Morphological and binding properties of interleukin-6 on thin ZnO films grown on (100) silicon substrates for biosensor applications

To develop effective protein immobilization technology with minimal amounts of protein for high sensitivity surface acoustic wave biosensors, we determined the binding properties, and morphological characteristics of human interleukin-6 (IL-6), a pro-inflammatory cytokine, on the surface of ZnO, and SiO(2) films grown onto (100) Si substrates, for the first time. Interleukin-6 was immobilized in the range of 0.276-10 pg/ml on the surface of ZnO and SiO(2), and visualized at each stage, while protein-protein interactions were measured with the antigen/antibody immunoassay of solid-phase ELISA, which we modified for these types of substrates. A relative mass value was determined in each case. ELISA detected upward of 1 and 6 ng/ml of protein applied on ZnO and SiO(2), respectively. It is concluded that the more reactive ZnO surface is a new and more effective template for protein immobilization.     

ProteoChip: a highly sensitive protein microarray prepared by a novel method of protein immobilization for application of protein-protein interaction studies

We have developed a highly sensitive microarray protein chip, ProteoChip, coated with ProLinker, novel calixcrown derivatives with a bifunctional coupling property that permits efficient immobilization of capture proteins on solid matrixes and makes high-throughput analysis of protein-protein interactions possible. The analysis of quartz crystal microbalance showed that both monoclonal antibody (mAb) and antigen (Ag) bound to the gold film of the sensor surface coated with ProLinker B and that it is useful for studies of Ab-Ag interactions. ProteoChip, aminated glass slide coated with ProLinker A, was also demonstrated to be useful for preparation of high-density array spots by using a microarrayer and for analysis of analyte Ags either by direct or sandwich methods of fluorescence immunoassay. The detection sensitivity of ProteoChip was as low as 1-10 femtogram/mL of analyte protein, useful for detection of tumor markers. ProteoChip was also useful for studies of direct protein-protein interactions as demonstrated by analysis of integrin-extracellular matrix protein interaction. These experimental results suggest that ProteoChip is a powerful tool for development of chip-based lead screening microarrays to monitor protein-protein interactions (i.e. drug target) as well as for biomarker assays which require high detection sensitivity.     

Novel and Efficient Protocol for DNA Coating-Based Identification of DNA-Protein Interaction by Antibody-Mediated Immunodetection

Background:Studying protein-protein and protein-DNA interactions are prerequisites for the identification of function and mechanistic role of various proteins in the cell. Protocols for analyzing DNA-based Protein-Protein and Protein-DNA interactions are complicated and need to be simplified for efficient tracking of binding capabilities of various proteins to specific DNA molecules. Here, we demonstrated a simple yet efficient protocol for the identification of DNA coating-based Protein-DNA interaction using antibodymediated immunodetection.Methods:Briefly, we have coated specific DNA in the microtiter plate followed by incubating with protein lysate. Specific protein-DNA and/or protein-protein bind with DNA interactions are identified using specific fluorophore-conjugated antibodies. Antibodies are used to detect a protein that is bound to the DNA.Results:Fluorescent-based detection identifies the specific interaction between Protein-DNA with respect to coated DNA fragments. The protocol uses indirect conjugated antibodies and hence the technique is sensitive for effective identification of Protein-DNA interactions.Conclusion:Based on the results we conclude that the demonstrated protocol is simple, efficient and sensitive for identification of Protein-DNA interactions.Key Words: DNA coating, Lamin A, Protein-DNA interaction.     

Fluorescence correlation spectroscopy as a method for assessment of interactions between phage displaying antibodies and soluble antigen

Phage display is widely used for expression of combinatorial libraries, not least for protein engineering purposes. Precise selection at the single molecule level will provide an improved tool for generating proteins with complex and distinct properties from large molecular libraries. To establish such an improved selection system, we here report the detection of specific interactions between phage with displayed antibody fragments and fluorescently labeled soluble antigen based on Fluorescence Correlation Spectroscopy (FCS). Our novel strategy comprises the use of two separate fluorochromes for detection of the phage-antigen complex, either with labeled antiphage antibody or using a labeled antigen. As a model system, we studied a human monoclonal antibody to the hepatitis-C virus (HCV) envelope protein E2 and its cognate antigen (rE2 or rE1/E2). We could thus assess the specific interactions and determine the fraction of specific versus background phage (26% specific phage). Aggregation of these particular antigens made it difficult to reliably utilize the full potential of cross-correlation studies using the two labels simultaneously. However, with true monomeric proteins, this will certainly be possible, offering a great advantage in a safer and highly specific detection system.     

Continuum electrostatic methods applied to pH-dependent properties of antibody-antigen association

Protein association events are a critical component of the functioning of biological systems. Antibody/antigen association, which involves extraordinarily specific interactions, has been a paradigm for the study of structural factors and intermolecular forces controlling protein-protein association. As new experimental approaches to the study of antibody/antigen affinity have become routine, and as more structures of complexes of antibodies and their antigens have become available, it has become possible to use computational approaches to study these interactions. Electrostatic interactions are known to play an important role in protein complex formation. In this review, we focus on the use of continuum electrostatic methods to compute pH-dependent properties of proteins and discuss the use of these methods in the study of antibody/antigen complexes.     

Microfluidic systems integrated with two-dimensional surface plasmon resonance phase imaging systems for microarray immunoassay

This study reports a microfluidic chip integrated with an arrayed immunoassay for surface plasmon resonance (SPR) phase imaging of specific bio-samples. The SPR phase imaging system uses a surface-sensitive optical technique to detect two-dimensional (2D) spatial phase variation caused by rabbit immunoglobulin G (IgG) adsorbed on an anti-rabbit IgG film. The microfluidic chip was fabricated by using micro-electro-mechanical-systems (MEMS) technology on glass and polydimethylsiloxane (PDMS) substrates to facilitate well-controlled and reproducible sample delivery and detection. Since SPR detection is very sensitive to temperature variation, a micromachine-based temperature control module comprising micro-heaters and temperature sensors was used to maintain a uniform temperature distribution inside the arrayed detection area with a variation of less than 0.3 degrees C. A self-assembled monolayer (SAM) technique was used to pattern the surface chemistry on a gold layer to immobilize anti-rabbit IgG on the modified substrates. The microfluidic chip is capable of transporting a precise amount of IgG solution by using micropumps/valves to the arrayed detection area such that highly sensitive, highly specific bio-sensing can be achieved. The developed microfluidic chips, which employed SPR phase imaging for immunoassay analysis, could successfully detect the interaction of anti-rabbit IgG and IgG. The interactions between immobilized anti-rabbit IgG and IgG with various concentrations have been measured. The detection limit is experimentally found to be 1 x 10(-4)mg/ml (0.67 nM). The specificity of the arrayed immunoassay was also explored. Experimental data show that only the rabbit IgG can be detected and the porcine IgG cannot be adsorbed. The developed microfluidic system is promising for various applications including medical diagnostics, microarray detection and observing protein-protein interactions.     

Purification and characterization of E. histolytica antigen for diagnosis of amoebiasis

Entamoeba histolytica soluble crude antigen was fractionated by gel filtration on Sephacryl S-300 into four fractions, viz. F1(669 kDa); F2(51.2 kDa); F3(25.1 kDa) and F4(10.5 kDa). F1 fraction was observed to be more sensitive and specific for the detection of antibody in amoebiasis than the crude and other fractions of purified antigens employing IHAT and ELISA. ELISA was found to be better than IHAT since it could detect antibody in the sera (3/6) of asymptomatic cyst passers. The cross reaction of crude antigen with toxocariasis (1/4) and toxoplasmosis (2/5) sera were associated with F4 fraction. F3 and F4 were having low molecular weight and were not sensitive in detection of antibody in amoebiasis. Biochemical characterization revealed glycoprotein nature of the specific (F1) antigen fraction.     

Ag/SiO2 core-shell nanoparticle-based surface-enhanced Raman probes for immunoassay of cancer marker using silica-coated magnetic nanoparticles as separation tools

A simple, sensitive and highly specific immunoassay has been developed based on surface-enhanced Raman scattering for human alpha-fetoprotein (AFP), a tumor marker for the diagnosis of hepatocellular carcinoma. This strategy combines the Ag/SiO2 core-shell nanoparticles embedded with rhodamine B isothiocyanate dye molecules as Raman tags and the amino group modified silica-coated magnetic nanoparticle as immobilization matrix and separation tool. In the proposed system, a sandwich-type immunoassay was performed between polyclonal antibody functionalized Ag/SiO2 nanoparticle-based Raman tags and monoclonal antibody modified silica-coated magnetic nanoparticles. The presence of the analyte and the reaction between the antigen and antibody can be monitored by the Raman spectra of the Ag/SiO2 tags. Compared to the previous surface-enhanced Raman immunoassays, the main advantage of this strategy lies in two aspects. One is the high stability of Raman tags derived from the silica shell-coated silver core-shell nanostructure. The other is the use of silica-coated magnetic nanoparticles as immobilization matrix and separation tool, thus avoiding complicated pretreatment and washing steps. We have studied in detail the experimental parameters such as the effects of the antibody concentration modified on the Raman tags and on the magnetic particles, and the immunoreaction time. Using this strategy, concentration of human AFP up to 0.12 microg/ml was detected with a detection limit of 11.5 pg/ml.     

Detection of insulin-antibody binding on a solid surface using imaging ellipsometry

Imaging ellipsometry (IE) was used to detect the binding of insulin to its antibody on a solid surface. The modification of a gold surface with 11-mecaptoundecanoic acid (11-MUA), the adsorption of protein G, and antibody immobilization onto the protein G layer were confirmed by surface plasmon resonance. Ellipsometric images and ellipsometric angles of the surface antibody were acquired using the IE system by off-null ellipsometry. Ellipsometric images of antigen binding to the antibody were acquired, and their mean optical intensities estimated. Changes in mean optical intensity indicated that the detection range for insulin was from 10 ng/ml to 100 microg/ml.     

Study of antibody/antigen binding kinetics by total internal reflection ellipsometry

Total internal reflection ellipsometry (TIRE) has been applied for the investigation of (i) kinetics of biosensing layer formation, which was based on the immobilization of fragmented and intact antibodies, and (ii) kinetics of antigen interaction with the immobilized antibodies. It has been demonstrated that ellipsometric parameter Δ(t) showed much higher sensitivity at the initial phase of Au-protein and protein-protein interaction, while the parameter Ψ(t) was more sensitive when the steady-state conditions were established. A new method, which taking into consideration this feature and nonlinear change of Δ(t) and Ψ(t) parameters during various stages of biological layer formation process, was used for the calculation of antibody and antigen adsorption/interaction kinetics. The obtained results were analyzed using a model, which took into account partial reversibility during the formation of both antibody and antigen based monolayers. It was shown that the immobilization rate of antibody during the preparation of the sensing layer was similar for the formation of both intact and fragmented antibody based layers; however, the residence time was 25 times longer for intact antibody based layer formation in comparison to that of fragmented antibody based layer formation. On the contrary, residence time of antigen interaction with immobilized antibodies was about 8 times longer for the sensor based on fragmented antibodies. Moreover, it has been determined that the structural differences of immobilized antibodies (fragmented or intact) significantly influence antibody-antigen interaction rate, the major difference being in the residence time of antigen interaction with both types of immobilized antibodies.     

用于丙型肝炎病毒抗原测定的SiO_2载体的制备及其性质分析

为了优化将抗体偶联在二氧化硅试管表面上以便进行丙型肝炎抗原检测的分析系统,本研究通过氨基硅烷的活化作用,在玻璃表面形成活化的氨基,以戊二醛作为化学交联试剂,在已硅化的玻璃表面固定丙肝单克隆抗体,并进行丙肝抗原(HCAg)的测定。结果显示,通过条件优化实验,发现以10%(V/V)的氨基硅烷水溶液处理玻璃试管3h后,再用3%(V/V)的戊二醛水溶液交联丙肝单克隆抗体2h,可以得到固定效果较好,非特异性较低的玻璃载体,对HCAg可测至1μg/L。可见,用该方法制备的玻璃载体可为进一步建立新的HCAg磁性免疫检测系统提供理论和实验依据。     

Ultrasensitive detection of cellular protein interactions using bioluminescence resonance energy transfer quantum dot-based nanoprobes

Sensitive detection of protein interactions is a critical step toward understanding complex cellular processes. As an alternative to fluorescence-based detection, Renilla reniformis luciferase conjugated to quantum dots results in self-illuminating bioluminescence resonance energy transfer quantum dot (BRET-Qdot) nanoprobes that emit red to near-infrared bioluminescence light. Here, we report the development of an ultrasensitive technology based on BRET-Qdot conjugates modified with streptavidin ([BRET-Qdot]-SA) to detect cell-surface protein interactions. Transfected COS7 cells expressing human cell-surface proteins were interrogated with a human Fc tagged protein of interest. Specific protein interactions were detected using a biotinylated anti-human Fc region specific antibody followed by incubation with [BRET-Qdot]-SA. The luciferase substrate coelenterazine activated bioluminescence light emission was detected with an ultra-fast and -sensitive imager. Protein interactions barely detectable by the fluorescence-based approach were readily quantified using this technology. The results demonstrate the successful application and the flexibility of the BRET-Qdot-based imaging technology to the ultrasensitive investigation of cell-surface proteins and protein-protein interactions.     

Electrochemical DNA base pairs quantification and endonuclease cleavage detection

A label-free multiplexed immunoassay strategy was proposed for the simultaneous detection of two tumor markers, carcinoembryonic antigen (CEA) and α-fetoprotein (AFP). Monoclonal antibody of CEA was co-immobilized with ferrocenecarboxylic acid (FCA) inside the channels of mesoporous silica (MPS) to prepare the label-free probe for CEA. Also, monoclonal antibody of AFP was co-immobilized with horseradish peroxidase (HRP) inside the channels of MPS to prepare the label-free probe for AFP by using o-phenylenediamine (OPD) and H(2)O(2) as the electrochemical substrates. Thus, the multianalyte immunosensor was constructed by coating the probes of CEA and AFP respectively onto the different areas of indium-tin oxide (ITO) electrode. When the immunosensor was incubated with sample antigens, CEA and AFP antigens were introduced into the mesopores of MPS after the immunoassay reaction. Because all of the Si-OH groups on the external surface of MPS were blocked with Si(CH(3))(3), the proteins and substrates were limited to be embedded on the internal pore walls. Therefore, the electric response transfer was confined inside the pore channels. The nonconductive immunoconjugates blocked the electron transfer and the peak responses changed on the corresponding surface respectively. Then, the simultaneous detection of CEA and AFP achieved. The linear ranges of CEA and AFP were 0.5-45ngmL(-1) and 1-90ngmL(-1) with the detection limits of 0.2ngmL(-1) and 0.5ngmL(-1) (S/N=3), respectively. The fabricated immunosensor shows appropriate sensitivity and offers an alternative to the multianalyte detection of antigens or other bioactive molecules.     

The intrinsic contributions of tyrosine, serine, glycine and arginine to the affinity and specificity of antibodies

Synthetic antibody libraries with restricted chemical diversity were used to explore the intrinsic contributions of four amino acids (Tyr, Ser, Gly and Arg) to the affinity and specificity of antigen recognition. There was no correlation between nonspecific binding and the content of Tyr, Ser or Gly in the antigen-binding site, and in fact, the most specific antibodies were those with the highest Tyr content. In contrast, Arg content was clearly correlated with increased nonspecific binding. We combined Tyr, Ser and Gly to generate highly specific synthetic antibodies with affinities in the subnanomolar range, showing that the high abundance of Tyr, Ser and Gly in natural antibody germ line sequences reflects the intrinsic capacity of these residues to work together to mediate antigen recognition. Despite being a major functional contributor to co-evolved protein-protein interfaces, we find that Arg does not contribute generally to the affinity of naïve antigen-binding sites and is detrimental to specificity. Again, this is consistent with studies of natural antibodies, which have shown that nonspecific, self-reactive antibodies are rich in Arg and other positively charged residues. Our findings suggest that the principles governing naïve molecular recognition differ from those governing co-evolved interactions. Analogous studies can be designed to explore the roles of the other amino acids in molecular recognition. Results of such studies should illuminate the basic principles underlying natural protein-protein interactions and should aid the design of synthetic binding proteins with functions beyond the scope of natural proteins.     

Detection of protein-protein interactions using nonimmune IgG and BirA-mediated biotinylation

Detection of protein-protein interactions in cells is crucial for understanding the biological functions of proteins, including their roles in signal transduction. However, current methods require specific antibodies both for immunoprecipitation and detection, making them expensive and sometimes unreliable. Here we describe protocols for protein-protein interaction assays that use nonimmune IgG-conjugated Sepharose to precipitate the IgG binding domain (ZZ) fused to the bait protein; the interaction partner is fused to Avitag and biotinylated by BirA so that it can be detected by a one-step blot with Dylight 680 streptavidin to detect the Avitag fusion protein. Since this method does not require specific antibodies and is inexpensive, sensitive, and reliable, it should be useful for detecting protein-protein interactions in cells.     

New reactive polymer for protein immobilisation on sensor surfaces

Immobilisation of biorecognition elements on transducer surfaces is a key step in the development of biosensors. The immobilisation needs to be fast, cheap and most importantly should not affect the biorecognition activity of the immobilised receptor. A novel protocol for the covalent immobilisation of biomolecules containing primary amines using an inexpensive and simple polymer is presented. This tri-dimensional (3D) network leads to a random immobilisation of antibodies on the polymer and ensures the availability of a high percentage of antibody binding sites. The reactivity of the polymer is based on the reaction between primary amines and thioacetal groups included in the polymer network. These functional groups (thioacetal) do not need any further activation in order to react with proteins, making it attractive for sensor fabrication. The novel polymer also contains thiol derivative groups (disulphide groups or thioethers) that promote self-assembling on a metal transducer surface. For demonstration purposes the polymer was immobilised on Au Biacore chips. The resulting polymer layer was characterised using contact angle meter, atomic force microscopy (AFM) and ellipsometry. A general protocol suitable for the immobilisation of bovine serum albumin (BSA), enzymes and antibodies such as polyclonal anti-microcystin-LR antibody and monoclonal anti-prostate specific antigen (anti-PSA) antibody was then optimised. The affinity characteristics of developed immunosensors were investigated in reaction with microcystin-LR, and PSA. The calculated detection limit for analytes depended on the properties of antibodies. The detection limit for microcystin-LR was 10 ngmL(-1) and for PSA 0.01 ngmL(-1). The non-specific binding of analytes to synthesised polymers was very low. The polymer-coated chips were stored for up to 2 months without any noticeable deterioration in their ability to react with proteins. These findings make this new polymer very promising for the development of low-cost, easy to prepare and sensitive biosensors.     

An interleukin-6 ZnO/SiO(2)/Si surface acoustic wave biosensor

A novel high sensitivity ZnO/SiO(2)/Si Love mode surface acoustic wave (SAW) biosensor for the detection of interleukin-6 (IL-6), is reported. The biosensors operating at 747.7MHz and 1.586GHz were functionalized by immobilizing the monoclonal IL-6 antibody onto the ZnO biosensor surface both through direct surface adsorption and through covalent binding on gluteraldehyde. The morphology of the IL-6 antibody-protein complex was studied using scanning electron microscopy (SEM), and the mass of the IL-6 protein immobilized on the surface was measured from the frequency shift of the SAW resonator biosensor. The biosensor was shown to have extended linearity, which was observed to improve with higher sensor frequency and for IL-6 immobilization through the monoclonal antibody. Preliminary results of biosensor measurements of low levels of IL-6 in normal human serum are reported. The biosensor can be fully integrated with CMOS Si chips and developed as a portable real time detection system for the interleukin family of proteins in human serum.     

Label free novel electrical detection using micromachined PZT monolithic thin film cantilever for the detection of C-reactive protein

In this paper, we report the novel electrical measurement for the label-free detection of C-reactive protein (CRP) using resonant frequency shift in the monolithic thin film cantilever of micromachined Pb(Zr0.52Ti0.48)O3 (PZT) which was fabricated with the composition of SiO2/Ta/Pt/PZT/Pt/SiO2 on silicon nitride (SiNx) supporting layer for the dual purpose of electrical self-excitation and sensing. The specific binding characteristics of CRP antigen to its antibody, which is immobilized with Calixcrown SAMs on Au surface deposited on microcantilever, is determined in high sensitivity to the nanogram level per milliliter by measuring the resonant frequency shift. The nanomechanical PZT cantilever turns out a robust platform for the highly specific antigen-antibody interaction and provides with the novel tool for qualification and quantification of biomolecules without any sample labeling and bulky optical apparatus.     

An impedimetric immunosensor for the label-free detection of bisphenol A

Label-free detection of bisphenol A based on the impedance measurement was achieved with an impedimetric immunosensor. The immunosensor was fabricated by the covalent bond formation between a polyclonal antibody and a carboxylic acid group functionalized onto a nano-particle comprised conducting polymer. By using a commercial reagent 4,4-bis(4-hydroxyphenyl) valeric acid (BHPVA), which has an analogous structure of BPA, we have prepared the antigen through the conjugation of BHPVA with bovine serum albumin (BSA) and then produced a specific polyclonal antibody. The immobilization of antibody and the interaction between antibody and antigen were studied using quartz crystal microbalance (QCM) and electrochemical impedance spectroscopic (EIS) techniques. The impedance and mass changes due to the specific immuno-interaction at the sensor surface were utilized to detect antigen and bisphenol A (BPA). The immunosensor showed specific recognition of BPA with less interference than 4.5% from other common phenolic compounds. Under an optimized condition, the linear dynamic range of BPA detection was between 1 and 100 ng/ml. The detection limit of bisphenol A was determined to be 0.3+/-0.07 ng/ml. The proposed immunosensor was applied to a human serum sample and the BPA concentration was determined by the standard addition method.     

HIV感染早期病毒p24蛋白的检测

目的:建立敏感、特异的检测血清中HIV-p24蛋白的方法,作为HIV感染早期即窗口期的监测手段。方法:用纯化的p24蛋白免疫小鼠及家兔,获得单克隆及多克隆抗体,经DEAE-52阴离子交换柱纯化后,标记辣根过氧化物酶,建立ELISA双抗体夹心及间接双抗体夹心方法,检测HIV-p24蛋白。结果:包被单抗、标记多抗或包被多抗、标记单抗,均能特异地检出系列稀释的p24蛋白,包被混合单抗较包被多抗更敏感;经标记的抗种属抗体放大可明显提高检测的敏感性。结论:建立了敏感、特异的检测p24蛋白的双抗体夹心法,间接放大方法可检出50pg/mL的HIV-p24蛋白,检测敏感性与国际同类产品相似。