Antibody-coated Surface Research Articles

Development of a universal, oriented antibody immobilization method to functionalize vascular prostheses for enhanced endothelialization for potential clinical application

BackgroundThrombosis is a common cause of vascular prosthesis failure. Antibody coating of prostheses to capture circulating endothelial progenitor cells to aid endothelialization on the device surface appears a promising solution to prevent thrombus formation. Compared with random antibody immobilization, oriented antibody coating (OAC) increases antibody-antigen binding capacity and reduces antibody immunogenicity in vivo. Currently, few OAC methods have been documented, with none possessing clinical application potential.ResultsDopamine and the linker amino-PEG8-hydrazide-t-boc were successfully deposited on the surface of cobalt chromium (CC) discs, CC stents and expanded polytetrafluoroethylene (ePTFE) grafts under a slightly basic condition. CD34 antibodies were immobilized through the reaction between aldehydes in the Fc region created by oxidation and hydrazides in the linker after t-boc removal. CD34 antibody-coated surfaces were integral and smooth as shown by scanning electron microscopy (SEM), had significantly reduced or no substrate-specific signals as revealed by X-ray photoelectron spectroscopy, were hospitable for HUVEC growth as demonstrated by cell proliferation assay, and specifically bound CD34 + cells as shown by cell binding testing. CD34 antibody coating turned hydrophobic property of ePTFE grafts to hydrophilic. In a porcine carotid artery interposition model, a confluent monolayer of cobblestone-shaped CD31 + endothelial cells on the luminal surface of the CD34 antibody coated ePTFE graft were observed. In contrast, thrombi and fibrin fibers on the bare graft, and sporadic cells on the graft coated by chemicals without antibodies were seen.ConclusionA universal, OAC method was developed. Our in vitro and in vivo data suggest that the method can be potentially translated into clinical application, e.g., modifying ePTFE grafts to mitigate their thrombotic propensity and possibly provide for improved long-term patency for small-diameter grafts.

Immunocapturing rare cells from blood: A simple and robust microsystem approach

Cell immunocapture microsystems are a fast-emerging field with several potential medical diagnostic applications. Isolation and quantification of circulating rare cells (CRCs) show great importance in the early stages of disease diagnostics and prognostics. Here, we present a simple and robust stop-flow microsystem (fabricated by a combination of glass microblasting and 3D printing) based on a planar antibody-coated surface that is effective in the immunocapture of the model as well as naturally occurring rare cells. A chip with a planar immunocapture channel working in the so-called stop-flow dynamic regime was designed to enable monitoring the efficiency of the cell capture by fluorescence microscopy. Up to 90% immunocapture efficiency of MCF-7 cells spiked into whole blood on CD326 antibody-coated planar surfaces was achieved. We discuss the role of the planar surface modifications, the influence of the set stop-flow dynamic conditions, and medium complexity on the efficiency of cell immunocapture. The presented results could be further employed in the design of microsystems for cell-size-independent isolation and identification of rare cells from blood.

Mechanisms of frustrated phagocytic spreading of human neutrophils on antibody-coated surfaces

Complex motions of immune cells are an integral part of diapedesis, chemotaxis, phagocytosis, and other vital processes. To better understand how immune cells execute such motions, we present a detailed analysis of phagocytic spreading of human neutrophils on flat surfaces functionalized with different densities of immunoglobulin G (IgG) antibodies. We visualize the cell-substrate contact region at high resolution and without labels using reflection interference contrast microscopy and quantify how the area, shape, and position of the contact region evolves over time. We find that the likelihood of the cell commitment to spreading strongly depends on the surface density of IgG, but the rate at which the substrate-contact area of spreading cells increases does not. Validated by a theoretical companion study, our results resolve controversial notions about the mechanisms controlling cell spreading, establishing that active forces generated by the cytoskeleton rather than cell-substrate adhesion primarily drive cellular protrusion. Adhesion, on the other hand, aids phagocytic spreading by regulating the cell commitment to spreading, the maximum cell-substrate contact area, and the directional movement of the contact region.

A Novel Methodology for Detecting Variations in Cell Surface Antigens Using Cell-Tearing by Optical Tweezers.

The quantitative analysis of cell surface antigens has attracted increasing attention due to the antigenic variation recognition that can facilitate early diagnoses. This paper presents a novel methodology based on the optical “cell-tearing” and the especially proposed “dilution regulations” to detect variations in cell surface antigens. The cell attaches to the corresponding antibody-coated slide surface. Then, the cell-binding firmness between a single cell and the functionalized surface is assayed by optically tearing using gradually reduced laser powers incorporated with serial antibody dilutions. Groups B and B3 of red blood cells (RBCs) were selected as the experiment subject. The results indicate that a higher dilution called for lower power to tear off the cell binding. According to the proposed relative-quantitative analysis theory, antigenic variation can be intuitively estimated by comparing the maximum allowable dilution folds. The estimation result shows good consistency with the finding in the literature. This study suggests a novel methodology for examining the variation in cell surface antigens, expected to be widely capable with potential sensor applications not only in biochemistry and biophysics, but also in the micro-/nano- engineering field.

A Transparent Semiconducting Surface for Capturing and Releasing Single Cells from a Complex Cell Mixture.

Selective isolation of individual target cells from a heterogeneous population is technically challenging; however, the ability to retrieve single cells can have high significance in various aspects of biological research. Here, we present a new photoelectrochemical surface based on a transparent electrode that is compatible with high-resolution fluorescence microscopy for isolating individual rare cells from complex biological samples. This is underpinned by two important factors: (i) careful design of the electrode by patterning discrete Au disks of micron dimension on amorphous silicon-indium tin oxide films and (ii) orthogonal surface chemistry, which modifies the patterned electrode with self-assembly layers of different functionalities, to selectively capture target cells on the Au disks and resist cell binding to the amorphous silicon surface. The co-stimulation of the surface using light from a microscope and an electric potential triggers the reductive desorption of the alkanethiol monolayer from the Au disks to release the single cells of interest from the illuminated regions only. Using circulating tumor cells as a model, we demonstrate the capture of cancer cells on an antibody-coated surface and selective release of single cancer cells with low expression of epithelial cell adhesion molecules.

A simple immunoassay for extracellular vesicle liquid biopsy in microliters of non-processed plasma

BackgroundExtracellular vesicles (EVs), released by most cell types, provide an excellent source of biomarkers in biological fluids. However, in order to perform validation studies and screenings of patient samples, it is still necessary to develop general techniques permitting rapid handling of small amounts of biological samples from large numbers of donors.ResultsHere we describe a method that, using just a few microliters of patient’s plasma, identifies tumour markers exposed on EVs. Studying physico-chemical properties of EVs in solution, we demonstrate that they behave as stable colloidal suspensions and therefore, in immunocapture assays, many of them are unable to interact with a stationary functionalised surface. Using flocculation methods, like those used to destabilize colloids, we demonstrate that cationic polymers increase EV ζ-potential, diameter, and sedimentation coefficient and thus, allow a more efficient capture on antibody-coated surfaces by both ELISA and bead-assisted flow cytometry. These findings led to optimization of a protocol in microtiter plates allowing effective immunocapture of EVs, directly in plasma without previous ultracentrifugation or other EV enrichment. The method, easily adaptable to any laboratory, has been validated using plasma from lung cancer patients in which the epithelial cell marker EpCAM has been detected on EVs.ConclusionsThis optimized high throughput, easy to automate, technology allows screening of large numbers of patients to phenotype tumour markers in circulating EVs, breaking barriers for the validation of proposed EV biomarkers and the discovery of new ones.Graphical

Development and In-Depth Characterization of Bacteria Repellent and Bacteria Adhesive Antibody-Coated Surfaces Using Optical Waveguide Biosensing.

Bacteria repellent surfaces and antibody-based coatings for bacterial assays have shown a growing demand in the field of biosensors, and have crucial importance in the design of biomedical devices. However, in-depth investigations and comparisons of possible solutions are still missing. The optical waveguide lightmode spectroscopy (OWLS) technique offers label-free, non-invasive, in situ characterization of protein and bacterial adsorption. Moreover, it has excellent flexibility for testing various surface coatings. Here, we describe an OWLS-based method supporting the development of bacteria repellent surfaces and characterize the layer structures and affinities of different antibody-based coatings for bacterial assays. In order to test nonspecific binding blocking agents against bacteria, OWLS chips were coated with bovine serum albumin (BSA), I-block, PAcrAM-g-(PMOXA, NH2, Si), (PAcrAM-P) and PLL-g-PEG (PP) (with different coating temperatures), and subsequent Escherichia coli adhesion was monitored. We found that the best performing blocking agents could inhibit bacterial adhesion from samples with bacteria concentrations of up to 107 cells/mL. Various immobilization methods were applied to graft a wide range of selected antibodies onto the biosensor’s surface. Simple physisorption, Mix&Go (AnteoBind) (MG) films, covalently immobilized protein A and avidin–biotin based surface chemistries were all fabricated and tested. The surface adsorbed mass densities of deposited antibodies were determined, and the biosensor;s kinetic data were evaluated to divine the possible orientations of the bacteria-capturing antibodies and determine the rate constants and footprints of the binding events. The development of affinity layers was supported by enzyme-linked immunosorbent assay (ELISA) measurements in order to test the bacteria binding capabilities of the antibodies. The best performance in the biosensor measurements was achieved by employing a polyclonal antibody in combination with protein A-based immobilization and PAcrAM-P blocking of nonspecific binding. Using this setting, a surface sensitivity of 70 cells/mm2 was demonstrated.

T cell stiffness is enhanced upon formation of immunological synapse.

T cells are activated by target cells via an intimate contact, termed immunological synapse (IS). Cellular mechanical properties, especially stiffness, are essential to regulate cell functions. However, T cell stiffness at a subcellular level at the IS still remains largely elusive. In this work, we established an atomic force microscopy (AFM)-based elasticity mapping method on whole T cells to obtain an overview of the stiffness with a resolution of ~60 nm. Using primary human CD4+ T cells, we show that when T cells form IS with stimulating antibody-coated surfaces, the lamellipodia are stiffer than the cell body. Upon IS formation, T cell stiffness is enhanced both at the lamellipodia and on the cell body. Chelation of intracellular Ca2+ abolishes IS-induced stiffening at the lamellipodia but has no influence on cell-body-stiffening, suggesting different regulatory mechanisms of IS-induced stiffening at the lamellipodia and the cell body.

Entangled Nanoplasmonic Cavities for Estimating Thickness of Surface-Adsorbed Layers.

Plasmonic sensors provide real-time and label-free detection of biotargets with unprecedented sensitivity and detection limit. However, they usually lack the ability to estimate the thickness of the target layer formed on top of the sensing surface. Here, we report a sensing modality based on reflection spectroscopy of a nanoplasmonic Fabry-Perot cavity array, which exhibits characteristics of both surface plasmon polaritons and localized plasmon resonances and outperforms its conventional counterparts by providing the thickness of the surface-adsorbed layers. Through numerical simulations, we demonstrate that the designed plasmonic surface resembles two entangled Fabry-Perot cavities excited from both ends. Performance of the device is evaluated by studying sensor response in the refractive index (RI) measurement of aqueous glycerol solutions and during formation of a surface-adsorbed layer consisting of protein (i.e., NeutrAvidin) molecules. By tracking the resonance wavelengths of the two modes of the nanoplasmonic surface, it is therefore possible to measure the thickness of a homogeneous adsorbed layer and RI of the background solution with precisions better than 4 nm and 0.0001 RI units. Using numerical simulations, we show that the thickness estimation algorithm can be extended for layers consisting of nanometric analytes adsorbed on an antibody-coated sensor surface. Furthermore, performance of the device has been evaluated to detect exosomes. By providing a thickness estimation for adsorbed layers and differentiating binding events from background RI variations, this device can potentially supersede conventional plasmonic sensors.

One-Step Digital Immunoassay for Rapid and Sensitive Detection of Cardiac Troponin I.

A rapid and sensitive method to detect cardiac troponin I (cTnI) in human blood is critical to the diagnosis and treatment of acute myocardial infarction (AMI). Here, we describe a simple one-step digital immunoassay for single-molecule detection without washing steps. A sample containing cTnI mixed with detection antibody-conjugated gold nanoparticles (AuNPs) is added to a capture antibody-coated sensor surface and the formation of the antibody-cTnI-antibody sandwich is detected by digitally counting the binding of the individual gold nanoparticles to the sensor surface in real time using a bright-field optical imaging setup together with a differential imaging algorithm. The digital immunoassay detects cTnI in undiluted human plasma, which achieves a detection limit of 5.7 ng/L within a detection time of only 10 min, which meets the requirement of current clinical high-sensitivity troponin assay (∼70 ng/L cutoff). We anticipate that the one-step and real-time digital immunoassay can be applied to the detection of other disease biomarkers in blood.

Detection of Brucella abortus by a platform functionalized with protein A and specific antibodies IgG.

Oriented immobilization of antibodies on a sensor surface is critical for enhancing both the antigen-binding capacity and the sensitivity of immunosensors. In this study, we describe a strategy to adsorb immunoglobulin G (IgG) anti-Brucella antibodies onto a silicon surface, oriented by protein A obtained from Staphylococcus aureus (SpA). X-ray photoelectron spectroscopy and atomic force microscopy were used to characterize topographically, morphologically, and chemical changes of the sensor functionalization. The activity of the biosensor was assessed by confocal microscopy, scanning electronic microscopy, and bacteria capture assays (BCA). According to the BCA, the efficiency of Brucella abortus detection with the SpA-IgG anti Brucella biosensor was three-fold higher than that of the random orientated IgG anti Brucella biosensor. The limit of detection was 1 × 106 CFU/ml. These data show that the orientation of antibodies immobilization is crucial to developing immunosensors for bacterial antigen detection as Brucella spp and improve its sensibility level. Functionalization with protein A increases Brucella detection by an antibody-coated surface. Functionalized silicon surface for Brucella detection was characterized by atomic force microscopy, X-ray photoelectron spectroscopy and confocal microscopy.

Tumor cell capture from blood by flowing across antibody-coated surfaces.

The load of circulating tumor cells (CTC) is related to poor outcomes in cancer patients. A sufficient number of these cells would enable a full characterization of the cancer. An approach to probe larger blood volumes, allowing for the detection of more of these very rare CTC, is the use of leukapheresis. Currently available techniques allow only the analysis of a small portion of leukapheresis products. Here, we present a method that uses flow rather than static conditions which allows processing of larger volumes. We evaluated the conditions needed to isolate tumor cells from blood while passing antibody coated surfaces. Results show that our set-up efficiently captures cancer cells from whole blood. Results show that the optimal velocity at which cells are captured from blood is 0.6 mm s-1. Also, it can be concluded that the VU1D9 antibody targeting the EpCAM antigen has very high capture efficiency. When using an antibody that does not capture 100% of all cells, combining multiple antibodies on the capture surface is very beneficial leading to an increase in cell capture and is therefore worthwhile considering in any cancer cell capture methodology.

Interferometric plasmonic imaging and detection of single exosomes

Exosomes play an important role in numerous cellular processes. Fundamental study and practical use of exosomes are significantly constrained by the lack of analytical tools capable of physical and biochemical characterization. In this paper, we present an optical approach capable of imaging single exosomes in a label-free manner, using interferometric plasmonic microscopy. We demonstrate monitoring of the real-time adsorption of exosomes onto a chemically modified Au surface, calculating the image intensity, and determining the size distribution. The sizing capability enables us to quantitatively measure the membrane fusion activity between exosomes and liposomes. We also report the recording of the dynamic interaction between exosomes and antibodies at the single-exosome level, and the tracking of hit-stay-run behavior of exosomes on an antibody-coated surface. We anticipate that the proposed method will contribute to clinical exosome analysis and to the exploration of fundamental issues such as the exosome-antibody binding kinetics.

Spatio-temporal image correlation spectroscopy and super-resolution microscopy to quantify molecular dynamics in T cells.

Many cellular processes are regulated by the spatio-temporal organisation of signalling complexes, cytoskeletal components and membranes. One such example is at the T cell immunological synapse where the retrograde flow of cortical filamentous (F)-actin from the synapse periphery drives signalling protein microclusters towards the synapse centre. The density of this mesh however, makes visualisation and analysis of individual actin fibres difficult due to the resolution limit of conventional microscopy. Recently, super-resolution methods such as structured illumination microscopy (SIM) have surpassed this resolution limit. Here, we apply SIM to better visualise the dense cortical actin meshwork in T cell synapses formed against activating, antibody-coated surfaces and image under total-internal reflection fluorescence (TIRF) illumination. To analyse the observed molecular flows, and the relationship between them, we apply spatio-temporal image correlation spectroscopy (STICS) and its cross-correlation variant (STICCS). We show that the dynamic cortical actin mesh can be visualised with unprecedented detail and that STICS/STICCS can output accurate, quantitative maps of molecular flow velocity and directionality from such data. We find that the actin flow can be disrupted using small molecule inhibitors of actin polymerisation. This combination of imaging and quantitative analysis may provide an important new tool for researchers to investigate the molecular dynamics at cellular length scales. Here we demonstrate the retrograde flow of F-actin which may be important for the clustering and dynamics of key signalling proteins within the plasma membrane, a phenomenon which is vital to correct T cell activation and therefore the mounting of an effective immune response.

One-step direct immunoassay with horseradish peroxidase as antigen for studying the functionality of antibody surfaces

Antibody-coated surfaces (Ab surfaces) play a key role in bioanalytical tools developed for biosensors and diagnostics. Therefore, a high and well-defined functional activity of the Ab surface is required. The functional activity of the Ab surface depends on its available binding sites i.e. “the active sites” that are able to capture antigen (Ag). The number of active binding sites strongly depends on the immobilization strategy used to fix the Ab on the solid surface. Determination of layer thickness or surface topology are often used to characterize the Ab surfaces but there is no gold standard method for the study of the functionality of the Ab surfaces.For that purpose, we aim at developing an assay allowing to determine the performances of Ab surfaces. In the present study, anti-horseradish peroxidase antibody (anti-HRP Ab) is used as capture Ab covalently bound to the surface and enzyme HRP as Ag. This direct assay permits, in one-step, to generate a signal utilizing the catalytic properties of HRP. The signal is directly proportional to the amount of HRP bound on the Ab surface, and therefore to the active binding sites of immobilized Ab. The HRP/anti-HRP Ab interactions may be a useful indicator to construct accurate and reproducible active Ab surfaces and also to improve their performances in term of stability and sensitivity.Optimization of the assay parameters and quality of the results are presented. A good repeatability and an acceptable inter-day precision (RSD < 10%) are reported.

Growth of Escherichia coli on the GaAs (001) surface

Detection of pathogenic bacteria and monitoring their susceptibility to antibiotics are of great importance in the fields of medicine, pharmaceutical research, as well as water and food industries. In order to develop a photonic biosensor for detection of bacteria by taking advantage of photoluminescence (PL) of GaAs-based devices, we have investigated the capture and growth of Escherichia coli K12 on bare and biofunctionalized surfaces of GaAs (001) – a material of interest for capping different semiconductor microstructures. The results were compared with the capture and growth of Escherichia coli K12 on Au surfaces that have commonly been applied for studying a variety of biological and biochemical reactions. We found that neither GaAs nor Au-coated glass wafers placed in Petri dishes inoculated with bacteria inhibited bacterial growth in nutrient agar, regardless of the wafers being bare or biofunctionalized. However, the capture and growth of bacteria on biofunctionalized surfaces of GaAs and Au wafers kept in a flow cell and exposed to different concentrations of bacteria and growth medium revealed that the initial surface coverage and the subsequent bacterial growth were dependent on the biofunctionalization architecture, with antibody-coated surfaces clearly being most efficient in capturing bacteria and offering better conditions for growth of bacteria. We have observed that, as long as the GaAs wafers were exposed to bacterial suspensions at concentrations of at least 105 CFU/mL, bacteria could grow on the surface of wafers, regardless of the type of biofunctionalization architecture used to capture the bacteria. These results provide important insight towards the successful development of GaAs-based devices designed for photonic monitoring of bacterial reactions to different biochemical environments.

Investigation of Utilizing Fluent MPM in Microscopic Biological Applications

The isolation of rare cells (those infrequently seen in the medium of interest) is important in diagnostic medicine. The use of antibody-coated surfaces to bind and capture cells of specific types has been extensively studied but collision frequency and probability of detachment before capture is complete are less studied and more important for the capture of rare cells. For surface capture to be effective, cells need to make physicochemical contact with the antibody coated entities. Because cells suspended in medium cannot be considered as point masses due to their large volumes, the usual computational fluid dynamics (CFD) techniques of tracking particle movement are insufficient to predict the frequency and duration of contact. The novel Macroscopic Particle Model (MPM) recently proposed by developers of the Fluent CFD package is used and extended to relate its parameters to collision frequencies and patterns known to occur in experimentally observed systems. This study explores and evaluates many of MPM’s parameters according to their effects on the numerical accuracy and overall similarity of the simulations to experimentally observed systems. The current MPM model shows some limitations when simulating real three dimensional systems; nevertheless, with the ongoing revisions to MPM by Fluent developers, we are optimistic about its accuracy and utility in the future.

Microfluidic PMMA-based microarray sensor chip with imaging analysis for ABO and RhD blood group typing.

Solid phase microarrays have been described for use in blood typing; red blood cells (RBCs) captured on immobilized antibodies were detected using surface plasmon resonance or fluorescence. We present antibody microarray on Poly (methylmethacrylate) (PMMA) surface coupled with microfluidic system for ABO and RhD blood typing. After immobilized by antigen-antibody interaction, the RBCs were detected by image recognition. The sensor surface was produced from grafted aminopropyltriethoxysilane (APTES) on photochemical modified PMMA surface by UV irradiation and subsequently reacted with glutaraldehyde cross-linking. The amine group of monoclonal antibody of anti-A, anti-B and anti-D was reacted with an aldehyde group on the glutaraldehyde modified surface, forming an imine linkage. RBCs were captured by the coated antibody via antigen-antibody interaction, and blood grouping was determined by microarray image cell counting. Suitable condition for RBC detection was 10% RBC concentration at 10μl/min flow rate. This setting eliminated non-specific RBC binding resulting in correct blood groups identification of all 136 samples tested. The platform showed good reproducibility with coefficient of variation of 2·17%, 3·62% and 2·51% for anti-A, anti-B and anti-D respectively. The antibody-coated surface can be stabilized by stabilizer coating and stored for long-term use. The PMMA array chip demonstrated its good accuracy and precision in rapid blood group testing. For its high throughput, the method has potential for use in large blood donation centre.

Membrane tension drives ligand-independent integrin signaling.

Integrins anchor cells to the extracellular matrix (ECM) and control a multitude of essential cellular functions by activating a variety of signaling pathways. In this issue of The EMBO Journal, a study conducted by Ferraris and colleagues report that binding of urokinase-type plasminogen activator receptor (uPAR) to vitronectin is sufficient to trigger ligand-independent β1 and β3 integrin signaling. The coupling of uPAR and integrins occurs independent of lateral interaction between the two receptors, but relies on membrane tension (Ferraris et al, 2014).

Capture, release and culture of circulating tumor cells from pancreatic cancer patients using an enhanced mixing chip

Circulating tumor cells (CTCs) from peripheral blood hold important information for cancer diagnosis and disease monitoring. Analysis of this "liquid biopsy" holds the promise to usher in a new era of personalized therapeutic treatments and real-time monitoring for cancer patients. But the extreme rarity of CTCs in blood makes their isolation and characterization technologically challenging. This paper reports the development of a geometrically enhanced mixing (GEM) chip for high-efficiency and high-purity tumor cell capture. We also successfully demonstrated the release and culture of the captured tumor cells, as well as the isolation of CTCs from cancer patients. The high-performance microchip is based on geometrically optimized micromixer structures, which enhance the transverse flow and flow folding, maximizing the interaction between CTCs and antibody-coated surfaces. With the optimized channel geometry and flow rate, the capture efficiency reached >90% with a purity of >84% when capturing spiked tumor cells in buffer. The system was further validated by isolating a wide range of spiked tumor cells (50-50,000) in 1 mL of lysed blood and whole blood. With the combination of trypsinization and high flow rate washing, captured tumor cells were efficiently released. The released cells were viable and able to proliferate, and showed no difference compared with intact cells that were not subjected to the capture and release process. Furthermore, we applied the device for detecting CTCs from metastatic pancreatic cancer patients' blood; and CTCs were found from 17 out of 18 samples (>94%). We also tested the potential utility of the device in monitoring the response to anti-cancer drug treatment in pancreatic cancer patients, and the CTC numbers correlated with the clinical computed tomograms (CT scans) of tumors. The presented technology shows great promise for accurate CTC enumeration, biological studies of CTCs and cancer metastasis, as well as for cancer diagnosis and treatment monitoring.