The high sensitivity of optical detection techniques and the highly specific reactions between antibodies and antigens mean that optical immunoassays have attracted much interest in the fields of protein, hormone, drug, and microorganism detection, without the need for complex separation and extraction steps. The immobilization of an antibody on a solid support is a crucial step for optical immunoassays. This review surveys the latest advances in current antibody immobilization techniques in detail, including physical adsorption, covalent attachment, bioaffinity immobilization, and some recently developed methods. Furthermore, specific consideration is given to oriented immobilization, which may improve the homogeneous surface covering the accessibility of the active site and surface coverage, and the analytical performance of immunoassays. Finally, new perspectives for antibody immobilization techniques in optical immunoassays are outlined.

With the success of high-throughput DNA microarrays, protein biochips have been intensively investigated and broadly used in bioscience research, clinic diagnosis, drug discovery, and other applications. However, there is great need to significantly improve the sensitivity of protein chips, especially in early diagnosis. A major challenge of improving sensitivity is that protein detection does not have an effective amplification method, such as PCR for DNA microarrays. Construction of unique biofilms for efficient immobilization of protein probes and innovation of new amplification schemes could play a critical role in performance improvement of protein biochips. With dramatic developments in microfabrication, nanotechnologies, and biotechnologies, enormous progress has been made, particularly in improving biosensing sensitivity. This article reviews new advances in protein biochip technologies with emphasis on novel approaches for efficient probe immobilization and nanomaterials-assisted signal amplification for high performance protein chips. Prominent progress in integration of protein microarrays with microfluidic platforms is briefly discussed. The major challenges and perspectives on the future of protein biochips are also addressed.

In this review, the authors will discuss novel and prospective antibody nanosensors for the detection of specific analytes used in a number of fields of analytical chemistry. Biosensors—transducers that incorporate biological molecules for recognition—have been found to be fundamental in a number of chemical, clinical, and environmental analyses. Antibody nanosensors make up a large area of this research, as the antibodies' specific recognition elements make them highly selective and sensitive. These biological molecules can also be tailored to recognize any single analyte or group of analytes, and can be easily functionalized to a number of nanomaterial substrates. Herein, a number of antibody nanosensor transduction methods will be examined, including electrochemical, optical, magnetic, and piezoelectric, among others that fall into multiple categories. This review will show that it is clear that antibody nanosensors—and nanosensors in general—are highly sensitive no matter the transduction method, and that various transduction methods can be suited for a number of different applications.

Orientation of surface immobilized capture proteins, such as antibodies, plays a critical role in the performance of immunoassays. The sensitivity of immunodiagnostic procedures is dependent on presentation of the antibody, with optimum performance requiring the antigen binding sites be directed toward the solution phase. This review describes the most recent methods for oriented antibody immobilization and the characterization techniques employed for investigation of the antibody state. The introduction describes the importance of oriented antibodies for maximizing biosensor capabilities. Methods for improving antibody binding are discussed, including surface modification and design (with sections on surface treatments, three-dimensional substrates, self-assembled monolayers, and molecular imprinting), covalent attachment (including targeting amine, carboxyl, thiol and carbohydrates, as well as “click” chemistries), and (bio)affinity techniques (with sections on material binding peptides, biotin-streptav...

Abstract Detection elements play a key role in analyte recognition in biosensors. Therefore, detection elements with high analyte specificity and binding strength are required. While antibodies (Abs) have been increasingly used as detection elements in biosensors, a key challenge remains - the immobilization on the biosensor surface. This minireview highlights recent approaches to immobilize and study Abs on surfaces. We first introduce Ab species used as detection elements, and discuss techniques recently used to elucidate Ab orientation by determination of layer thickness or surface topology. Then, several immobilization methods will be presented: non-covalent and covalent surface attachment, yielding oriented or random coupled Abs. Finally, protein modification methods applicable for oriented Ab immobilization are reviewed with an eye to future application.

Abstract Microparticulate glass represents a potential contamination to protein formulations that may occur as a result of processing conditions or glass types. The effect of added microparticulate glass to formulations of three humanized antibodies was tested. Under the three formulation conditions tested, all three antibodies adsorbed irreversibly at near monolayer surface coverages to the glass microparticles. Analysis of the secondary structure of the adsorbed antibodies by infrared spectroscopy reveal only minor perturbations as a result of adsorption. Likewise, front-face fluorescence quenching measurements reflected minimal tertiary structural changes upon adsorption. In contrast to the minimal effects on protein structure, adsorption of protein to suspensions of glass microparticles induced significant colloidal destabilization and flocculation of the suspension. Copyright 漏 2010 Wiley-Liss, Inc. and the American Pharmacists Association

Protein adsorption onto membrane surfaces is important in fields related to separation science and biomedical research. This study explored the molecular interactions between protein, bovine serum albumin (BSA), and nitrocellulose films (NC) using electrokinetic phenomena and the effects of these interactions on the streaming potential measurements for different membrane pore morphologies and pH conditions. The data were used to calculate the streaming ratios of membranes-to-proteins and to compare these values to the electrostatic or hydrophobic attachment of the protein molecules onto the NC membranes. The results showed that different pH and membrane pore morphologies contributes to different protein adsorption mechanisms. The protein adsorption was significantly reduced under conditions where the membrane and protein have like-charges due to electrostatic repulsion. At the isoelectric point (IEP) of the protein, the repulsion between the BSA and the NC membrane was at the lowest; thus, the BSA could be easily attached onto the membrane/solution interface. In this case, the protein was considered to be in a compact layer without intermolecular protein repulsions.

Understanding and controlling protein adsorption on surfaces is critical to a range of biological and materials applications. Kinetic details that provide the equilibrium and nonequilibrium mechanisms are difficult to acquire. In this work, single-molecule fluorescence microscopy was used to study the adsorption of Alexa 555 labeled α-lactalbumin (α-LA) on two chemically identical but morphologically different polymer surfaces: flat and porous nylon-6,6 thin films. The adsorption kinetics of spatially resolved single molecule α-LA binding to nylon films were quantified by a monolayer adsorption model. The surface morphology of the porous nylon-6,6 films increased the number of adsorption sites but decreased the binding affinity compared to the flat films. Such single-molecule based kinetic studies may be extended to various protein-polymer interactions.

Abstract Nanoparticles are the simplest form of structures with sizes in the nanometer (nm) range. In principle any collection of atoms bonded together with a structural radius of < 100 nm can be considered nano particles. Nanotechnology offers unique approaches to probe and control a variety of biological and medical processes that occur at nanometer scales, and is expected to have a revolutionary impact on biology and medicine. Among the approaches for exploiting nanotechnology in medicine, nanoparticles offer some unique advantages as sensing, image enhancement, and delivery agents. Several varieties of nanoparticles with biomedical relevance are available including, polymeric nanoparticles, metal nanoparticles, liposomes, micelles, quantum dots, dendrimers, and nanoassemblies. To further the application of nanoparticles in disease diagnosis and therapy, it is important that the systems are biocompatible and capable of being functionalized for recognition of specific target sites in the body after systemic administration. In this review, we have explained some important applications of gold nanoparticles.

Background: Functional antibody surfaces were prepared on ultraflat polystyrene surfaces by physical adsorption, and the uniform distribution of monoclonal antibodies against hepatitis B surface antigen (anti-HBs) on such surfaces and the presence of dense hepatitis B surface antigen (HBsAg) particles captured by immobilized antibodies were identified. Methods: A model polystyrene film was spin-coated directly onto a silicon wafer surface. Atomic force microscopy was used to directly monitor the immobilization of anti-HBs antibodies and their specific molecular interaction with HBsAg. Enzyme immunoassay was also used to characterize functional antibody surfaces. Results: A mean roughness of 2 A for areas of 25 [micro][m.sup.2] was produced. We found a uniform distribution of anti-HBs antibodies on ultraflat polystyrene surfaces and the presence of dense HBsAg particles bound to such anti-HBs surfaces after incubation with HBsAg. Conclusions: This study confirmed the potential of preparing dense, homogeneous, highly specific, and highly stable antibody surfaces by immobilizing antibodies on polystyrene surfaces with controlled roughness. It is expected that such biofunctional surfaces could be of interest for the development of new solid-phase immunoassay techniques and biosensor techniques.

Herein we reported that a hydrophobin film was used as a solid support on the polystyrene surface for immobilizing antibodies in the time-resolved immunofluorometric assay (TR-IFMA). Quartz crystal microbalance with dissipative monitoring (QCM-D), X-ray photoelectron spectroscopy (XPS) and water contact angle (WCA) measurements, as well as atomic force microscope (AFM) were used to characterize the hydrophilic modification of polystyrene surface with Class I hydrophobin isolated from Grifola frondosa (HGFI). The performance of HGFI-modified polystyrene was evaluated by TR-IFMA of carcinoembryonic antigen (CEA). QCM-D revealed that HGFI formed an intact monolayer on the polystyrene at pH 5. XPS and WCA measurements showed that self-assembling HGFI could render polystyrene surface hydrophilic for three months. AFM indicated that an end-on antibody monolayer was adsorbed on the HGFI film rather than multilayers on the polystyrene in a side-on orientation. Furthermore, a linear calibration curve (from 5 to 600 ng/mL) of CEA showed HGFI-modified polystyrene had higher detection sensitivity than unmodified ones in TR-IFMA. This present method for modifying polystyrene is simple without severe chemical treatment and may have wide applicability to functionalize other supports for immobilizing biomolecules.

Abstract The adsorption of mouse monoclonal immunoglobulin G (IgG) on negatively charged polystyrene microparticles was studied by the laser Doppler velocimetry (LDV) electrophoretic mobility measurements. The dependence of the electrophoretic mobility of microparticles on the IgG concentration in the suspension was determined for different ionic strengths and pHs (3.5, 7.4). The increase in the electrophoretic mobility was quantitatively interpreted in terms of the 3D electrokinetic model. The maximum coverage of IgG on latex was determined by the depletion method aided by AFM imaging. It was shown by monitoring the electrophoretic mobility and hydrodynamic dimeter of IgG covered microparticles over prolonged time periods that IgG adsorption was irreversible. The acid-base properties of the IgG monolayers were also determined in pH cycling experiments. It was also confirmed that the adsorption of human serum albumin (HSA) on saturated IgG monolayers, often referred to as blocking, was negligible at pH 7.4. Copyright 2016 Elsevier Inc. All rights reserved.

A novel and simple approach for oriented immobilization of antibody (Ab) was proposed through carbodiimide reaction and controlling electric field (CEF). The electrode was modified by self-assembled...

A general label-free photoelectrochemical (PEC) platform was manufactured by assembly of CdSe nanoparticles (NPs) sensitized anatase TiO 2 -functionalized electrode via layer-by-layer (LBL) strategy. CdSe NPs were assembled on anatase TiO 2 -functionalized electrode through dentate binding of TiO 2 NPs to –COOH groups. Ascorbic acid (AA) was used as an efficient electron donor for scavenging photogenerated holes under visible-light irradiation. The photocurrent response of the CdSe NPs modified electrode was significantly enhanced as a result of the band alignment of CdSe and TiO 2 in electrolyte. Ochratoxin A (OTA), as model analyte, was employed to investigate the performance of the PEC platform. Antibodies of OTA were immobilized on CdSe sensitized electrode by using the classic 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride coupling reactions between –COOH groups on the surfaces of CdSe NPs and –NH 2 groups of the antibody. Under the optimized conditions, the photocurrent was proportional to OTA concentration range from 1002pg/mL to 5002ng/mL with detection limit of 2.002pg/mL. The employed PEC platform established a simple, fast and inexpensive strategy for fabrication of label-free biosensor, which might be widely applied in bioanalysis and biosensing in the future.

Abstract--Herein we report a rapid and sensitive surface Plasmon resonance (SPR) biosensor to detect progesterone (P4), a model analyte of small molecules. A terthiophene scaffold with an oligoethylene glycol (OEG) linker is used as an anchor for the P4-ovalbumin (OVA) immobilization on gold to form a stable, regenerable SPR surface. Subsequently, inhibition SPR immunoassays based on this terthiophene/OVA-P4 surface have demonstrated that P4 limit of detection (LOD) in buffer solution was 0.13 ng ml-1. Due to the rapidity of each measurement (less than 5 min per measurement), and the high sensitivity of the assay, we believe the sensing platform may hold great potential for P4 detections.飥

Infrared spectroscopy is used to investigate the transformation of carboxyl-terminated alkyl chains immobilized on a surface into succinimidyl ester-terminated chains by reaction with an aqueous solution of N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The acid chains are covalently grafted at the surface of hydrogenated porous silicon whose large specific surface area allows for assessing the activation yield in a semiquantitative way by infrared (IR) spectroscopy and detecting trace amounts of surface products and/or reaction products of small IR cross section. In this way, we rationalize the different reaction paths and optimize the reaction conditions to obtain as pure as possible succinimidyl ester-terminated surfaces. A diagram mapping the surface composition after activation was constructed by systematically varying the solution composition. Results are accounted for by NHS surface adsorption and a kinetic competition between the various EDC-induced surface reactions.

1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) alone, and in combination with N-hydroxysuccinimide (NHS) or sulfoNHS were employed for crosslinking anti-human fetuin A (HFA) antibodies on 3-aminopropyltriethoxysilane (APTES)-functionalized surface plasmon resonance (SPR) gold chip and 96-well microtiter plate. The SPR immunoassay and sandwich enzyme linked immunosorbent immunoassay (ELISA) for HFA clearly demonstrated that EDC crosslinks anti-HFA antibodies to APTES-functionalized bioanalytical platforms more efficiently than EDC/NHS and EDC/sulfoNHS at a normal pH of 7.4. Similar results were obtained by sandwich ELISAs for human Lipocalin-2 and human albumin, and direct ELISA for horseradish peroxidase. The more efficient crosslinking of antibodies by EDC to the APTES-functionalized platforms increased the cost-effectiveness and analytical performance of our immunoassays. This study will be of wide interest to researchers developing immunoassays on APTES-functionalized platforms that are being widely used in biomedical diagnostics, biosensors, lab-on-a-chip and point-of-care-devices. It stresses a critical need of an intensive investigation into the mechanisms of EDC-based amine-carboxyl coupling under various experimental conditions.

A novel protein immobilization method based on plasma treatment of paper on the low-cost paper-based immunodevice was established in this work. By using a benchtop plasma cleaner, the paper microzone was treated by oxygen plasma treatment for 402min and then the antibody can be directly immobilized on the paper surface. Aldehyde group was produced after the plasma treatment, which can be verified from the fourier transform infrared spectroscopy (FT-IR) spectra and x-ray photoelectron spectroscopy (XPS) spectra. By linked to aldehyde group, the antibody can be immobilized on the paper surface without any other pretreatment. A paper-based immunodevice was introduced here through this antibody immobilization method. With sandwich chemiluminescence (CL) immunoassay method, the paper-based immunodevice was successfully performed for carcinoembryonic antigen (CEA) detection in human serum with a linear range of 0.1–80.002ng/mL. The detection limit was 0.0302ng/mL, which was 30 times lower than the clinical CEA level. Comparing to the other protein immobilization methods on paper-based device, this strategy was faster and simpler and had potential applications in point-of-care testing, public health and environmental monitoring.

In the present work, we developed and optimized a technique to produce a thin, stable silane layer on silicon substrate in a controlled environment using (3-aminopropyl)triethoxysilane (APTES). The effect of APTES concentration and silanization time on the formation of silane layer is studied using spectroscopic ellipsometry and Fourier transform infrared spectroscopy (FTIR). Biomolecules of interest are immobilized on optimized silane layer formed silicon substrates using glutaraldehyde linker. Surface analytical techniques such as ellipsometry, FTIR, contact angle measurement system, and atomic force microscopy are employed to characterize the bio-chemically modified silicon surfaces at each step of the biomolecule immobilization process. It is observed that a uniform, homogenous and highly dense layer of biomolecules are immobilized with optimized silane layer on the silicon substrate. The developed immobilization method is successfully implemented on different silicon substrates (flat and pillar). Also, different types of biomolecules such as anti-human IgG (rabbit monoclonal to human IgG), Listeria monocytogenes, myoglobin and dengue capture antibodies were successfully immobilized. Further, standard sandwich immunoassay (antibody鈥揳ntigen鈥揳ntibody) is employed on respective capture antibody coated silicon substrates. Fluorescence microscopy is used to detect the respective FITC tagged detection antibodies bound to the surface after immunoassay.

We report the fabrication of a patterned protein array using three orthogonal methods of immobilization that are detected exploiting a fluorogenic surface. Upon reaction of thiols, the fluorogenic tether reports the bond formation by an instantaneous rise in (blue) fluorescence intensity providing a means to visualize the immobilization even of nonfluorescent biomolecules. First, the covalent, oriented immobilization of a visible fluorescent protein (TFP) modified to display a single cysteine residue was detected. Colocalization of the fluorescence of the immobilized TFP and the fluorogenic group provided a direct tool to distinguish covalent bond formation from physisorption of proteins. Subsequent orthogonal immobilization of thiol-functionalized biomolecules could be conveniently detected by fluorescence microscopy using the fluorogenic surface. A thiol-modified nitrilotriacetate ligand was immobilized for binding of hexahistidine-tagged red-fluorescing TagRFP, while an appropriately modified biotin was immobilized for binding of Cy5-labeled streptavidin.

Antibody microarrays have the potential to revolutionize protein diagnostics. The major problems in the fabrication of antibody arrays, however, concern the reproducibility and homogeneity of the attachment of the proteins on the solid substrate. We here compare the DNA-directed immobilization (DDI) method with two conventional strategies for immobilization of antibodies on glass substrates. DDI is based on the self-assembly of semisynthetic DNA-streptavidin conjugates which converts an array of DNA oligomers into an antibody microarray. DDI was compared with direct spotting of antibodies on chemically activated glass slides and with immobilization of biotinylated antibodies on streptavidin-coated slides. The immobilized antibodies were used as capture reagents in a two-sided (sandwich) immunoassay for the quantification of rabbit IgG as a model antigen. Detection limits down to 0.001nM (150 pg/mL) were attained with all three array formats; however, DDI and direct spotting of the antibodies led to the highest fluorescence intensities. DDI led to the best spot homogeneity and intra- and interexperimental reproducibility. Moreover, DDI allowed highly economical use of antibody materials; that is, at least 100-fold less antibody is needed for preparing an array by DDI instead of by direct spotting. Taking into account the greater versatility and convenience of handling of the self-assembly approach, this study demonstrates that DDI is an advantageous alternative for generating versatile and robust protein arrays.

This study presents two-step and multistep reactions for modifying the surface of plasma-functionalized poly(tetrafluoroethylene)(PTFE)surfaces for subsequent conjugation of biologically relevant molecules.First,PTFE films were treated by a radiofrequency glow discharge(RFGD)ammonia plasma to introduce amino groups on the fluoropolymer surface.This plasma treatment is well optimized and allows the incorporation of a relative surface concentration of approximately 2-3.5% of amino groups,as assessed by chemical derivatization followed by X-ray photoelectron spectroscopy(XPS).In a second step,these amino groups were further reacted with various chemical reagents to provide the surface with chemical functionalities such as maleimides,carboxylic acids,acetals,aldehydes,and thiols,that could be used later on to conjugate a wide variety of biologically relevant molecules such as proteins,DNA,drugs,etc.In the present study,glutaric and cis-aconitic anhydrides were evaluated for their capability to provide carboxylic functions to the PTFE plasma-treated surface.Bromoacetaldehyde diethylacetal was reacted with the aminated PTFE surface,providing a diethylacetal function,which is a latent form of aldehyde functionality.Reactions with cross-linkers such as sulfo-succinimidyl derivatives(sulfo-SMCC,sulfo-SMPB)were evaluated to provide a highly reactive maleimide function suitable for further chemical reactions with thiolated molecules.Traut reagent(2-iminothiolane)was also conjugated to introduce a thiol group onto the fluoropolymer surface.PTFE-modified surfaces were analyzed by XPS with a particular attention to quantify the extent of the reactions that occurred on the polymer.Finally,surface immobilization of fibronectin performed using either glutaric anhydride or sulfo-SMPB activators demonstrated the importance of selecting the appropriate conjugation strategy to retain the protein biological activity.

Abstract Bispecific antibodies (BsAbs) are designed to engage two antigens simultaneously, thus, effectively expanding the ability of antibody-based therapeutics to target multiple pathways within the same cell, engage two separate soluble antigens, bind the same antigen with distinct paratopes, or crosslink two different cell types. Many recombinant BsAb formats have emerged, however, expression and purification of such constructs can often be challenging. To this end, we have developed a chemical strategy for generating BsAbs using native IgG2 architecture. Full-length antibodies can be conjugated via disulfide bridging with linkers bearing orthogonal groups to produce BsAbs. We report that an 伪HER2/EGFR BsAb was successfully generated by this approach and retained the ability to bind both antigens with no significant loss of potency

We report on in situ fluorescent quantification of the conjugation efficiency between azide‐terminated synthetic polymers/ imaging probes and thiol‐functionalized antibodies/proteins/peptides, by utilizing a doubly caged profluorescent and heterodifunctional core molecule (C1) as the self‐sorting bridging unit. Orthogonal dual 'click' coupling of C1 with azide‐ and thiol‐functionalized precursors leads to highly fluorescent bioconjugates, whereas single click products of C1 remain essentially nonfluorescent. This 'AND' logic gate‐type fluorogenic feature also enables further integration with FRET processes. For the construction of antibody‐probe conjugates from an anti‐carcinoembryonic antigen and a quinone‐caged profluorescent naphthalimide derivative, the dual 'click' coupling process with C1 can be conveniently monitored via emission turn‐on of C1, whereas prominent changes in FRET ratios occur for antibody‐probe conjugates when triggered by specific tumor‐associated enzymes.

A strategy for the preparation of homogeneous antibody–drug conjugates (ADCs) containing multiple payloads has been developed. This approach utilizes sequential unmasking of cysteine residues with orthogonal protection to enable site‐specific conjugation of each drug. In addition, because the approach utilizes conjugation to native antibody cysteine residues, it is widely applicable and enables high drug loading for improved ADC potency. To highlight the benefits of ADC dual drug delivery, this strategy was applied to the preparation of ADCs containing two classes of auristatin drug‐linkers that have differing physiochemical properties and exert complementary anti‐cancer activities. Dual‐auristatin ADCs imparted activity in cell line and xenograft models that are refractory to ADCs comprised of the individual auristatin components. This work presents a facile method for construction of potent dual‐drug ADCs and demonstrates how delivery of multiple cytotoxic warheads can lead to improved ADC activities. Lastly, we anticipate that the conditions utilized herein for orthogonal cysteine unmasking are not restricted to ADCs and can be broadly utilized for site‐specific protein modification.

Dengue Virus (DENV) is a flavivirus spread by mosquitoes that are endemic in tropic and subtropic climates. It is the causative pathogen of Dengue Fever, Dengue Hemorrhagic Fever, and Dengue Shock Syndrome. Besides mosquito transmission, DENV can also be transmitted through blood transfusion. Due to the high expense and expertise needed to run tests like ELISA and Polymerase Chain Reaction (PCR) based-assays for DENV detection, a rapid, sensitive and cost effective gold nanoparticles mediated surface-enhanced Raman spectroscopy (SERS) assay has been developed as an alternative assay to detect DENV. In this project, I successfully produced a monoclonal antibody that specifically binds to DENV-2 from the HB46-ATCC cell line. Using HiTrap鈩 Protein G HP columns, the antibodies were purified and concentrated by SpinX centrifuge filters. Bradford assay was used to measure antibody concentration, SDS-PAGE was used to detect possible protein contamination, and immunostaining confirmed antibody specificity for DENV 2. The SERS results show that this antibody can be used for specifically detection of DENV-2.

Tris(2‐carboxyethyl)phosphine (TCEP) is an often used reducing agent in biochemistry due to its selectivity towards disulfide bonds. As TCEP causes undesired consecutive side reactions in various analysis methods (i.e. gel electrophoresis, protein labeling), it is usually removed via dialysis or gel filtration. Herein, an alternative method of separation is presented, namely the immobilization of TCEP on magnetic nanoparticles. This magnetic reagent provides a simple and rapid approach to remove the reducing agent after successful reduction. High reducing capacities up to 70 μmol per gramm of particles were achieved by using surface‐initiated atom transfer polymerization.

Abstract Protein chip technology is essential for high-throughput functional proteomics. We developed a novel protein tag consisting of five tandem cysteine repeats (Cys-tag) at termini of proteins. The Cys-tag was designed to allow covalent attachment of proteins to the surface of a maleimide-modified, diamond-like, carbon-coated silicon substrate. As model proteins, we created an enhanced green fluorescent protein (EGFP) and an EGFP-stathmin fusion protein, both of which contained a Cys-tag. We also included an oligo-histidine tag to allow its purification by the use of Ni beads, and we expressed the protein in Escherichia coli. The purified Cys-tagged EGFP could be captured on the maleimide-coated substrate efficiently so that 50 pg of the fusion protein was detected by fluorescence, and as little as 5 pg was immunodetected by combination with enhanced chemiluminescence. This highly sensitive immunodetection may be due to the strong covalent binding of the Cys-tag to the substrate combined with efficient exposure of the protein to the surrounding solution. Thus, the Cys-tag should be useful for developing a novel protein printing method for protein chips that requires very low amounts of protein and can be used for high-performance analysis of protein-ligand interactions.

Keep still! A general strategy has been developed for the site-specific and covalent immobilization of biomolecules. The applicability of this protocol was demonstrated by using the industrially important lipase CalB. Two mutants that possess a free cysteine (Cys293 or C-terminal) were prepared and immobilized in an active conformation at the desired position.

Protein immobilization on surfaces is of great importance in numerous applications in biology and biophysics. The key for the success of all these applications relies on the immobilization technique employed to attach the protein to the corresponding surface. Protein immobilization can be based on covalent or noncovalent interaction of the molecule with the surface. Noncovalent interactions include hydrophobic interactions, hydrogen bonding, van der Waals forces, electrostatic forces, or physical adsorption. However, since these interactions are weak, the molecules can get denatured or dislodged, thus causing loss of signal. They also result in random attachment of the protein to the surface. Siteu2013specific covalent attachment of proteins onto surfaces, on the other hand, leads to molecules being arranged in a definite, orderly fashion and uses spacers and linkers to help minimize steric hindrances between the protein and the surface. This work reviews in detail some of the methods most commonly used as well as the latest developments for the site-specific covalent attachment of protein to solid surfaces.

In this article we describe a new, convenient procedure to carry out intramolecular (cyclization) and intermolecular native chemical ligations of unprotected peptides directly from a solid support. Our solid-phase ligation approach eliminates the need to manipulate peptide 090000thioacid and peptide thioester intermediates in aqueous solution before the ligation step, thereby leading to a reduction in handling losses and significantly increasing the overall efficiency of the chemical ligation strategy. A key step in our ligation scheme is the ability to generate fully unprotected peptides tethered to a solid support through an 090009thioester linkage. This can be achieved efficiently using optimized Boc-solid-phase peptide synthesis on a 3-mercaptopropionamide-polyethylene glycol-poly-(N,N-dimethylacrylamide) copolymer support (HS-PEGA). Once the synthesis is complete, the fully protected peptide 090000thioester resin is treated with HF to give the corresponding fully unprotected peptide 090000thioester resin. Using this procedure several polypeptides ranging from 15 to 47 residues were synthesized successfully. These peptide-resins were then used to perform both intramolecular (head-to-tail cyclizations) and intermolecular solid-phase ligations. The intramolecular solid-phase ligations proceeded much faster than their intermolecular counterparts, but in both cases the reactions were observed to be remarkably clean. The presence of aromatic thiol cofactors significantly accelerated the relatively slow intermolecular ligations. This novel methodology was then extended to provide a general method for performing sequential intermolecular ligations, allowing easy access to much larger polypeptide and protein systems.

As prepared nanomaterials of metals, semiconductors, polymers and carbon often need surface modifications such as ligand exchange, and chemical and bioconjugate reactions for various biosensor, bioanalytical, bioimaging, drug delivery and therapeutic applications. Such surface modifications help us to control the physico-chemical, toxicological and pharmacological properties of nanomaterials. Furthermore, introduction of various reactive functional groups on the surface of nanomaterials allows us to conjugate a spectrum of contrast agents, antibodies, peptides, ligands, drugs and genes, and construct multifunctional and hybrid nanomaterials for the targeted imaging and treatment of cancers. This tutorial review is intended to provide an introduction to newcomers about how chemical and bioconjugate reactions transform the surface of nanomaterials such as silica nanoparticles, gold nanoparticles, gold quantum clusters, semiconductor quantum dots, carbon nanotubes, fullerene and graphene, and accordingly formulate them for applications such as biosensing, bioimaging, drug and gene delivery, chemotherapy, photodynamic therapy and photothermal therapy. Nonetheless, controversial reports and our growing concerns about toxicity and pharmacokinetics of nanomaterials suggest the need for not only rigorous in vivo experiments in animal models but also novel nanomaterials for practical applications in the clinical settings. Further reading of original and review articles cited herein is necessary to buildup in-depth knowledge about the chemistry, bioconjugate chemistry and biological applications of individual nanomaterials.

Abstract Much effort has been put into the optimization of the functional activity of proteins. For biosensors this protein functional optimization will increase the biosensor's sensitivity and/or selectivity. However, the strategy chosen for the immobilization of the proteins to the sensor surface might be equally important for the development of sensor surfaces that are optimally biologically active. Several studies published in recent years show that the oriented immobilization of the bioactive molecules improves the sensor's properties. In this review, we discuss the state of the art of the different protein immobilization strategies that are commonly used today with a special focus on biosensor applications. These strategies include nonspecific immobilization techniques either by physical adsorption, by covalent coupling, or by specific immobilization via site-specifically introduced tags or bio-orthogonal chemistry. The different tags and bio-orthogonal chemistry available and the techniques to site-specifically introduce these groups in proteins are also discussed.

This paper reports a convenient method for immobilizing biologically active ligands to self-assembled monolayers of alkanethiolates on gold (SAMs). This methodology is based on monolayers that present maleimide and penta(ethylene glycol) groups. The maleimide groups react efficiently with thiol-terminated ligands, whereas the penta(ethylene glycol) groups prevent the nonspecific adsorption of protein to the substrate. The rate and selectivity of the immobilization of a ferrocene-thiol conjugate were characterized using cyclic voltammetry. This paper presents three examples of biochips prepared using this methodology. In the first example, four carbohydrate-thiol conjugates were immobilized to monolayers and the lectin-binding properties of the substrates were examined using fluorescence and surface plasmon resonance spectroscopy. The second biochip was used to study the enzymatic phosphorylation of the immobilized peptide IYGEFKKKC by the tyrosine kinase c-src. Monolayers presenting this peptide were then...

This paper describes the exohedral N-decoration of multi-walled carbon nanotubes (MWCNTs) with NH-aziridine groups via [2+1] cycloaddition of a tert-butyl-oxycarbonyl nitrene followed by controlled thermal decomposition of the cyclization product. The chemical grafting with N-containing groups deeply modifies the properties of the starting MWCNTs, generating new surface microenvironments with specific base (Br03nsted) and electronic properties. Both these features translate into a highly versatile single-phase heterogeneous catalyst (MW@NAz) with remarkable chemical and electrochemical performance. Its surface base character promotes the Knoevenagel condensation with superior activity to that of related N-doped and N-decorated carbon nanomaterials of the state-of-the-art; the N-induced electronic surface redistribution drives the generation of high energy surface “C” sites suitable for O2 activation and its subsequent electrochemical reduction (ORR).

Abstract The goal of this work is to develop an innovative approach for the coating of gold nanoparticles (AuNPs) with a synthetic functional copolymer. This stable coating with a thickness of few nanometers provides, at the same time, stabilization and functionalization of the particles. The polymeric coating consists of a backbone of polydimethylacrylamide (DMA) functionalized with an alkyne monomer that allows the binding of azido modified molecules by Cu(I)-catalyzed azide/alkyne 1,3-dipolar cycloaddition (CuAAC, click chemistry). The thin polymer layer on the surface stabilizes the colloidal suspension whereas the alkyne functions pending from the backbone are available for the reaction with azido-modified proteins. The reactivity of the coating is demonstrated by immobilizing an azido modified anti-mouse IgG antibody on the particle surface. This approach for the covalent binding of antibody to a gold-NPs is applied to the development of gold labels in biosensing techniques.

Abstract Sensitivity of biosensors depends on the orientation of bio-receptors on the sensor surface. The objective of this study was to organize bio-receptors on surfaces in a way that their analyte binding site is exposed to the analyte solution. VHH proteins recognizing foot-and-mouth disease virus (FMDV) were used for making biosensors, and azides were introduced in the VHH to function as bioorthogonal reactive groups. The importance of the orientation of bio-receptors was addressed by comparing sensors with randomly oriented VHH (with multiple exposed azide groups) to sensors with uniformly oriented VHH (with only a single azide group). A surface plasmon resonance (SPR) chip exposing cyclooctyne was reacted to azide functionalized VHH domains, using click chemistry. Comparison between randomly and uniformly oriented bio-receptors showed up to 800-fold increase in biosensor sensitivity. This technique may increase the containment of infectious diseases such as FMDV as its strongly enhanced sensitivity may facilitate early diagnostics. Copyright 2014 Elsevier B.V. All rights reserved.

The oxidation of antibody carbohydrate residues by periodate is a common approach for the site-specific immobilization or modification of antibodies for use in various bioanalytical methods. This study examined the time dependence of this oxidation process under a variety of pH, temperature, and concentration conditions. Polyclonal rabbit immunoglobulin G (IgG) was used as the model system for these studies. Flow-injection analysis and a hydrazide label (Lucifer yellow CH) were used to monitor the progress of the oxidation reaction. It was found that the number of oxidized sites that were available for labeling could be varied between one and eight groups per antibody by adjusting the time, pH, periodate concentration, or reaction temperature. In each case, most of these groups were produced during the first 30-60 min of the reaction. A comparison was made between these results and those of previous studies that have examined the effects of periodate treatment on amino acid residues and antibody activity. From this work, general guidelines were developed for the control and optimization of antibody oxidation for use with assays that require either high or low levels of antibody modification.

There is a growing interest in the use of magnetic nanoparticles (MNPs) for their application in quantitative and highly-sensitive biosensors. The use of them as labels of biological recognition events and their detection by means of some magnetic method constitutes a very promising strategy for quantitative high-sensitive lateral-flow assays. In the present article, we report the importance of nanoparticle functionalization for the improvement of sensitivity for a lateral flow immunoassay. More precisely, we have found that immobilization of IgG anti-hCG through its polysaccharide moieties on magnetic nanoparticles allows more successful recognition of the hCG hormone. Although we used the detection of hCG as a model in this work, the strategy of binding antibodies to MNPs through its sugar chains reported here is applicable to other antibodies. Its potential is huge as it will be very useful for the development of quantitative and high-sensitive lateral-flow assays for its use on human and veterinary, medicine, food and beverage manufacturing, pharmaceutical, medical biologics and personal care product production, environmental remediation, etc.

Antibody microarrays have important applications for the sensitive detection of biologically important target molecules and as biosensors for clinical applications. Microarrays produced by oriented immobilization of antibodies generally have higher antigen-binding capacities than those in which antibodies are immobilized with random orientations. Here, we present a UV photo-cross-linking approach that utilizes boronic acid to achieve oriented immobilization of an antibody on a surface while retaining the antigen-binding activity of the immobilized antibody. A photoactive boronic acid probe was designed and synthesized in which boronic acid provided good affinity and specificity for the recognition of glycan chains on the Fc region of the antibody, enabling covalent tethering to the antibody upon exposure to UV light. Once irradiated with optimal UV exposure (16 mW/cm(2)), significant antibody immobilization on a boronic acid-presenting surface with maximal antigen detection sensitivity in a single step was achieved, thus obviating the necessity of prior antibody modifications. The developed approach is highly modular, as demonstrated by its implementation in sensitive sandwich immunoassays for the protein analytes Ricinus communis agglutinin 120, human prostate-specific antigen, and interleukin-6 with limits of detection of 7.4, 29, and 16 pM, respectively. Furthermore, the present system enabled the detection of multiple analytes in samples without any noticeable cross-reactivities. Antibody coupling via the use of boronic acid and UV light represents a practical, oriented immobilization method with significant implications for the construction of a large array of immunosensors for diagnostic applications.

Selective chemical reactions enacted within a cellular environment can be powerful tools for elucidating biological processes or engineering novel interactions. A chemical transformation that permits the selective formation of covalent adducts among richly functionalized biopolymers within a cellular context is presented. A ligation modeled after the Staudinger reaction forms an amide bond by coupling of an azide and a specifically engineered triarylphosphine. Both reactive partners are abiotic and chemically orthogonal to native cellular components. Azides installed within cell surface glycoconjugates by metabolism of a synthetic azidosugar were reacted with a biotinylated triarylphosphine to produce stable cell-surface adducts. The tremendous selectivity of the transformation should permit its execution within a cell's interior, offering new possibilities for probing intracellular interactions.

Abstract The Staudinger ligation between an azido-protein and a phosphinothioester-derivatized surface is demonstrated to be an effective means for the site-specific, covalent immobilization of a protein. Immobilization yields of >50% are obtained in 80% of their expected activity. No other method enables more rapid immobilization or a higher yield of active protein. Because azido-peptides and azido-proteins are readily attainable by synthesis, biosynthesis, or semisynthesis, the Staudinger ligation could be of unsurpassed utility in creating microarrays of functional peptides and proteins.

Protein, peptide and small molecule microarrays are valuable tools in biological research. In the last decade, substantial progress has been achieved to make these powerful technologies more reliable and available for researchers. This review describes chemical preparation methods for these microarrays with focus on site-selective and bioorthogonal immobilization reactions, particularly the Staudinger ligation and the thiol-ene reaction. In addition, the application of peptide microarrays, which were prepared by Staudinger ligation, to substrate specificity mapping is illustrated. Copyright 2009 European Peptide Society and John Wiley &amp; Sons, Ltd.

Author information: (1)Department of Chemical Engineering and Applied Chemistry, University of Toronto, 160 College Street, Toronto, ON M5S 3E1, Canada.

Abstract A simple technique has been devised that allows the direct synthesis of native backbone proteins of moderate size. Chemoselective reaction of two unprotected peptide segments gives an initial thioester-linked species. Spontaneous rearrangement of this transient intermediate yields a full-length product with a native peptide bond at the ligation site. The utility of native chemical ligation was demonstrated by the one-step preparation of a cytokine containing multiple disulfides. The polypeptide ligation product was folded and oxidized to form the native disulfide-containing protein molecule. Native chemical ligation is an important step toward the general application of chemistry to proteins.

Abstract Liposomes have become popular drug delivery vehicles and have more recently also been applied as contrast agents for molecular imaging. Most current methods for functionalization of liposomes with targeting proteins rely on reactions of amine or thiol groups at the protein exterior, which generally result in nonspecific conjugation at multiple sites on the protein. In this study, we present native chemical ligation (NCL) as a general method to covalently couple recombinant proteins in a highly specific and chemoselective way to liposomes containing cysteine-functionalized phospholipids. A cysteine-functionalized phospholipid (Cys-PEG-DSPE) was prepared and shown to readily react with the MESNA thioester of EYFP, which was used as a model protein. Characterization of the EYFP-liposomes using fluorescence spectroscopy showed full retention of the fluorescent properties of conjugated EYFP and provides a lower limit of 120 proteins per liposome. The general applicability of NCL was further tested using CNA35, a collagen-binding protein recently applied in fluorescent imaging of collagen. NCL of CNA35 thioester yielded liposomes containing approximately 100 copies of CNA35 per liposome. The CNA35-liposomes were shown to be fully functional and bind collagen with a 150-fold higher affinity compared to CNA35. Our results show that NCL is an attractive addition to existing conjugation methods that allows direct, covalent, and highly specific coupling of recombinant proteins to liposomes and other lipid-based assemblies.

61A photoactivable ZBpa–Biotin was fabricated by aaRS/tRNA and Avitag/BirA techniques.61A approach for Fc-specific photo-biotinylated IgG via ZBpa–Biotin was proposed.61The photo-biotinylated IgG was used to fabricate an immunosensor for detecting CEA.61It gave a LOD of 202ng02mL-1CEA, was 5-fold lower than that of NHS-biotinylated IgG.

Abstract A method is described by which an immunoaffinity matrix was constructed by binding antibody directly or indirectly to protein A-Sepharose 4B followed by cross-linking of the complex with dimethyl pimelimidate. This allows optimal spatial orientation of antibodies and, thus, maximum antigen binding efficiency. The affinity matrices were stable to high and low pH buffers without any significant antibody loss. The optimal conditions of antibody saturation, cross-linker concentration, and elution system were established and affinity columns made with the monoclonal antibodies J5, W6/32, and OKT9 for one-step isolation of the common acute lymphoblastic leukemia-associated antigen, HLA-AB antigens, and transferrin receptor, respectively, from cell lysates. The same methodology was also applied to immobilize transferrin with polyvalent anti-transferrin antibodies. This was then used to isolate the transferrin receptor from cell lysates.

Design of an optimal surface biofunctionalization still remains an important challenge for the application of biosensors in clinical practice and therapeutic follow-up. Optical biosensors offer real-time monitoring and highly sensitive label-free analysis, along with great potential to be transferred to portable devices. When applied in direct immunoassays, their analytical features depend strongly on the antibody immobilization strategy. A strategy for correct immobilization of antibodies based on the use of ProLinker64 has been evaluated and optimized in terms of sensitivity, selectivity, stability and reproducibility. Special effort has been focused on avoiding antibody manipulation, preventing nonspecific adsorption and obtaining a robust biosurface with regeneration capabilities. ProLinker64-based approach has demonstrated to fulfill those crucial requirements and, in combination with PEG-derivative compounds, has shown encouraging results for direct detection in biological fluids, such as pure urine or diluted serum. Furthermore, we have implemented the ProLinker64 strategy to a novel nanoplasmonic-based biosensor resulting in promising advantages for its application in clinical and biomedical diagnosis.

Antibody immobilization on a solid surface is inevitable in the preparation of immunochips/sensors. Antibody-binding proteins such as proteins A and G have been extensively employed to capture antibodies on sensor surfaces with right orientations, maintaining their full functionality. Because of their synthetic versatility and stability, in general, small molecules have more advantages than proteins. Nevertheless, no small molecule has been used for oriented and specific antibody immobilization. Here is described a novel strategy to immobilize an antibody on various sensor surfaces by using a small antibody-binding peptide. The peptide binds specifically to the Fc domain of immunoglobulin G (IgG) and, therefore, affords a properly oriented antibody surface. Surface plasmon resonance analysis indicated that a peptide linked to a gold chip surface through a hydrophilic linker efficiently captured human and rabbit IgGs. Moreover, antibodies captured by the peptide exhibited higher antigen binding capacity compared with randomly immobilized antibodies. Peptide-mediated antibody immobilization was successfully applied on the surfaces of biosensor substrates such as magnetic particles and glass slides. The antibody-binding peptide conjugate introduced in this work is the first small molecule linker that offers a highly stable and specific surface platform for antibody immobilization in immunoassays.

Here, we describe the use of DNA-conjugated antibodies for rapid and sensitive detection of whole viruses using a single-particle interferometric reflectance imaging sensor (SP-IRIS), a simple, label-free biosensor capable of imaging individual nanoparticles. First, we characterize the elevation of the antibodies conjugated to a DNA sequence on a three-dimensional (3-D) polymeric surface using a fluorescence axial localization technique, spectral self-interference fluorescence microscopy (SSFM). Our results indicate that using DNA linkers results in significant elevation of the antibodies on the 3-D polymeric surface. We subsequently show the specific detection of pseudotyped vesicular stomatitis virus (VSV) as a model virus on SP-IRIS platform. We demonstrate that DNA-conjugated antibodies improve the capture efficiency by achieving the maximal virus capture for an antibody density as low as 0.72 ng/mm2, whereas for unmodified antibody, the optimal virus capture requires six times greater antibody densit...

The conserved nucleotide binding site (NBS), found on the Fab variable domain of all antibody isotypes, remains a not-so-widely known and unutilized site. Here, we describe a UV photo-cross-linking method (UV-NBS) that utilizes the NBS for oriented immobilization of antibodies onto surfaces, such that the antigen binding activity remains unaffected. Indole-3-butyric acid (IBA) has an affinity for the NBS with a Kd ranging from 1 to 8 渭M for different antibody isotypes and can be covalently photo-cross-linked to the antibody at the NBS upon exposure to UV light. Using the UV-NBS method, antibody was successfully immobilized on synthetic surfaces displaying IBA via UV photo-cross-linking at the NBS. An optimal UV exposure of 2 J/cm2 yielded significant antibody immobilization on the surface with maximal relative antibody activity per immobilized antibody without any detectable damage to antigen binding activity. Comparison of the UV-NBS method with two other commonly used methods, 蔚-NH3+ conjugation and phy...

Abstract As methods to orient proteins are conceived, techniques must also be developed that provide an accurate characterization of immobilized protein orientation. In this study, X-ray photoelectron spectroscopy (XPS), surface plasmon resonance (SPR), and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were used to probe the orientation of a surface immobilized variant of the humanized anti-lysozyme variable fragment (HuLys Fv, 26 kDa). This protein contained both a hexahistidine tag and a cysteine residue, introduced at opposite ends of the HuLys Fv, for immobilization onto nitrilotriacetic acid (NTA) and maleimide oligo(ethylene glycol) (MEG)-terminated substrates, respectively. The thiol group on the cysteine residue selectively binds to the MEG groups, while the his-tag selectively binds to the Ni-loaded NTA groups. XPS was used to monitor protein coverage on both surfaces by following the change in the nitrogen atomic %. SPR results showed a 10-fold difference in lysozyme binding between the two different HuLys Fv orientations. The ToF-SIMS data provided a clear differentiation between the two samples due to the intensity differences of secondary ions originating from asymmetrically located amino acids in HuLys Fv (histidine: 81, 82, and 110 m/z; phenylalanine: 120 and 131 m/z). An intensity ratio of the secondary ion peaks from the histidine and phenylalanine residues at either end of the protein was then calculated directly from the ToF-SIMS data. The 45% change in this ratio, observed between the NTA and MEG substrates with similar HuLys Fv surface coverages, indicates that the HuLys Fv fragment has opposite orientations on two different surfaces. Copyright 2011 Wiley Periodicals, Inc.

A novel strategy for site-specific and covalent attachment of proteins has been developed, intended for robust and controllable immobilization of histidine (His)-tagged ligands in protein microarrays. The method is termed chelation assisted photoimmobilization (CAP) and was demonstrated using human IgG-Fc modified with C-terminal hexahistidines (His-IgGFc) as the ligand and protein A as the analyte. Alkanethiols terminated with either nitrilotriacetic acid (NTA), benzophenone (BP), or oligo(ethylene glycol) were synthesized and mixed self-assembled monolayers (SAMs) were prepared on gold and thoroughly characterized by infrared reflection absorption spectroscopy (IRAS), ellipsometry, and contact angle goniometry. In the process of CAP, NTA chelates Ni(2+) and the complex coordinates the His-tagged ligand in an oriented assembly. The ligand is then photoimmobilized via BP, which forms covalent bonds upon UV light activation. In the development of affinity biosensors and protein microarrays, site-specific attachment of ligands in a fashion where analyte binding sites are available is often preferred to random coupling. Analyte binding performance of ligands immobilized either by CAP or by standard amine coupling was characterized by surface plasmon resonance in combination with IRAS. The relative analyte response with randomly coupled ligand was 2.5 times higher than when site-specific attachment was used. This is a reminder that also when immobilizing ligands via residues far from the binding site, there are many other factors influencing availability and activity. Still, CAP provides a valuable expansion of protein immobilization techniques since it offers attractive microarraying possibilities amenable to applications within proteomics.

A robot-assisted high-throughput methodology was employed to produce chromium(III) complexes suitable for the surface modification of the commercially available PerkinElmer Optiplate96 well plate for use in enzyme-linked immunosorbent assays (ELISAs). The complexes were immobilized to the native functionality of the well plate and first screened using a horseradish-peroxidase-tagged (HRP) mouse antibody to quantify binding. The top “hits” were further assessed for their ability to present the antibody in a functional state using an ELISA. “Hits” from the second screen yielded four complexes capable of improving the signal intensity of the ELISA by greater than 500%. The metal/base ratio of these complexes was also investigated, and we isolated the most stable and reproducible candidate, [Cr(OH)6]3–, which was formed from chromium(III) perchlorate and pH adjusted with ethylenediamine. This chromium solution was employed in a clinically relevant setting for the detection of bovine TNFα producing up to a 200...

Enzyme linked immunosorbent assays (ELISAs) are employed for the detection and quantification of antigens from biological sources such as serum and cell culture media. A sandwich ELISA is dependent on the immobilization of a capture antibody, or antibody fragment, and the effective presentation of its antigen binding sites. Immobilization to common microtitre plates relies on non-specific interactions of the capture protein with a surface that may result in unfavourable orientation and conformation, compromising ELISA signal strength and performance. We have developed a wet chemical surface activation method that utilizes a chromium (III) solution to immobilize native, non-tagged, capture antibodies on commercially available microtitre plates. Antibodies captured by this method had increased antigen binding, particularly from dilute antibody solutions, relative to antibodies adsorbed directly to the plate surface. A variety of monoclonal antibodies with complementary antigen systems were used to demonstrate improvements in ELISA signal and reproducibility. The simplicity and versatility of this method should enable ELISA enhancement in assays where chemiluminescence is used as the detection method.

Protein microarrays, on which thousands of discrete proteins are printed, provide a valuable platform for functional analysis of the proteome. They have been widely used for biomarker discovery and to study protein-protein interactions. The accomplishments of DNA microarray technology, which had enabled massive parallel studies of gene expression, sparked great interest for the development of protein microarrays to achieve similar success at the protein level. Protein microarray detection techniques are often classified as being label-based and label-free. Most of the microarray applications have employed labelled detection such as fluorescent, chemiluminescent and radioactive labelling. These labelling strategies have synthetic challenges, multiple label issues and may exhibit interference with the binding site. Therefore, development of sensitive, reliable, high-throughput, label-free detection techniques are now attracting significant attention. Label-free detection techniques monitor biomolecular interactions and simplify the bioassays by eliminating the need for secondary reactants. Moreover, they provide quantitative information for the binding kinetics. In this article, we will review several label-free techniques, which offer promising applications for the protein microarrays, and discuss their prospects, merits and challenges.

Total internal reflection ellipsometry (TIRE) technique was used to investigate biological recognition layers of immunosensors in order to estimate orientation of immobilized intact- and fragmented-antibodies. Two differently prepared biological recognition layers formed on gold-substrate were investigated: one layer was based on intact-antibodies (intact-Ab), second layer was based on chemically fragmented specific-antibodies (frag-Ab), which were obtained by reduction of intact-Ab. It was shown that TIRE enables to resolve differences in nanostructures of intact-Ab and frag-Ab layers. A multilayer model applied in our calculations shows that the distance between fragmented-antibody active sites and gold-surface after the immobilization process is lower than theoretical dimension of fragmented-antibodies. Moreover, it was calculated that analytical sensitivity of the Δ( λ) parameter of TIRE was 5.89 times better if compared to the sensitivity of the Ψ( λ) parameter, which is in fact similar to the sensitivity of surface plasmon resonance (SPR) based immunosensors.

End-on immobilization of antibody is an important technology to greatly improve the sensitivity of immuno-sensing. The end-on, or constant fragment (Fc) site-specifically oriented antibodies expose more active antigen binding sites (Fab) and tend to capture more analytes from the solution. In this study, dual polarization interferometry (DPI), which is the only available equipment that can concurrently obtain the mass, thickness, density, and refractive index in real time, was applied to investigate two end-on antibody immobilization methods, as exemplified by prostate-specific antigen (PSA) antibody. In the first method, antibody was immobilized via the saccharide chain linked to Fc portion of the antibody, by chelation to the surface-bound boronic acid. In the second method, antibody was partially reduced by tris(2-carboxyethyl) phosphine (TCEP) under mild conditions, followed by covalent conjugation to the surface via thiol-maleimide reaction. Time-resolved measurement from DPI verifies the end-on conformation of the antibody on the sensor surface. The second method shows better detection performance, with enhanced sensitivity and reproducibility than the first method, due to the optimal alignment of the antibody. Finally, these two methods were compared with the protein G-based antibody orientation control and random antibody immobilization in terms of the mass, thickness, density, refractive index, reproducibility, and stability. The detailed antibody immobilization parameters obtained in this paper are of great importance in the developing of solid鈥搇iquid phase immunosensors with enhanced detection sensitivity.

[EN] The challenging lecture given in 1959 by physicist and Nobel Prize awarded R. P. Feynman:鈥淭here's plenty of room at the bottom鈥 is considered to be the starting point fornanotechnology. With this peculiar title, Feynman encouraged researchers to explorebeyond the atomic level and predicted exciting new phenomena that might revolutionizescience and technology. Among these pioneering researchers are Eric Betzig, Stefan W. Helland William E. Moerner, who have been awarded with the Nobel Prize in Chemistry 2014for developing the super-resolved fluorescence microscopy. However, it is important toremark that the exploration of this amazing nanomolecular world began in the early 1980swith the invention of the scanning tunneling1 and the atomic force microscopes2 (Figure 1).Figure 1. Measurement scales.The study and manipulation of interactions of nanometric dimensions could begin assoon as measuring tools became more efficient. The last decades have witnessed thedevelopment of techniques able to obtain information at the sub-molecular level, and theirapplications especially on the biomedical field. As an example, the stimulating labor ofstudying the role of conformational dynamics in reaction mechanisms has resulted inNanometers10-1 10 102 103 1 104 105 106 107 108DPI WORLDAtom IgG Erythrocyte Grain of salt Tennis ballGlucose Virus Amoeba Pleanumerous advances in life sciences.3 In this regard, the precise knowledge about molecularinteractions and their effects on protein function has greatly aided the discovery of newtargets in medical chemistry.Figure 2. General scheme of different DPI applications.The role of the structure in the protein behavior is a fundamental step in theutilization, characterization and understanding of numerous biological processes.Consequently, highly advanced functional and structural measurement tools have beendeveloped over the last decadess.4 Particularly, nuclear magnetic resonance (NMR), X-raycrystallography and neutron reflectivity (NR) provide structural measurements, whereastools such as microcalorimetry or surface plasmon resonance (SPR) provide functional data.Furthermore, the more recent approach based on Dual Polarization Interferometry (DPI), isallowing the molecular interactions to be quantitatively measured at nanometric dimensions.DPI is currently one of the most powerful label-free biosensing techniques inheterogeneous format to record real-time data of conformational dynamics, which isefficiently employed in different applications, such as bionanotechnology, surface science,BiotechnologyDrugDiscoveryLipidStudiesCrystallographySurfaceScienceDPIand crystallography or drug discovery (Figure 2). Their measurements can provideinformation about the connection between the biomolecule function and its structuralchanges. This technique is, to our knowledge, the most well-built and well-thought throughsuch waveguide sensor. It has its weaknesses, e.g. the necessity of using a relatively longsensor element, but the information it delivers is the interaction of two polarization modesof the propagating light with a molecular film at the top of the waveguide with which itinteracts through the evanescent field. It is well known that the use of an interferometricreadout and a long waveguide makes the measurements very stable and more accurate thanthose of the competitor techniques. Accordingly, DPI can be described as a molecular rulerwhose quantitative values can be correlated directly with those from other usual techniques,such as NMR, X-ray crystallography and NR, providing higher sensitivity and accuracy thanthe classical acoustic and optical biosensors.In 1996, Dr Neville Freeman conceived of the idea behind DPI as a robust andreproducible biosensing technology and, together with Dr Graham Cross, developed theconcept and filed the original patent.5,6 This novel technique has gained popularity amongthe scientific community in the last decade and the number of publications dealing with thistechnique has increased considerably since the initial report in 2003.7 In this review, DPI iscompared with other techniques, and its theoretical basis and applications are outlined.The fundamentals are specified together with strategies for chip functionalization andapplications of the aforementioned technology in a wide variety of research areas. All thisgives a unique chance to learn from this sensing technique, which may be an essentialreference to facilitate the work of future users.

Surface biofunctionalization, including chemical activation and attachment of the bioreceptor, is an essential step to provide reliable detection of biomolecular binding events monitored by Surface Plasmon Resonance (SPR), the most employed optical biosensor, and other biosensor techniques. Recent progress in the area of immobilization procedures are aimed at producing reproducible interfacial surfaces that enable the sensitive and specific recognition of the analyte. Antibodies are still the most employed bioreceptors for SPR assays. A wide range of strategies have been proposed to maximize the SPR immunosensor performance by controlling the stability and orientation of the immobilized antibody. This article reviews the most recent advancements in random and oriented antibody immobilization approaches for SPR biosensing applications, with a special focus on the research that have been done to find universal linkers, which can allow the use of the same functionalized surface for different applications.

A sensitive and selective wavelength modulation surface plasmon resonance (SPR) biosensor is reported with Au nanoparticle decorated graphene oxide (GO) as an enhanced sensing platform. GO sheets possess favourable water dispersibility, good biocompatibility and high loading capacity. An Au-GO composite with the Au spheres size of 15-20 nm was synthesized and modified with staphylococcal protein A (SPA). The as-prepared composite assembles directly onto the Au film surface of the SPR sensor. Meanwhile, SPA specifically recognizes and binds the Fc portion of antibodies, contributing to highly oriented antibody immobilization on the chip surface without any antibody modification. Consequently, the biosensor based on the SPA modified Au-GO composite exhibits a satisfactory response to rabbit IgG in the concentration range of 0.1-50 渭g mL(-1), while the biosensor based on the sole SPA layer for antibody immobilization shows a response in the concentration range of 1.6-50 渭g mL(-1). Experimental results show that the SPA modified Au-GO composite can be successfully used for the signal amplification of immunosensors, thereby improving the sensitivity and obviating the need of chemical modification of the antibody.

Abstract The specific recognition between monoclonal antibody (anti-human prostate-specific antigen, anti-hPSA) and its antigen (human prostate-specific antigen, hPSA) has promising applications in prostate cancer diagnostics and other biosensor applications. However, because of steric constraints associated with interfacial packing and molecular orientations, the binding efficiency is often very low. In this study, spectroscopic ellipsometry and neutron reflection have been used to investigate how solution pH, salt concentration and surface chemistry affect antibody adsorption and subsequent antigen binding. The adsorbed amount of antibody was found to vary with pH and the maximum adsorption occurred between pH 5 and 6, close to the isoelectric point of the antibody. By contrast, the highest antigen binding efficiency occurred close to the neutral pH. Increasing the ionic strength reduced antibody adsorbed amount at the silica-water interface but had little effect on antigen binding. Further studies of antibody adsorption on hydrophobic C8 (octyltrimethoxysilane) surface and chemical attachment of antibody on (3-mercaptopropyl)trimethoxysilane/4-maleimidobutyric acid N-hydroxysuccinimide ester-modified surface have also been undertaken. It was found that on all surfaces studied, the antibody predominantly adopted the 'flat on' orientation, and antigen-binding capabilities were comparable. The results indicate that antibody immobilization via appropriate physical adsorption can replace elaborate interfacial molecular engineering involving complex covalent attachments.

Neutron reflectometry provides evidence of ternary protein adsorption within polyethylene glycol (PEG) brushes. Anti-PEG Immunoglobulin G antibodies (Abs) binding the methoxy terminated PEG chain segment specifically adsorb onto PEG brushes grafted to lipid monolayers on a solid support. The Abs adsorb at the outer edge of the brush. The thickness and density of the adsorbed Ab layer, as well as its distance from the grafting surface grow with increasing brush density. At high densities most of the protein is excluded from the brush. The results are consistent with an inverted “Y” configuration with the two F AB segments facing the brush. They suggest that increasing the grafting density favors narrowing of the angle between the F AB segments as well as overall orientation of the bound Abs perpendicular to the surface.

The interaction between proteins and solid surfaces can influence their conformation and therefore also their activity and affinity. These interactions are highly specific for the respective combination of proteins and solids. Consequently, it is desirable to investigate the conformation of proteins on technical surfaces, ideally at single molecule level, and to correlate the results with their activity. This is in particular true for biosensors where the conformation-dependent target affinity of an immobilized receptor determines the sensitivity of the sensor. Here, we investigate for the first time the immobilization and orientation of antibodies (Abs) photoactivated by a photonic immobilization technique (PIT), which has previously demonstrated to enhance binding capabilities of antibody receptors. The photoactivated immunoglobulins are immobilized on ultrasmooth template stripped gold films and investigated by atomic force microscopy (AFM) at the level of individual molecules. The observed protein ori...

尾 2 -Microglobulin (B2M) is a human protein involved in the regulation of immune response and represents a useful biomarker for several diseases. Recently, anti-B2M monoclonal antibodies have been introduced as innovative therapeutic agents. A deeper understanding of the molecular interaction between the two partners could be of utmost relevance for both designing array-based analytical devices and improving current immunotherapies. A visualization at the nanoscale performed by Atomic Force Microscopy revealed that binding of B2M to the antibody occurred according to two preferred interaction geometries. Additionally, Atomic Force Spectroscopy and Surface Plasmon Resonance provided us with detailed information on the binding kinetics and the energy landscape of the complex, both at the single molecule level and in bulk conditions. Combination of these complementary techniques contributed to highlight subtle differences in the kinetics behaviour characterizing the complexes. Collectively, the results may deserve significant interest for designing, development and optimization of novel generations of nanobiosensor platforms.

Abstract Large-scale molecular dynamics (MD) simulations and atomic force microscopy (AFM) in liquid are combined to characterize the adsorption of Immunoglobulin G (IgG) antibodies over a hydrophobic surface modeled with a three-layer graphene slab. We consider explicitly the water solvent, simulating systems with massive sizes (up to 770芒聙聣000 atoms), for four different adsorption orientations. Protocols based on steered MD to speed up the protein diffusion stage and to enhance the dehydration process are combined with long simulation times (>150 ns) in order to make sure that the final adsorption states correspond to actual stable configurations. Our MD results and the AFM images demonstrate that the IgG antibodies are strongly adsorbed, do not unfold, and retain their secondary and tertiary structures upon deposition. Statistical analysis of the AFM images shows that many of the antibodies adopt vertical orientations, even at very small coverages, which expose at least one Fab binding site for recognition events. Single molecule force spectroscopy experiments demonstrate the immunological response of the deposited antibodies by recognizing its specific antigens. The above properties together with the strong anchoring and preservation of the secondary structure, make graphene an excellent candidate for the development of immunosensors.

The preparation and performance of a suitable chimeric biosensor based on antibodies (Abs) immobilized on lipase-coated magnetic particles by means of a standing orienting strategy are presented. This novel system is based on hydrophobic magnetic particles coated with modified lipase molecules able to orient and further immobilize different Abs in a covalent way without any previous site-selective chemical modification of biomacromolecules. Different key parameters attending the process were studied and optimized. The optimal preparation was performed using a controlled loading (1 nmol Ab g(-1) chimeric support) at pH 9 and a short reaction time to recover a biological activity of about 80%. AFM microscopy was used to study and confirm the Abs-oriented immobilization on lipase-coated magnetic particles and the final achievement of a highly active and recyclable chimeric immune sensor. This direct technique was demonstrated to be a powerful alternative to the indirect immunoactivity assay methods for the study of biomacromolecule-oriented immobilizations.

A high-sensitivity flow-based immunoassay is reported based on a gold-coated quartz crystal microbalance (QCM) chip functionalized directly in the QCM without requiring covalent conjugation steps. Specifically, the irreversible adsorption of a biotinylated graphene oxide-avidin complex followed by loading of a biotinylated capture antibody is applied to avoid more complex conventional surface modification chemistries and enable chip functionalization and sensing all within the QCM instrument. The resulting immunosensors exhibit significantly lower nonspecific protein adsorption and stronger signal for antigen sensing relative to simple avidin-coated sensors. Reproducible quantification of rabbit IgG concentrations ranging from 0.1 ng/mL to 10 渭g/mL (6 orders of magnitude) can be achieved depending on the approach used to quantify the binding with simple mass changes used to detect higher concentrations and a horseradish peroxidase-linked detection antibody that converts its substrate to a measurable preci...

61A sensitive mass enhanced quartz crystal microbalance immunosenor was fabricated.61Magnetic bead supported bienzymes were used as biocatalyst.61Bienzymes catalyzed precipitation reaction was used for protein detection.

The adsorption of single-component bovine serum albumin (BSA), bovine fibrinogen (Fgn) and bovine immunoglobulin G (IgG) films, as well as mult--component bovine plasma films onto bare and sodium styrene sulfonate (NaSS)-grafted gold substrates was characterized. The adsorption isotherms, measured via X-ray photoelectron spectroscopy showed that at low solution concentrations all three single-component proteins adsorb with higher affinity onto gold surfaces compared to NaSS surfaces. However, at higher concentrations, NaSS surfaces adsorb the same or more total protein than gold surfaces. This may be because proteins that adsorb onto NaSS undergo structural rearrangements, resulting in a larger fraction of irreversibly adsorbed species over time. Still, with the possible exception of BSA adsorbed onto gold, neither surface appeared to have saturated at the highest protein solution concentration studied. Principal component analysis of amino acid mass fragments from time-of-flight secondary ion mass spectr...

Artificial neural networks (ANNs) form a class of powerful multivariate analysis techniques, yet their routine use in the surface analysis community is limited. Principal component analysis (PCA) is more commonly employed to reduce the dimensionality of large data sets and highlight key characteristics. Herein, we discuss the strengths and weaknesses of PCA and ANNs as methods for investigation and interpretation of a complex multivariate sample set. Using time-of-flight secondary ion mass spectrometry (ToF-SIMS) we acquired spectra from an antibody and its proteolysis fragments with three primary-ion sources to obtain a panel of 72 spectra and a characteristic peak list of 775 fragment ions. We describe the use of ANNs as a means to interpret the ToF-SIMS spectral data, highlight the optimal neural network design and computational parameters, and discuss the technique limitations. Further, employing Bi3+ as the primary-ion source, ANNs can accurately classify antibody fragments from the parent antibody b...