Study of cell-matrix adhesion dynamics using surface plasmon resonance imaging ellipsometry

The interaction of cells with extracellular matrix, termed cell-matrix adhesions, importantly governs multiple cellular phenomena. Knowledge of the functional dynamics of cell-matrix adhesion could provide critical clues for understanding biological phenomena. We developed surface plasmon resonance imaging ellipsometry (SPRIE) to provide high contrast images of the cell-matrix interface in unlabeled living cells. To improve the contrast and sensitivity, the null-type imaging ellipsometry technique was integrated with an attenuated total reflection coupler. We verified that the imaged area of SPRIE was indeed a cell-matrix adhesion area by confocal microscopy imaging. Using SPRIE, we demonstrated that three different cell types exhibit distinct features of adhesion. SPRIE was applied to diverse biological systems, including during cell division, cell migration, and cell-cell communication. We imaged the cell-matrix anchorage of mitotic cells, providing the first label-free imaging of this interaction to our knowledge. We found that cell-cell communication can alter cell-matrix adhesion, possibly providing direct experimental evidence for cell-cell communication-mediated changes in cell adhesion. We also investigated shear-stress-induced adhesion dynamics in real time. Based on these data, we expect that SPRIE will be a useful methodology for studying the role of cell-matrix adhesion in important biological phenomena.     

Detection of biospecific interactions using amplified ellipsometry

Amplified detection of biomolecules and biological interactions using an optical surface technique, ellipsometry, is demonstrated for two biosystems--immunoglobulin G with anti-immunoglobulin G (IgG) and the lectin concanavalin A (Con A) with yeast cells. In order to improve the sensitivity of the ellipsometer signal, an amplifier conjugate is formed by binding the affinity ligand to a 12-nm silica particle which is readily detected by the ellipsometer. Thus by using conjugates of IgG-silica and Con A-silica, amplifications of five to seven times have been obtained enabling detection of less than 20 pg/mm2 of biomolecular material.     

Feasibility of protein A for the oriented immobilization of immunoglobulin on silicon surface for a biosensor with imaging ellipsometry

The feasibility of using protein A to immobilize antibody on silicon surface for a biosensor with imaging ellipsometry was presented in this study. The amount of human IgG bound with anti-IgG immobilized by the protein A on silicon surface was much more than that bound with anti-IgG immobilized by physical adsorption. The result indicated that the protein A could be used to immobilize antibody molecules in a highly oriented manner and maintain antibody molecular functional configuration on the silicon surface. High reproducibility of the amount of antibody immobilization and homogenous antibody adsorption layer on surfaces could be obtained by this immobilization method. Imaging ellipsometry has been proven to be a fast and reliable detection method and sensitive enough to detect small changes in a molecular monolayer level. The combination of imaging ellipsometry and surface modification with protein A has the potential to be further developed into an efficient immunoassay protein chip.     

Characterization of physical properties of supported phospholipid membranes using imaging ellipsometry at optical wavelengths

Subnanometer-scale vertical z-resolution coupled with large lateral area imaging, label-free, noncontact, and in situ advantages make the technique of optical imaging ellipsometry (IE) highly suitable for quantitative characterization of lipid bilayers supported on oxide substrates and submerged in aqueous phases. This article demonstrates the versatility of IE in quantitative characterization of structural and functional properties of supported phospholipid membranes using previously well-characterized examples. These include 1), a single-step determination of bilayer thickness to 0.2 nm accuracy and large-area lateral uniformity using photochemically patterned single 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers; 2), hydration-induced spreading kinetics of single-fluid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers to illustrate the in situ capability and image acquisition speed; 3), a large-area morphological characterization of phase-separating binary mixtures of 1,2-dilauroyl-sn-glycero-3-phosphocholine and galactosylceramide; and 4), binding of cholera-toxin B subunits to GM1-incorporating bilayers. Additional insights derived from these ellipsometric measurements are also discussed for each of these applications. Agreement with previous studies confirms that IE provides a simple and convenient tool for a routine, quantitative characterization of these membrane properties. Our results also suggest that IE complements more widely used fluorescence and scanning probe microscopies by combining large-area measurements with high vertical resolution without the use of labeled lipids.     

Detection of creatinine enriched on a surface imprinted polystyrene film using FT-ATR-IR

The surface of polystyrene (PS) was chemically modified by coating a thin layer of polyaniline (PANI) by oxidizing aniline using ammonium persulfate. Affinity sites for creatinine, a clinically relevant molecule, were created in the coated layer by adding creatinine as print molecules during the oxidation. The imprinted layer adsorbed creatinine was compared to non-imprinted surface reflecting the creation of creatinine-specific sites on the surface. The equilibrium was attained rapidly, indicating that a material of this kind is suitable for sensing applications. The adsorbed creatinine on the surface was detected using the technique of Fourier transform attenuated total internal reflection infra red spectroscopy (FT-ATR-IR). The results show that molecularly imprinted surface can enrich molecules of interest and the enriched molecules can be detected using FT-IR.     

Development of a protein microarray using sequence-specific DNA binding domain on DNA chip surface

A protein microarray based on DNA microarray platform was developed to identify protein-protein interactions in vitro. The conventional DNA chip surface by 156-bp PCR product was prepared for a substrate of protein microarray. High-affinity sequence-specific DNA binding domain, GAL4 DNA binding domain, was introduced to the protein microarray as fusion partner of a target model protein, enhanced green fluorescent protein. The target protein was oriented immobilized directly on the DNA chip surface. Finally, monoclonal antibody of the target protein was used to identify the immobilized protein on the surface. This study shows that the conventional DNA chip can be used to make a protein microarray directly, and this novel protein microarray can be applicable as a tool for identifying protein-protein interactions.     

Protein microarray biosensors based on imaging ellipsometry techniques and their applications

After years of development, biosensors based on imaging ellipsometry and biosensors based on total internal reflection imaging ellipsometry have been successfully implemented in various engineering systems. Their experimental setups, detection principles, and biological and clinical applications are briefly reviewed.     

Detection of dysplastic intestinal adenomas using a fluorescent folate imaging probe

Macrophages have long been recognized as a prominent component of tumors. Activated macrophages overexpress folate receptors and we used this phenomenon to image inflammatory reactions in colon dysplasia using a fluorescent folate probe (FFP). APC(Delta468) mice injected with FFP showed fluorescent adenomas (target-to-background ratio, adenoma vs. adjacent normal mucosa, of 2.46 +/- 0.41), significantly higher (p < .001) than adenomas in animals injected with a non-folate-containing control probe. Fluorescence-activated cell-sorting analysis revealed a 3-fold higher content of Mac1-positive cells in colonic adenomas compared with normal adjacent mucosa (6.8% vs. 2.2%), and confirmed the source of FFP-positive cells to be primarily an F4/80-positive macrophage subpopulation. Taken together, these results indicate that probe potentially can be used to image dysplastic intestinal adenomas in vivo.     

Detection of porphyrin using a short peptide immobilized on a surface plasmon resonance sensor chip

In this paper the development and feasibility of a novel detection system for a low molecular weight chemical, in which a peptide was utilized as a binding molecule, are described. Surface plasmon resonance (SPR) apparatus was used as a transducer. The porphyrin binding peptide, PSP2, was used as a model peptide ligand, while a porphyrin derivative, H₂TMpyP, was used as a model low-molecular-weight chemical. PSP2 was covalently immobilized onto the SPR sensor chip and SPR measurement using the PSP2-immobilized chip for various concentrations of porphyrin was carried out. H₂TMpyP was detectable in the range from 100 ng ml⁻¹ to 10 μg ml⁻¹ with a linear correlation and good precision and the PSP2-immobilized chip could be regenerated within 1 min after measurement in this system. From comparison of the detection manners of three porphyrin derivatives, the ability of a short peptide to discriminate between differences in molecular structure was demonstrated. Moreover, the self-assembled monolayer (SAM) of PSP2 was successfully prepared on the gold substrate and H₂TMpyP could be detected using the PSP2-SAM chip.     

Detection of fungal spores using a generic surface plasmon resonance immunoassay

This paper describes a biosensor-based method for detection of fungal spores using surface plasmon resonance (SPR). The approach involves the use of a mouse monoclonal antibody (Pst mAb8) and a SPR sensor for label-free detection of urediniospores from the model organism Puccinia striiformis f.sp. tritici (Pst). In the subtractive inhibition assay, urediniospores and Pst mAb8 were mixed, urediniospore-bound Pst mAb8 removed by centrifugation and the remaining Pst mAb8 quantified using the SPR sensor. Assay conditions were optimised and a detection limit of 3.1 x 10(5)urediniospores/ml was achieved. Spiked Pst samples were further examined in a background of a related spore and it was found that Pst detection was possible in this mixture. This study represent the first use of SPR technology for fungal spore detection as well as the first report of a successful biosensor-based detection strategy for Pst.     

Detection of DNA hybridization on a liposome surface using ultrasound velocimetry and turbidimetry methods

19-mer oligonucleotides with oleylamine tethered at 3' and 5' terminal, respectively, were incorporated into unilamellar liposomes of dioleoylphosphatidylcholine (DOPC). Addition of complementary nucleotide resulted in hybridization with oligonucleotides located on different liposomes and caused liposome aggregation. Significant changes of sound velocimetry and turbidity were readily observed at 10 nM concentration of the complementary chain.     

Detection of human proteins using arrayed imaging reflectometry

Assays built upon protein arrays are critical tools in determining the basic nature of biology, and have considerable promise in diagnosing human disease. These protein arrays aid in the elucidation of mapping pathway interactions, disease biomarker discovery, and regulatory processes. The solutions used in these experiments, including cellular lysate and serum, are inherently complex mixtures and are high in total protein content. Therefore, array-based assays must be robust and maintain a high level of selectivity and sensitivity. We report herein that arrayed imaging reflectometry (AIR), a label-free biosensing platform we have previously disclosed, is highly suitable for the detection of human proteins in complex solutions. In particular, we demonstrate array-based detection of cytokines in buffered solutions, and in undiluted human serum.     

Functional demonstration of connexin-protein binding using surface plasmon resonance

Surface plasmon resonance (SPR) allows examination of protein-protein interactions in real time, from which both binding affinities and kinetics can be directly determined. We have used the SPR technique to search for proteins in heart tissue that would be candidate binding partners for the cardiac gap junction protein, connexin43 (Cx43). Heart lysate showed a strong, pH-dependent binding to the carboxyl terminus (CT) of Cx43 (amino acids 254-382) covalently linked to an SPR cuvette. Binding was inhibited by the presence of v-src transfected 3T3 cell lysate, suggesting that binding partners in these two lysates may compete for overlapping epitopes on Cx43CT. The combined application of proteomic and functional studies is expected to identify which proteins within heart tissue interact with Cx43 and what roles they may play in gap junction function.     

Detection of viral bioagents using a shear horizontal surface acoustic wave biosensor

Viruses are of high medical and biodefense concern and their detection at concentrations well below the threshold necessary to cause health hazards continues to be a challenge with respect to sensitivity, specificity, and selectivity. Ideally, assays for accurate and real time detection of viral agents would not necessitate any pre-processing of the analyte, which would make them applicable for example to bodily fluids (blood, sputum) and man-made as well as naturally occurring bodies of water (pools, rivers). We describe herein a robust biosensor that combines the sensitivity of surface acoustic waves (SAW) generated at a frequency of 325MHz with the specificity provided by antibodies for the detection of viral agents. A lithium tantalate-based SAW transducer with silicon dioxide waveguide sensor platform featuring three test and one reference delay lines was used to adsorb antibodies directed against either Coxsackie virus B4 or the category A bioagent Sin Nombre virus (SNV), a member of the genus Hantavirus, family Bunyaviridae, negative-stranded RNA viruses. Rapid detection (within seconds) of increasing concentrations of viral particles was linear over a range of order of magnitude for both viruses, although the sensor was approximately 5 x 10(5)-fold more sensitive for the detection of SNV. For both pathogens, the sensor's selectivity for its target was not compromised by the presence of confounding Herpes Simplex virus type 1. The biosensor was able to detect SNV at doses lower than the load of virus typically found in a human patient suffering from hantavirus cardiopulmonary syndrome (HCPS). Further, in a proof-of-principle real world application, the SAW biosensor was capable to selectively detect SNV agents in complex solutions, such as naturally occurring bodies of water (river, sewage effluent) without analyte pre-processing. This is the first study that reports on the detection of viral agents using an antibody-based SAW biosensor that has the potential to be used as a hand-held and self-contained device for rapid viral detection in the field.     

Antifreeze glycopeptide adsorption on single crystal ice surfaces using ellipsometry

Antarctic fishes synthesise antifreeze proteins which can effectively inhibit the growth of ice crystals. The mechanism relies on adsorption of these proteins to the ice surface. Ellipsometry has been used to quantify glycopeptide antifreeze adsorption to the basal and prism faces of single ice crystals. The rate of accumulation was determined as a function of time and at concentrations between 0.0005 and 1.2 mg/ml. Estimates of packing density at saturation coverage have been made for the basal and prism faces.     

Detection of phosphatidylserine surface exposure on human erythrocytes using annexin V-ferrofluid

The asymmetric transbilayer distribution of phospholipids in the plasma membrane and the regulation of phosphatidylserine (PS) exposure at the cell surface of animal cells are of high physiological significance. It has been shown previously that annexin V is one of the most sensitive tools with which the presence of small amounts of PS on the outer surface of eukaryotic cells can be detected. We present here the covalent coupling of annexin V molecules to magnetic nanoparticles of maghemite. The resulting annexin V-ferrofluid is used in the magnetic separation of PS exposing cells, as illustrated for human erythrocytes modified in their phospholipid transbilayer asymmetry by the use of a calcium ionophore. Results on stored human erythrocytes and comparison with results obtained using iodinated and fluorescein-labeled annexin V are also presented.     

Detection of protein-protein interactions on SiO2/Si surfaces by spectroscopic ellipsometry

We have applied spectroscopic ellipsometry to sensitive detection of specific protein-protein interactions on SiO2/Si substrates. First, the change of ellipticity of the reflected polarized light (600-1100 nm) was correlated with the thickness of the protein layer immobilized on SiO2/Si surfaces by measuring monomeric (myoglobin) and homotetrameric (hemoglobin) proteins with a similar monomer size. Protein-protein interactions were then measured with the antigen/antibody and cell-surface receptor/ligand systems; in each system either of the two proteins was bound to SiO2/Si substrates. Consequently, significant ellipticity changes were observed only for the cases where the interactions were specific. A specific antibody binding was also detectable with an antigen displayed on the surface of bacteriophage particles. These results show the usefulness of spectroscopic ellipsometry for sensitive detection of protein-protein interactions and its applicability to a detection method for the protein-based biochips to be developed in the future.     

Non-invasive imaging of cysteine cathepsin activity in solid tumors using a 64Cu-labeled activity-based probe

The papain family of cysteine cathepsins are actively involved in multiple stages of tumorigenesis. Because elevated cathepsin activity can be found in many types of human cancers, they are promising biomarkers that can be used to target radiological contrast agents for tumor detection. However, currently there are no radiological imaging agents available for these important molecular targets. We report here the development of positron emission tomography (PET) radionuclide-labeled probes that target the cysteine cathepsins by formation of an enzyme activity-dependent bond with the active site cysteine. These probes contain an acyloxymethyl ketone (AOMK) functional group that irreversibly labels the active site cysteine of papain family proteases attached to a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) tag for labeling with (64)Cu for PET imaging studies. We performed biodistribution and microPET imaging studies in nude mice bearing subcutaneous tumors expressing various levels of cysteine cathepsin activity and found that the extent of probe uptake by tumors correlated with overall protease activity as measured by biochemical methods. Furthermore, probe signals could be reduced by pre-treatment with a general cathepsin inhibitor. We also found that inclusion of a Cy5 tag on the probe increased tumor uptake relative to probes lacking this fluorogenic dye. Overall, these results demonstrate that small molecule activity-based probes carrying radio-tracers can be used to image protease activity in living subjects.     

Study of binding between protein A and immunoglobulin G using a surface tension probe

Molecular interactions and binding are one of the most important and fundamental properties in the study of biochemical and biomedical systems. The understanding of such interactions and binding among biomolecules forms the basis for the design and processing of many biotechnological applications, such as bioseparation and immunoadsorption. In this study, we present a novel method to probe molecular interactions and binding based on surface tension measurement. This method complements conventional techniques, which are largely based on optical, spectroscopic, fluorescence polarization, chromatographic or atomic force microscopy measurements, by being definite in determining molecular binding ratio and flexible in sample preparation. Both dynamic and equilibrium (or quasi-equilibrium) information on molecular binding can be obtained through dynamic and equilibrium surface tension measurements. For an important pair of biological ligand and ligate, Protein A and immunoglobulin G (IgG), the existence of molecular interactions and the binding ratio of 1:2 have been determined unequivocally with the proposed surface tension method. These results are confirmed/supported by a mass balance calculation and spectrophotometry experiment. In addition, adsorption isotherms for Protein A and IgG separately at the air/water interface have been established with the dynamic surface tension measurements. The results show that the Langmuir isotherm equation can describe the adsorption data satisfactorily for both Protein A and IgG solutions.