,通过物理吸附将兔抗人免疫球蛋白G(IgG)抗体固定于微孔板上,与相应抗原IgG发生免疫反应后,再通过夹心模式捕获相应的纳米金标记的羊抗人IgG抗体,然后再与金标羊抗人IgG抗体和金标兔抗羊二抗形成的免疫复合物反应,在微孔板上进一步引入大量的纳米金,将金溶解后,在碳糊电极上用阳极溶出伏安法(ASV)对金离子进行检测,溶出峰电流的大小间接与待分析物IgG的浓度成正比。对免疫分析的一些实验条件进行了优化。阳极溶出峰电流与IgG的对数浓度在1.1~1 143 ng/mL范围内呈良好的线性关系,检出限为1 ng/mL。将该方法应用于人血清中IgG浓度的测定,取得了满意结果。     

A galactose polyacrylate-based hydrogel scaffold for the detection of cholera toxin and staphylococcal enterotoxin B in a sandwich immunoassay format

A galactoside-based polyacrylate hydrogel was used as a scaffold to immobilize antibodies for the development of a sandwich immunoassay to detect cholera toxin (CT) and staphylococcal enterotoxin B (SEB). The hydrogel possesses large pores and simulates a solution-like environment allowing easy penetration of large biomolecules. Highly crosslinked hydrogels containing pendant amine or carboxyl functionalities were polymerized through a free-radical polymerization process. Covalent crosslinking of the antibodies on hydrogel films was accomplished using a homobifunctional crosslinker or carbodiimide chemistry. Utilizing the two different crosslinking methodologies, our results demonstrated the effectiveness of repetitive additions of crosslinker reactant into a single location on the gel surface. This approach in fact increased the amount of immobilized antibody. Patterned arrays of the immobilized antibodies for sandwich immunoassay development were achieved using a PDMS template containing micro-channels. This template provided a suitable means for applying reagents in multiple cycles. Fluorescence and three-dimensional (3D) imaging by confocal microscopy and laser scanning confocal microscopy of Cy3-labeled anti-CT and/or Cy3-anti-SEB tracer molecules provided qualitative and quantitative measurements on the efficiency of protein immobilization, detection sensitivity and signal-to-noise ratios. As a result of using the galactose polyacrylate-base hydrogel as a platform for immunoassay development, we have successfully been able to achieve low limits of detection for SEB and cholera toxins (1.0 ng mL(-1)). Repetitive additions (>3 cycles) of the crosslinker and antibody have also shown a dramatic increase in the immobilization of antibody resulting in improved immunoassay sensitivity. Fluorescence signal-to-noise ratios using the hydrogel-based immunoassays have been observed as high a 40:1.     

Surface Plasmon Resonance Immunoassay for Ochratoxin A Based on Nanogold Hollow Balls with Dendritic Surface

Abstract

A surface plasmon resonance (SPR)‐immunosensor based on nano‐size gold hollow ball (GHB) with dendritic surface has been developed for detection of Ochratoxin A (OTA). A thionine thin film was initially electropolymerized onto the SPR‐probe surface, and then anti‐OTA monoclonal antibody (anti‐OTA) was immobilized onto the SPR‐probe surface by means of GHB conjugation. The binding of target molecules onto the immobilized antibodies causes an increase in the resonant angle of the sensor chip, and the resonant angle shift was proportional to the OTA concentration in the range of 0.05–7.5 ng/ml with a detection limit of 0.01 ng/ml at a signal/noise ration of 3. A glycine‐HCl solution (pH 2.8) was used to release antigen‐antibody complexes from the biorecognition surface. Good reusability was exhibited. Moreover, spiking various levels of OTA into three milk samples was assayed using the proposed immunoassay. Analytical results show the precision of the developed immunoassay is acceptable. Compared with the conventional enzyme‐linked immunosorbent assay, the proposed immunoassay system was simple and rapid without multiple labeling and separation steps. Importantly, the proposed immunoassay system could be further developed for the immobilization of other antigens or biocompounds.     

Polyelectrolyte complex membranes for specific cell adhesion

The presentation of bioactive ligands on biomaterial surfaces is often confounded by the adsorption of proteins present in the biological milieu, rendering any type of cellular response nonspecific. We have engineered a polyelectrolyte complex membrane that demonstrates specific adhesion of various cell types for both two-dimensional (2D) and three-dimensional (3D) cell culture systems. Specific cell adhesion is achieved by a three-tiered structure: a silica cross-linked polycation as the bottom (first) tier, a nonfouling polyanion-poly(ethylene glycol) (PEG) conjugate as the intermediate (second) tier, and the cell-adhesion ligand as the top (third) tier. Each tier of the membrane was characterized in terms of chemical composition and dimensions. Epithelial cells (primary human cortical renal cells and a hepatocellular carcinoma cell line) cultured on the membranes exhibited little cell attachment on the polyanion-PEG second tier and good cell adhesion on the RGD-modified third tier. Thus, the second tier allowed the effect of cell adhesion due to the ligand (third tier) to be isolated and distinguished from nonspecific cell attachment to the first tier. For the culturing of cells in three dimensions, the three-tiered membrane system was applied using a highly swellable chitosan membrane as the first tier. The resulting cell-membrane construct was uniformly dispersed and centrifuged to form a matrix that interacted intimately with cells in the form of a pellet. Presentation of RGD in the latter format enhanced the viability of human mesenchymal stem cells (hMSCs) over controls without RGD.     

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.