Molecular packing of lysozyme,fibrinogen, and bovine serum albumin on hydrophilic and hydrophobic surfaces studied by infrared-visible sum frequency generation and fluorescence microscopy

Infrared-visible sum frequency generation (SFG) vibrational spectroscopy, in combination with fluorescence microscopy, was employed to investigate the surface structure of lysozyme, fibrinogen, and bovine serum albumin (BSA) adsorbed on hydrophilic silica and hydrophobic polystyrene as a function of protein concentration. Fluorescence microscopy shows that the relative amounts of protein adsorbed on hydrophilic and hydrophobic surfaces increase in proportion with the concentration of protein solutions. For a given bulk protein concentration, a larger amount of protein is adsorbed on hydrophobic polystyrene surfaces compared to hydrophilic silica surfaces. While lysozyme molecules adsorbed on silica surfaces yield relatively similar SFG spectra, regardless of the surface concentration, SFG spectra of fibrinogen and BSA adsorbed on silica surfaces exhibit concentration-dependent signal intensities and peak shapes. Quantitative SFG data analysis reveals that methyl groups in lysozyme adsorbed on hydrophilic surfaces show a concentration-independent orientation. However, methyl groups in BSA and fibrinogen become less tilted with respect to the surface normal with increasing protein concentration at the surface. On hydrophobic polystyrene surfaces, all proteins yield similar SFG spectra, which are different from those on hydrophilic surfaces. Although more protein molecules are present on hydrophobic surfaces, lower SFG signal intensity is observed, indicating that methyl groups in adsorbed proteins are more randomly oriented as compared to those on hydrophilic surfaces. SFG data also shows that the orientation and ordering of phenyl rings in the polystyrene surface is affected by protein adsorption, depending on the amount and type of proteins.     

Characterization of bovine serum albumin adsorption on chromium and AISI 304 stainless steel, consequences for the Pseudomonas fragi K1 adhesion

Adsorption of BSA on the surface of chromium and 304 stainless steel, has been characterized by Contact Angle Measurements, X-ray Photoelectron Spectroscopy (XPS) and Infrared Reflection Absorption Spectroscopy (IRRAS). Bacterial adhesion has been tested and compared on these two materials before and after pre-conditioning the surface with BSA. Chromium and stainless steel surfaces, when covered by a natural oxide layer, exhibit different energetic characteristics as shown by their γ_s^- and γ_s^LW respective values. These data vary upon immersion in BSA solutions, tending towards common values for duration of immersions. After immersion in BSA solutions, the evolution of the N 1s XPS signal, specific for the BSA, suggests that the surface is nearly saturated in a few minutes. Longer times of immersion only lead to a re-ordering of the adsorbed layer. Immersion tests in dilute BSA solutions (0.01 g/l) enabled us to make clear a higher reactivity of chromium towards the protein compared to stainless steel. These differences are cancelled at higher BSA concentrations (1 g/l). IRRAS spectra of BSA adsorbed on the two substrates demonstrated the appearance of amide I and amide II bands with small shifts and intensity variations supporting orientation changes of the protein when the concentration or immersion time varies. A model for the building up of the BSA layer is proposed, which accounts for these data. Chromium and stainless steel surfaces, also have different behaviours towards adhesion of Pseudomonas fragi K1, whereas surfaces that are pre-conditioned by BSA behave in a similar way. The overall number of adherent bacteria is decreased on stainless steel, whereas it is hardly affected on chromium. On both surfaces, the fraction of viable cells is increased.     

The smart Petri dish: a nanostructured photonic crystal for real-time monitoring of living cells

The intensity of light scattered from a porous Si photonic crystal is used to monitor physiological changes in primary rat hepatocytes. The cells are seeded on the surface of a porous Si photonic crystal that has been filled with polystyrene and treated with an O2 plasma. Light resonant with the photonic crystal is scattered by the cell layer and detected as an optical peak with a charge-coupled-device spectrometer. It is demonstrated that exposure of hepatocytes to the toxins cadmium chloride or acetaminophen leads to morphology changes that cause a measurable increase in scattered intensity. The increase in signal occurs before traditional assays are able to detect a decrease in viability, demonstrating the potential of the technique as a complementary tool for cell viability studies. The scattering method presented here is noninvasive and can be performed in real time, representing a significant advantage compared to other techniques for in vitro monitoring of cell morphology.     

Three-Dimensional Aggregated Spheroid Model of Hepatocellular Carcinoma Using a 96-Pillar/Well Plate

A common method of three-dimensional (3D) cell cultures is embedding single cells in Matrigel. Separated cells in Matrigel migrate or grow to form spheroids but lack cell-to-cell interaction, which causes difficulty or delay in forming mature spheroids. To address this issue, we proposed a 3D aggregated spheroid model (ASM) to create large single spheroids by aggregating cells in Matrigel attached to the surface of 96-pillar plates. Before gelling the Matrigel, we placed the pillar inserts into blank wells where gravity allowed the cells to gather at the curved end. In a drug screening assay, the ASM with Hepatocellular carcinoma (HCC) cell lines showed higher drug resistance compared to both a conventional spheroid model (CSM) and a two-dimensional (2D) cell culture model. With protein expression, cytokine activation, and penetration analysis, the ASM showed higher expression of cancer markers associated with proliferation (p-AKT, p-Erk), tight junction formation (Fibronectin, ZO-1, Occludin), and epithelial cell identity (E-cadherin) in HCC cells. Furthermore, cytokine factors were increased, which were associated with immune cell recruitment/activation (MIF-3α), extracellular matrix regulation (TIMP-2), cancer interaction (IL-8, TGF-β2), and angiogenesis regulation (VEGF-A). Compared to CSM, the ASM also showed limited drug penetration in doxorubicin, which appears in tissues in vivo. Thus, the proposed ASM better recapitulated the tumor microenvironment and can provide for more instructive data during in vitro drug screening assays of tumor cells and improved prediction of efficacious drugs in HCC patients.     

Characterization of insulin adsorption in the presence of albumin by time-of-flight secondary ion mass spectrometry and X-ray photoelectron spectroscopy

With a view to develop an encapsulation membrane for a bioartificial pancreas, we have studied the adsorption of insulin and human serum albumin (HSA) on it. The aim of this study was to determine the possibility of insulin detection on a polycarbonate membrane surface in the presence of HSA, an abundant blood protein. The first step of the work consisted in the identification of time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS) specific signals for insulin and albumin. For this purpose, adsorption isotherms in physiological conditions (pH = 7.2, T = 37 degrees ) were established for the two proteins by looking at the SIMS intensity variations of the characteristic protein and substrate fragments when increasing the protein concentration in the solution. The CHS+ ToF-SIMS fragment and the S2p XPS peak were identified as representative insulin signals. The second step of the work consisted in performing simultaneous adsorption of the two proteins with increasing insulin concentration. We observed an increase of the insulin signal in ToF-SIMS and XPS for insulin concentration beyond 5 microg/mL. Principal component analysis (PCA) of the ToF-SIMS results permits us to obtain information about the protein layer composition. The results show that at low relative insulin concentration in solution, the mixed adsorbed layers are enriched in insulin compared to the solution.     

Microbicides for HIV/AIDS. 2. Electrophoretic fingerprinting of CD4+ T-cell model systems

New measurements of the dependence of the surface charge on the pH and electrolyte concentration for three living human white blood cell lines that are the principal targets of the HIV-1 virus are reported. Comparison of the electrophoretic fingerprint (EF) pattern, especially the line of zero mobility, with that of reference colloids establishes the separate individual identities and shows that all three exhibit a zwitterionic surface. With the EF results as a guide, preliminary biological infectivity measurements showed that small polyvalent cations modulate the negative charge on the T-cell surface in a way that strongly affects the infection kinetics. H9 cells were exposed to an infectious virus (X4), and the data showed that HIV interaction with target cells is enhanced by physiological fluids. The nondestructive methodology described is generally applicable to characterization of the surface charge and determination of the colloidal stability of any aqueous charged colloidal system without reference to any model of the double layer.     

The influence of electrolyte concentration on the adsorption of octadecanol on Au(111)

The influence of electrolyte concentration on the potential dependent adsorption and desorption of octadecanol to/from a Au(111) electrode was investigated utilizing electrochemical and elastically scattered light techniques. The electrolyte concentration was found to influence the potential driven changes of the adsorbed layer (adsorption and desorption). The capacitive changes in the adsorbed layer were found to occur at more negative potentials with lower electrolyte concentration. The changes in the optical measurement, used to measure the characteristics of the desorbed species, or aggregates, were also found to be affected similarly. The magnitude of the overall change in the scattered light intensity was slightly dependent on electrolyte concentration. The re-adsorption of the aggregates was influenced by electrolyte concentration. The scattered light signal for an intermediate adsorbed state (adsorbed aggregate) was more prevalent for higher electrolyte concentration, suggesting that these intermediates were somewhat different compared to lower electrolyte concentrations. The lower electrolyte concentration displayed a larger potential region where this intermediate was stable, but the intensity of the scattered light was much lower. The electrolyte concentration most strongly influenced the potentials of adsorption and desorption, as well as the potential region of stability for the adsorbed intermediates. The sweep rate also has an influence on the scattering characteristics of the desorbed species, suggesting a possible method for measuring the kinetics of the adsorption–desorption process or for controlling the character of the desorbed species. These changes were explained in terms of a mechanism for the wetting or de-wetting of a surface. The influence of electrolyte concentration provides another opportunity for investigating the dynamics of this adsorption–desorption process.     

Interaction of electromagnetic waves in planar waveguide with a nematic layer

This work is a theoretical study of energy exchange between two coupled TE-wave modes on director diffraction grating in a planar waveguide containing a layer of nematic liquid crystal. The diffraction grating is produced by an external electric field in the nematic layer with spatial periodic anchoring energy between director and waveguide surface. The intensity of a signal mode at the output of the nematic layer has been calculated in dependence of anchoring energy amplitude and modulation period, the size of nematic layer and electrical field value. The cases of co-propagating and oppositely propagating modes have been analysed. The analytical expressions that describe the maximum values of signal mode intensity have been derived. The maximum intensity value output from the nematic has been shown to depend monotonously on the anchoring energy parameters in the case of oppositely propagating wave modes and non-monotonously in the case of co-propagating wave modes. In both cases, the maximum value of signal mode intensity grows with the increase in electric field.     

The flowing mercury electrode in polarography. part I

A polarisable electrode of constant surface area is described in which the surface layer of a column of mercury is continuously renewed from an internal mercury supply. Reproducible diffusion currents are obtainable which do not exhibit maxima. Wave heights are proportional to the concentration of reducible ion. The mercury consumption is similar to that of the dropping mercury electrode. The electrode and cell are constructed throughout in polyethylene.     

A Rho Kinase (ROCK) Inhibitor,Y-27632, Inhibits the Dissociation-Induced Cell Death of Salivary Gland Stem Cells

Salivary gland stem cells (SGSCs) are potential cell sources for the treatment of salivary gland diseases. The control of cell survival is an essential factor for applying stem cells to regenerative medicine or stem cell-based research. The purpose of this study was to investigate the effects of the ROCK inhibitor Y-27632 on the survival of SGSCs and its underlying mechanisms. SGSCs were isolated from mouse submandibular glands and cultured in suspension. Treatment with Y-27632 restored the viability of SGSCs that was significantly decreased during isolation and the subsequent culture. Y-27632 upregulated the expression of anti-apoptotic protein BCL-2 in SGSCs and, in the apoptosis assay, significantly reduced apoptotic and necrotic cell populations. Matrigel was used to mimic the extracellular environment of an intact salivary gland. The expression of genes regulating apoptosis and the ROCK signaling pathway was significantly reduced when SGSCs were embedded in Matrigel. SGSCs cultured in Matrigel and treated with Y-27632 showed no difference in the total numbers of spheroids and expression levels of apoptosis-regulating genes. Matrigel-embedded SGSCs treated with Y-27632 increased the number of spheroids with budding structures and the expression of acinar cell-specific marker AQP5. We demonstrate the protective effects of Y-27632 against dissociation-induced apoptosis of SGSCs during their culture in vitro.     

Density control of poly(ethylene glycol) layer to regulate cellular attachment

A wide variety of cells usually integrate and respond to the microscale environment, such as soluble protein factors, extracellular matrix proteins, and contacts with neighboring cells. To gain insight into cellular microenvironment design, we investigated two-dimensional microarray formation of endothelial cells on a micropatterned poly(ethylene glycol) (PEG)-brushed surface, based on the relationship between PEG chain density and cellular attachment. The patterned substrates consisted of two regions: the PEG surface that acts as a cell-resistant layer and the exposed substrate surface that promotes protein or cell adsorption. A PEG-brushed layer was constructed on a gold substrate using PEG with a mercapto group at the end of the chain. The density of the PEG-brushed layer increased substantially with repetitive adsorption/rinse cycles of PEG on the gold substrate, allowing marked reduction of nonspecific protein adsorption. These repeated adsorption/rinse cycles were further regulated by using longer (5 kDa) and shorter (2 kDa) PEG to construct PEG layers with different chain density, and subsequent micropatterning was achieved by plasma etching through a micropatterned metal mask. The effects of PEG chain density on pattern formation of cell attachment were determined on micropatterning of endothelial cells. The results indicated that cell pattern formation was strongly dependent on the PEG chain density and on the extent of protein adsorption. Notably, a PEG chain density high enough to inhibit outgrowth of endothelial cells from the cell-adhering region in the horizontal direction could be obtained only by employing formation of a short filler layer of PEG in the preconstructed longer PEG-brushed layer, which prevented nonspecific protein adsorption almost completely. In this way, a completely micropatterned array of endothelial cells with long-term viability was obtained. This clearly indicated the importance of a short underbrushed PEG layer in minimizing nonspecific protein adsorption for long-term maintenance of the active cell pattern. The strategy for cell patterning presented here can be employed in tissue engineering to study cell-cell and cell-surface interactions. It is also applicable for high-throughput screening and clinical diagnostics, as well as interfacing cellular and microfabricated components of biomedical microsystems.     

Structure of Protein Layers during Competitive Adsorption

The formation of protein layers during competitive adsorption was studied with ellipsometry. Single, binary, and ternary protein solutions of human serum albumin (HSA), IgG, and fibrinogen (Fgn) were investigated at concentrations corresponding to blood plasma diluted 1/100. As a model surface, hydrophobic hexamethyldisiloxane (HMDSO) plasma polymer modified silica was used. By using multiambient media measurements of the bare substrate prior to protein adsorption the adsorbed amount as well as the thickness and refractive index of the adsorbed protein layer could be followedin situand in real time. Under conditions used in these experiments neither IgG nor fibrinogen could fully displace serum albumin from the interface. The buildup of the protein layer occurred via different mechanisms for the different protein systems. Fgn adsorbed in a rather flat orientation at low adsorbed amounts, while at higher surface coverage the protein reoriented to a more upright orientation in order to accommodate more molecules in the adsorbed layer. IgG adsorption proceeded mainly end-on with little reorientation or conformational change on adsorption. Finally, for HSA an adsorbed layer thickness greater than the molecular dimensions was observed at high concentrations (although not at low), indicating that aggregates or multilayers formed on HMDSO plasma polymer surfaces. For all protein mixtures the adsorbed layer structure and buildup indicated that Fgn was the protein dominating the adsorbed layer, although HSA partially blocked the adsorption of this protein. At high surface concentration, HSA/Fgn mixtures show an abrupt change in both adsorbed layer thickness and refractive index suggesting, e.g., an interfacial phase transition of the mixed protein layer. A similar but less pronounced behavior was observed for HSA/IgG. For IgG/Fgn and HSA/IgG/Fgn a buildup of the adsorbed layer similar to that displayed by Fgn alone was observed.     

A whole-cell assay for the high throughput screening of calmodulin antagonists

Cell-based screening systems for pharmaceuticals are desired over molecular biosensing systems because of the information they provide on toxicity and bioavailability. However, the majority of sensing systems developed are molecular biosensing type screening systems and cannot be easily adapted to cell-based screening. In this study, we demonstrate that protein-based molecular sensing systems that employ a fluorescent protein as a signal transducer are amenable to cell-based sensing by expressing the protein molecular sensing system in the cell and employing these cells for screening of desired molecules. To achieve this, we expressed a molecular sensing system based on the fusion protein of calmodulin (CaM) and enhanced green fluorescent protein (EGFP) in bacterial cells, and utilized these cells for the screening of CaM antagonists. In the presence of Ca²⁺, CaM undergoes a conformational change exposing a hydrophobic pocket that interacts with CaM-binding proteins, peptides, and drugs. This conformational change induced in CaM leads to a change in the microenvironment of EGFP, resulting in a change in its fluorescence intensity. The observed change in fluorescence intensity of EGFP can be correlated to the concentration of the analyte present in the sample. Dose-response curves for various tricyclic antidepressants were generated using cells containing CaM-EGFP fusion protein. Additionally, we demonstrate the versatility of our system for studying protein-protein interactions by using cells to study the binding of a peptide to CaM. The study showed that the CaM-EGFP fusion protein within the intact cells responds similarly to that of the isolated fusion protein, hence eliminating the need for any isolation and purification steps. We have demonstrated that this system can be used for the rapid screening of various CaM antagonists that are potential antipsychotic drugs.     

Dark-to-bright optical responses of liquid crystals supported on solid surfaces decorated with proteins

Protein assays are critical analytical tools performed in various biochemical laboratories to quantify the concentration of proteins. In this study, we report the optical responses of a thin layer of liquid crystals supported on glass slides decorated with proteins and the utility of this phenomenon as a new "all-or-nothing" type of protein assay. It was found that the orientations of liquid crystals are very sensitive to the concentration of protein solution applied to the surface. When the protein concentration exceeds a critical value (IgG 5.0 microg/mL, BSA 6.0 microg/mL, FTIC-anti-biotin 0.40 microg/mL, and FITC-anti-IgG 0.37 microg/mL), the thin layer of liquid crystals gives a very sharp dark-to-bright optical response within a small concentration range. This characteristic is not observed in any traditional protein assays, which are based on the adsorption of UV or visible light. The optical response is also very precise and reproducible. It is not affected by the thickness of the liquid crystal cell or the amount of organosilanes coated on the glass slides. The liquid crystal-based protein assay may be very useful for screening purposes, especially when a simple positive or negative answer is desired.     

Infrared spectral signatures of CDCP1-induced effects in colon carcinoma cells

Metastasis is the major cause of death by cancer. Indeed, metastatic colonies can reactivate and become life threatening, sometimes months or years after the initial diagnosis and surgery of the primary tumor. Therefore, there is a crucial need to develop methods for diagnosis of tumor cells that exhibit high metastatic potential. Here, we addressed the capability of vibrational spectroscopy for investigating the effects induced by CDCP1 expression in colon carcinoma cells. This transmembrane protein has been suggested to play a key role in metastasis by its pleiotropic function. We focused on a cellular model constituted by the cell lines SW480 and SW620 derived respectively from the primary tumor and a lymph node metastasis of the same patient. Induced CDCP1 expression in SW480 led to marked changes in cell morphology. Whereas SW480 form a cell layer, the SW480/CDCP1 cells exhibited reduced cell-to-cell contact. On collagen I, SW480 was more spread and filopodia were observed. In contrast, SW480/CDCP1 cells exhibited lower spreading with no formation of filopodia. Synchrotron Fourier transform infrared microspectroscopy experiments were performed on this cellular model. High quality spectroscopic information at sub-cellular resolution, provided by the use of the synchrotron source in infrared microspectroscopy, was recorded on numerous individual cells. Multivariate analysis of spectra recorded using principal component analysis indicated a highest intensity increase of the 970 and 1080 cm(-1) bands, and a modest intensity increase of the 1240 cm(-1) band in the SW480/CDCP1 cells. These bands were correlated with an increased content of phosphorylated proteins as confirmed by in situ labelling using a monoclonal antibody directed against phosphorylated tyrosines. Altogether, these results demonstrate that the vibrational technique used in this study exhibits the capability to characterize spectral signatures of CDCP1-induced effects in colon carcinoma cells. This study may open new avenues for rapid diagnosis of cells with a metastatic potential.     

Electrochemical diffusion potential in a flame plasma: theory and experiment

A flame doped with an appropriate additive to produce positive ions and free electrons is a quasineutral, weak, continuum plasma. When bounded by a metallic burner upstream and a metal plate downstream, the two electrodes and flame plasma can be viewed as a gas-phase electrochemical cell. When the ion (and electron) density varies continuously along the flame axis, an expression for the diffusion potential can be derived in terms of the concentration gradient. The familiar logarithmic dependence on the ion concentration is obtained. A plasma sheath develops at the metal plate electrode; it sustains a potential difference which can be modeled by a Boltzmann distribution of the electrons in the sheath. Since the plate has to be cooled in practice, the average sheath temperature is less than the flame temperature because the sheath occurs inside the thermal boundary layer which covers the plate electrode. Inevitably, the reduced sheath temperature affects the sheath voltage. Experimental measurements of the “cell” voltage are made for the two cases of a positive concentration gradient using a sodium plasma, and a negative gradient by doping the flame with methane. As predicted theoretically, the cell voltages have opposite signs. However, the magnitude of the cell voltage seems to depend significantly on the sheath temperature which appears to decrease steadily with increasing distance downstream from the burner. It is also possible that the measured cell voltages involve unknown surface contact potentials. When compared with solution concentration cells, gas-phase flame systems exhibit both similarities and differences.     

Semisynthesis of fluorescent metabolite sensors on cell surfaces

Progress in understanding signal transduction and metabolic pathways is hampered by a shortage of suitable sensors for tracking metabolites, second messengers, and neurotransmitters in living cells. Here we introduce a class of rationally designed semisynthetic fluorescent sensor proteins, called Snifits, for measuring metabolite concentrations on the cell surface of mammalian cells. Functional Snifits are assembled on living cells through two selective chemical labeling reactions of a genetically encoded protein scaffold. Our best Snifit displayed fluorescence intensity ratio changes on living cells significantly higher than any previously reported cell-surface-targeted fluorescent sensor protein. This work establishes a generally applicable and rational strategy for the generation of cell-surface-targeted fluorescent sensor proteins for metabolites of interest.     

Adsorption of fibrinogen on tantalum oxide, titanium oxide and gold studied by the QCM-D technique

The adsorption of human fibrinogen on tantalum oxide, titanium oxide and gold surfaces has been studied by quartz crystal microbalance with dissipation (QCM-D) at 37 degrees C. Two different protein concentrations have been used, one close to physiological concentration (1 mg/ml) and one significantly lower (0.033 mg/ml). To further characterize the adsorbed fibrinogen layer, the subsequent binding of both polyclonal and monoclonal antibodies of fibrinogen is studied. We found that the viscoelastic properties of the fibrinogen layer depends strongly on the initial protein concentration. The trend is generally seen for all three surfaces. The fibrinogen layer on gold and tantalum oxide is found to have the same viscoelastic properties, which are different from those found for the fibrinogen layer adsorbed on titanium oxide. The dependency of the surface chemistry on the viscoelastic properties of the fibrinogen layer is observed directly for the 0.033 mg/ml solution, and indirectly through the antibody response for the 1 mg/ml solution. From this we conclude that the orientation and/or denaturation of fibrinogen on a surface depends on the surface chemistry and the protein concentration in the solution, and that the binding of antibodies is a useful way to detect this difference.     

Evidence that Pulsed Electric Field Treatment Enhances the Cell Wall Porosity of Yeast Cells

The application of rectangular electric pulses, with 0.1–2 ms duration and field intensity of 2.5–4.5 kV/cm, to yeast suspension mediates liberation of cytoplasmic proteins without cell lysis. The aim of this study was to evaluate the effect of pulsed electric field with similar parameters on cell wall porosity of different yeast species. We found that electrically treated cells become more susceptible to lyticase digestion. In dependence on the strain and the electrical conditions, cell lysis was obtained at 2–8 times lower enzyme concentration in comparison with control untreated cells. The increase of the maximal lysis rate was between two and nine times. Furthermore, when applied at low concentration (1 U/ml), the lyticase enhanced the rate of protein liberation from electropermeabilized cells without provoking cell lysis. Significant differences in the cell surface of control and electrically treated cells were revealed by scanning electron microscopy. Data presented in this study allow us to conclude that electric field pulses provoke not only plasma membrane permeabilization, but also changes in the cell wall structure, leading to increased wall porosity.