Reduction of albumin adsorption onto silicon surfaces by Tween 20

The ability of Tween 20 to reduce the adsorption of albumin on silicon surfaces of different hydrophobicity was investigated by ellipsometry. As expected, protein adsorption was found to depend on the degree of hydrophobicity of the surfaces and on the concentration of the surfactant. A reduction of 90% in albumin adsorption on hydrophobic methylated surfaces by 0.05% Tween 20 was achieved, whereas a reduction of only 15% on hydrophilic surfaces was observed. Experiments of time-dependent protein adsorption in both pure protein and protein-surfactant mixtures were conducted to ascertain the stability of physically adsorbed Tween 20 films on intermediate silicon surfaces. It was found that the adsorbed Tween 20 film was robust and there was no evidence of exchange of the Tween molecules with albumin for up to 240 min exposure. Adsorption minima were confirmed to correlate with minima in contact angle and critical micelle concentration (CMC). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 618-625, 1997.     

Direct evidence for antifreeze glycoprotein adsorption onto an ice surface

Aqueous solutions of antifreeze glycoproteins (AFGP) exhibit hysteresis between the freezing and melting temperatures. Several recent studies on the mechanism of function of this protein system suggest that a likely model is for the antifreeze molecules to be adsorbed onto the surface of the crystals. However, direct proof of the presence of adsorbed AFGP has eluded previous researchers. In the present study, enhanced surface second-harmonic generation (SSHG) was observed in the presence of an active AFGP solution in contact with a pure single crystal of ice. The enhancement of SSHG is a positive indication that active AFGP molecules adsorb to the surface of ice crystals.     

Extracellular polymeric substances responsible for bacterial adhesion onto solid surface

The influence of extracellular polymeric substances (EPS) on bacterial cell adhesion onto solid surfaces was investigated using 27 heterotrophic bacterial strains isolated from a wastewater treatment reactor. Cell adhesion onto glass beads was carried out by the packed-bed method and the results were discussed in terms of the amount of each EPS component produced and cell surface characteristics such as zeta potential and hydrophobicity. Protein and polysaccharides accounted for 75-89% of the EPS composition, indicating that they are the major EPS components. Among the polysaccharides, the amounts of hexose, hexosamine and ketose were relatively high in EPS-rich strains. For EPS-poor strains, the efficiency of cell adhesion onto glass beads increased as the absolute values of zeta potential decreased, suggesting that electrostatic interaction suppresses cell adhesion efficiency. On the other hand, the amounts of hexose and pentose exhibited good correlations with cell adhesiveness for EPS-rich strains, indicating that polymeric interaction due to the EPS covering on the cell surface promoted cell adhesion. It was concluded that, if the EPS amount is relatively small, cell adhesion onto solid surfaces is inhibited by electrostatic interaction, and if it is relatively large, cell adhesion is enhanced by polymeric interaction.     

Surface-induced aggregation of ferritin. Concentration dependence of adsorption onto a hydrophobic surface

The isotherm of ferritin adsorption onto a hydrophobic surface was studied by transmission electron microscopy. Adsorbed ferritin was found to be distributed in molecular clusters. The adsorption process was diffusion-rate-limited after 20 h adsorption time at bulk concentrations below 1 mg/1. The clusters formed during the diffusion-rate-limited adsorption had a fractal dimension D approximately 1.0 when averaged over all clusters. The pair distribution function g(r) showed an increased probability of finding nearest neighbours at distances less than 30 nm. The surface concentration of adsorbed ferritin was weakly dependent on the bulk concentration of ferritin in the range 10 mg/1-10 g/1 and the average number of nearest neighbour molecules was constant in this concentration range. The mass distribution of adsorbed ferritin c(r) had a fractal dimension D = 1.8 at a bulk concentration of 10 g/l and a surface concentration corresponding to theta = 0.45 +/- 0.05. The pair correlation function g(r) showed decreasing probability of finding nearest neighbour molecules over long distances as in percolating clusters. The results indicate that ferritin adsorbs strongly to the surface at low surface concentrations and weakly at high surface concentrations. The stability of ferritin adsorption was correlated to the average number of nearest neighbour molecules, indicating a possibility that desorption is a critical supramolecular phenomenon.     

A poplar plastocyanin mutant suitable for adsorption onto gold surface via disulfide bridge

Aiming to achieve stable immobilization for a redox-active cupredoxin protein onto a gold substrate and its consequent molecular level monitoring by Scanning Tunnelling Microscopy (STM), we introduced a disulphide bridge within poplar plastocyanin, while avoiding the perturbation of its active site. We selected and modified residues Ile-21 to Cys and Glu-25 to Cys by structurally conservative mutagenesis. Optical absorption spectroscopy (UV-Vis), electron paramagnetic resonance (EPR), and resonance raman scattering (RRS) results indicate that the active site of the Ile21Cys, Glu25Cys plastocyanin (PCSS) to a large extent retains the spectroscopic properties of the wild-type protein. Furthermore, the redox midpoint potential of the couple CuII/CuI in PCSS, determined by cyclic voltammetry was found to be +348 mV close to the wild-type value. The STM images display self-assembled PCSS molecules immobilised onto gold substrate. Moreover, the full potentiostatic control of the electron transfer reaction during STM imaging, suggests that the adsorbed molecule maintains essentially its native redox properties.     

Use of a cyanine dye as a probe for albumin and collagen in the extracellular matrix

The aim of this work was to develop a quick method for analysis of macromolecules of the extracellular matrix. Of great interest are soluble components of the extracellular matrix, in particular, carrier proteins, whose variation dynamics can characterize the studied tissue in its development, adult stage, and aging. We suggest the method of analysis of the extracellular matrix to reveal the presence of albumin and collagen by using an anionic cyanine dye as a spectral and fluorescence probe. The method was applied for the analysis of the human vitreous body in the course of its development. Albumin was detected by the appearance of the trans monomer absorption and fluorescence bands in the dye spectra, and collagen was detected by the absorption and fluorescence bands of J aggregates. Hyaluronic acid present in the vitreous body does not interfere with the results of the analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis confirmed the presence of albumin in the vitreous body. We suppose that albumin as a protein carrying biologically active macromolecules plays an important role in the processes of differentiation and functional establishment of ocular tissues in the course of their prenatal development.     

In situ measurement of circular dichroism of DNA adsorbing onto a solid surface

The adsorption process of DNA dissolved in aqueous solutions onto the surface of polyethyleneimine (PEI) has been examined by the observation of in situ circular dichroism (CD), including time-resolved measurements, to elucidate the conformation of DNA at the liquid/solid interface. The adsorption process can be characterized by two stages that are characterized in terms of CD. In the first stage, time (t)<700 s, a slight change in the time-resolved CD spectra of DNA is observed, whereas the value of the induced CD of the dye intercalated in DNA is constant. This result can be explained by the interaction between DNA and PEI during the adsorption at the liquid/solid interface. The weakness of the interaction is attributed to the geometrical restriction of this interface. In the second stage, t>700 s, where no further adsorption occurs, a change in the induced CD as well as in the CD of DNA is observed. This change in the induced CD can be interpreted as a significant conformational change of DNA for stabilizing the ion complex with PEI.     

Tb(III) as a fluorescent probe for the structure of bovine serum albumin

Tb(III) was used as a fluorescent probe in the study of the calcium-binding sites on Bovine Serum Albumin (BSA). The fluorescence of Tb(III) is enhanced markedly when bound to BSA and nonradiative energy transfer between two fluorescent tryptophan(Trp) residues and Tb(III) bound to calcium-binding sites on BSA occurred. Experimental results show that the major groups in BSA bound to metal ion are the carboxyl side groups of glutamic acid (Glu) and aspartic acid (Asp). The average distance between the bound Tb(III) and the two tryptophan residues in BSA calculated by a F?ster dipole-dipole nonradiative energy transfer mechanism is 1.48 nm.     

A competitive inhibition ELISA as a probe for studying the refolding of human serum albumin

The refolding of iodoacetic acid-blocked human serum albumin (HSA) was studied using a modified competitive inhibition ELISA. A maximum of 89% native activity was detected 24 hours after initiating refolding using an albumin concentration of 600 micrograms/mL. The presence of both monomer and polymer HSA was studied using native polyacrylamide gel electrophoresis of thiol-blocked HSA samples. Monomer HSA was not detected until 2.5 hours after initiating refolding. Fractionated polymer and monomer HSA from a sample trapped at 72 hours after initiating refolding was determined to have 40% and 87% native activity respectively. Both polymer and monomer HSA fractions contribute to the overall immunological activity detected by the ELISA, at various times. The ELISA assay was able to detect the changing HSA conformation associated with refolding of totally reduced HSA.     

Determination of glucose oxidase immobilised as monolayer onto a flat surface

An assay to estimate the amount of glucose oxidase immobilised as a monolayer onto a flat surface is reported. This method is based on the electrochemical detection of the flavin adenine dinucleotide (FAD) cofactor released by the immobilised enzyme in acid solutions. FAD concentration in the acid solution was measured by amperometry, using a flow injection analysis (FIA) system equipped with a wall-jet electrode, and with a sensitivity of (9.2+/-2.0)x10(-2) nA/nM. By this method, the amount of glucose oxidase molecules present in a monolayer deposited on a silanised glass slide was easily detected, in which the detection limit is more than one order of magnitude lower than the maximum loading of the surface with an ordered monolayer of glucose oxidase.     

Macroporous hydrophobic cloth (polymacron) as a solid phase for nucleic acid probe hybridizations

Polyester cloth (polymacron) was employed as a solid phase onto which polymerase chain reaction (PCR) products from theListeria monocytogenes hlyA gene were dot blotted and sensitively detected by hybridization with nucleic acid probes. This inexpensive solid phase exhibited a high DNA binding capacity and provided ease of handling and washing between each reaction step, resulting in simple hybridization procedures for PCR product detection.Contribution No. 95-004     

Evaluation of a new annular capacitance probe for biomass monitoring in industrial pilot-scale fermentations

The four-pin electrode capacitance probe has already shown to be a valuable tool for on-line monitoring viable biomass concentration in industrial-type fermentations. A new prototype annular probe was developed and its performance in real-time monitoring the concentration of viable cells during industrial pilot-scale fermentation for the production of an Active Pharmaceutical Ingredient (API) was investigated and compared to the four-pin probe. A set of 14 fermentations was monitored on-line: four of them with the four-pin probe, the remaining with the annular probe. The performance of both the annular and the four-pin electrode probe were compared against each other and against off-line measurements (viscosity and packed mycelial volume). The prototype annular probe showed to have higher signal intensity and sensitivity than the standard four-pin probe, with higher signal-to-noise ratio. Furthermore, its new design and construction proved to be easier to handle in an industrial environment.     

Covalently coupling the antibody on an amine-self-assembled gold surface to probe hyaluronan-binding protein with capacitance measurement

Hyaluronan-binding proteins (HABPs), the important structural components of extracellular matrices, served important structural and regulatory functions during development and in maintaining adult tissue homestats. A sensitive, specific and rapid-responsing immunosensor to probe hyaluronan-binding cartilage protein was presented in this work. The novel immunosensor supplied a label-free detection method for HABP, which was based on measuring the capacitance change in-between the unlabeled HABP (antigen) and rabbit-anti-HABP (Ra-HABP, antibody). The HABP immunosensor was prepared by covalently coupling Ra-HABP on an amine-self-assembled gold surface with glutaraldehyde. The capacitance change corresponding to the concentration of HABP, the target antigen, was evaluated by an electrochemical approach called potentiostatic-step in microseconds. The immunosensor showed a specific response to HABP in the range 10-1000 ng/ml. The presented work supplied a promising clinical screening method.     

K-35, a fluorescent probe for albumin study: Optical properties

Fluorescent probe N-(carboxyphenyl)imide of 4-(dimethylamino)naphthalic acid, K-35, is used as an indicator of structural changes of human serum albumin molecules in pathology. The probe occupies albumin binding pockets where the probe environment is of very high polarity; probably, the pockets contain protein polar groups and also water molecules. At the same time the rather small Stokes shift of K-35 fluorescence spectrum shows that the polar group motion is one-two orders of magnitude lower than the mobility of polar molecules in polar fluids. K-35 fluorescence decay in HSA can be described as a sum of three exponentials with time constants close to τ₁ = 9 ns; τ₂ = 3.6 ns and τ₃ = 1.0 ns. The difference between excitation maxima of these three decay components shows that the environment of these three species of K-35 molecules has been different before excitation. Different τ values are probably a consequence of nonidentical structure of several binding sites, or the binding site(s) can have variable conformation.     

K-35, a fluorescent probe for albumin study: optical properties

Fluorescent probe N-(carboxyphenyl)imide of 4-(dimethylamino)naphthalic acid, K-35, is used as an indicator of structural changes of human serum albumin molecules in pathology. The probe occupies albumin binding pockets where the probe environment is of very high polarity; probably, the pocket(s) contains protein polar groups and water molecules. At the same time rather small Stokes shift of K-35 fluorescence spectrum shows that the polar group motion is of one-two order of value lower than mobility of polar molecules in polar fluids. K-35 fluorescence decay in HSA can be described as a sum of three exponentials with time constants close to tau1=9 ns; tau2=3.6 ns and tau3=1.0 ns. A difference between excitation maxima of these three decay components shows that environment of these three species of K-35 molecules has been different before excitation. Different r values are probably a consequence of non-identical structure of several binding sites, or a binding site(s) can have a variable conformation.     

Kinetic equations for surface adsorption

We investigate the time-dependent solution of a set of equations used to approximate the binding of flexible polymers to the surface of suspended targets. The equations account, for the possibility that the first adsorption step can be a rate limiting step. Rate constants for other steps of adsorption and desorption are proportional to the number of free and occupied polymer binding sites, respectively.     

Terbium as a solid-state probe for RNA

This paper continues previous work on the analysis of nucleic acid-terbium complexes in the solid state. The fluorescence excitation and emission spectra of the RNA-terbium(III) complex is reported. The fluorescence excitation and emission spectra of both the RNA-terbium(III) and DNA-terbium(III) complexes as trapped on millipore filters is reported. One hundred percent of the DNA combined with terbium was trapped on millipore filters. Deoxyribonucleic acid was recovered from DNA-terbium(III) complexes trapped on millipore filters using SDS-extraction. Energy transfer was shown to occur from the bases in nucleic acids to the terbium ion, whereas the actual binding of terbium to nucleic acids was due to phosphate groups. The relative fluorescence of homopolyribonucleotide-terbium complexes showed that the guanine moiety was responsible for most of the observed fluorescence. Binding studies showed an equal affinity of radioactive terbium for all the homopolyribonucleotides. The fluorescence of solid-state DNA and RNA terbium complexes was used to measure picomole quantities of DNA or RNA.     

Measuring adsorption of a hydrophobic probe with a surface plasmon resonance sensor to monitor conformational changes in immobilized proteins

Conformational changes of proteins immobilized on solid matrices were observed by measuring the adsorption of Triton X-100 (TX), a nonionic detergent, as a hydrophobic probe with BIACORE, a biosensor that utilizes the phenomenon of surface plasmon resonance (SPR). Two kinds of proteins, alpha-glucosidase and lysozyme, were covalently attached to dextran matrices on the sensor surface in the flow cell and then exposed to various concentrations of TX solution. We measured SPR signal changes derived from adsorption of TX to the immobilized proteins and calculated the monolayer adsorption capacity using the Brunauer-Emmett-Teller (BET) equation. The results demonstrated that monolayer adsorption capacity is proportional to the amount of immobilized proteins. Further, the unfolding process of immobilized proteins on the sensor surface induced by guanidine hydrochloride was investigated by monitoring SPR signal increases due to the adsorption of TX to the exposed hydrophobic region of the protein. Results strongly suggested that the increase in the SPR signal reflected the formation of the agglutinative unfolded state. We expect our measuring method using the SPR sensor and TX adsorption will be a novel tool to provide conformational information regarding various proteins on solid matrices.