Electrophoretic Studies on Plant Protoplasts I. pH Dependence of Zeta Potentials of Protoplasts from Various Sources

Electrophoretic mobilities of plant protoplasts from varioussources were measured, as a function of the pH of the medium,by a micro-electrophoresis technique to characterize the protoplastsin terms of curves of zeta potential vs. protoplast surfacepH (pH_s). The shape of the curves of zeta potential vs. pH_scurves differed among preparations of protoplasts isolated fromvarious species and strains. The isoelectric points (pI) ofthe protoplasts measured in this study were between 3.0 and4.0. These differences among the protoplasts suggest that itmay be possible to develop an electrophoretic method for theselection of protoplasts. The shape of the curves of zeta potentialvs. pH_s also indicated that carboxyl groups, as well as phosphategroups, may contribute to the negative charges on the surfaceof protoplasts. (Received October 14, 1988; Accepted February 22, 1989)     

The interaction between saliva and Actinobacillus actinomycetemcomitans influenced by the Zeta potential

The adhesion of Actinobacillus actinomycetemcomitans is a virulence factor in the aetiology of periodontitis and is determined by physico-chemical properties, e.g. surface charge and hydrophobicity, of the bacterial cell surface. Although oral surfaces are constantly coated with saliva, few studies have dealt with the binding of A. actinomycetemcomitans with saliva. In this report, the charge properties of A. actinomycetemcomitans have been studied through measurement of the zeta potential and the saliva-bacteria interaction investigated at different pH-values.At physiological conditions the zeta potential was negative, varying from -11 to -26 mV, for two laboratory and two fresh isolates of A. actinomycetemcomitans. Under these conditions, binding of the low-molecular-weight salivary mucin, lactoferrin, and S-IgA was confirmed using salivary samples and purified salivary fractions in liquid-phase and in ELISA. The iso-electric points of the laboratory and fresh clinical isolates of A. actinomycetemcomitans were determined at pH 4.6 and 3.8, respectively. At pH below the iso-electric point, giving positive values of the zeta potential, additional salivary protein species bound to A. actinomycetemcomitans, including the high-molecular-weight salivary mucin (MG1) and agglutinin. Binding of the low-molecular-weight salivary mucin (MG2), lactoferrin, and S-IgA, was hardly affected by this change in zeta potential. A salivary coating formed on the bacterium at pH 7 reduced the zeta potential of the laboratory strain Y4 greatly and an iso-electric point for the bacterium could not be determined. Overall, the study suggests that upon changes in environmental pH additional salivary attachment sites on the micro-organism are exposed.     

Surface pH controls purple-to-blue transition of bacteriorhodopsin. A theoretical model of purple membrane surface.

We have developed a surface model of purple membrane and applied it in an analysis of the purple-to-blue color change of bacteriorhodopsin which is induced by acidification or deionization. The model is based on dissociation and double layer theory and the known membrane structure. We calculated surface pH, ion concentrations, charge density, and potential as a function of bulk pH and concentration of mono- and divalent cations. At low salt concentrations, the surface pH is significantly lower than the bulk pH and it becomes independent of bulk pH in the deionized membrane suspension. Using an experimental acid titration curve for neutral, lipid-depleted membrane, we converted surface pH into absorption values. The calculated bacteriohodopsin color changes for acidification of purple, and titrations of deionized blue membrane with cations or base agree well with experimental results. No chemical binding is required to reproduce the experimental curves. Surface charge and potential changes in acid, base and cation titrations are calculated and their relation to the color change is discussed. Consistent with structural data, 10 primary phosphate and two basic surface groups per bacteriorhodopsin are sufficient to obtain good agreement between all calculated and experimental curves. The results provide a theoretical basis for our earlier conclusion that the purple-to-blue transition must be attributed to surface phenomena and not to cation binding at specific sites in the protein.     

An experimental test of new theoretical models for the electrokinetic properties of biological membranes. The effect of UO2++ and tetracaine on the electrophoretic mobility of bilayer membranes and human erythrocytes

For a large smooth particle with charges at the surface, the electrophoretic mobility is proportional to the zeta potential, which is related to the charge density by the Gouy-Chapman theory of the diffuse double layer. This classical model adequately describes the dependence of the electrophoretic mobility of phospholipid vesicles on charge density and salt concentration, but it is not applicable to most biological cells, for which new theoretical models have been developed. We tested these new models experimentally by measuring the effect of UO2++ on the electrophoretic mobility of model membranes and human erythrocytes in 0.15 M NaCl at pH 5. We used UO2++ for these studies because it should adsorb specifically to the bilayer surface of the erythrocyte and should not change the density of fixed charges in the glycocalyx. Our experiments demonstrate that it forms high-affinity complexes with the phosphate groups of several phospholipids in a bilayer but does not bind significantly to sialic acid residues. As observed previously, UO2++ adsorbs strongly to egg phosphatidylcholine (PC) vesicles: 0.1 mM UO2++ changes the zeta potential of PC vesicles from 0 to +40 mV. It also has a large effect on the electrophoretic mobility of vesicles formed from mixtures of PC and the negative phospholipid phosphatidylserine (PS): 0.1 mM UO2++ changes the zeta potential of PC/PS vesicles (10 mol % PS) from -13 to +37 mV. In contrast, UO2++ has only a small effect on the electrophoretic mobility of either vesicles formed from mixtures of PC and the negative ganglioside GM1 or erythrocytes: 0.1 mM UO2++ changes the apparent zeta potential of PC/GM1 vesicles (17 mol % GM1) from -11 to +5 mV and the apparent zeta potential of erythrocytes from -12 to -4 mV. The new theoretical models suggest why UO2++ has a small effect on PC/GM1 vesicles and erythrocytes. First, large groups (e.g., sugar moieties) protruding from the surface of the PC/GM1 vesicles and erythrocytes exert hydrodynamic drag. Second, charges at the surface of a particle (e.g., adsorbed UO2++) exert a smaller effect on the mobility than charges located some distance from the surface (e.g., sialic acid residues).     

Surface Properties of Bacteria from Different Wastewater Treatment Plants

The surface properties of the individual members of degradative biocommunities isolated from different laboratory and natural populations were characterized. The bacterial strains isolated from a given origin and degrading a given substrate varied with respect to their hydrophobic and electrostatic properties (e.g. contact angle, adsorption to hexadecane, isoelectric point, adsorption of anionic orcationic dyes). However, despite their specific surface characteristics, in most cases the net charge properties of different bacterial strains (characterized by the zeta potential profiles of the bacteria in relation to the pH) were found to be related to the substrate the bacteria were able to degrade as well as to the consortium the bacteria were isolated from. For one group of specialized bacteria, only oneor at most two characteristic zeta potential profiles were measured. Compared to the differences between different strains, the zeta potential profiles of individual strains were only slightly affected by either growth state or changes in the actual nutrient composition. Even if isolated strains were cultivated in standard nutrient broth for several months, only slight differences in the zeta potential profiles were measured. Only the isoelectric focusing experiments indicated thatcultivation in a complex medium favoured a progressively decreased uniformity of surface charge properties. Thus, measurement of zeta potential profiles under standardized conditions may be a useful means to compare the surface structures of bacteria from different origins.     

Zeta potential as a diagnostic tool to evaluate the biomass electrostatic adhesion during ion-exchange expanded bed application

Expanded bed adsorption is an integrative technology in downstream processing allowing the direct capture of target proteins from biomass (cells or cell debris) containing feedstocks. Potential adhesion of biomass on the surface of adsorbent, however, may hamper the application of this technique. Since the electrostatic forces dominate the interactions between biomass and adsorbent, the concept of zeta potential was introduced to characterize the biomass/adsorbent electrostatic interactions during expanded bed application. The criterion of zeta potential evaluation proposed in the previous paper (Biotechnol Bioeng, 83(2):149-157, 2003) was verified further with the experimental validation. The zeta potential of intact cells and homogenates of four microorganisms (Escherichia coli, Bacillus subtilis, Pichia pastoris, and S. cerevisiae) were measured under varying pH and salt concentration, and two ion-exchange adsorbents (Streamline DEAE and Streamline QXL) were investigated. The biomass transmission index (BTI) from the biomass pulse response experiments was used as the indicator of biomass adhesion in expanded bed. Combining the influences from zeta potential of adsorbent (zeta(a)), zeta potential of biomass (zeta(b)) and biomass size (d), a good relationship was established between the zeta potential parameter (-zeta(a)zeta(b)d) and BTI for all experimental conditions. The threshold value of parameter (-zeta(a)zeta(b)d) can be defined as 120 mV2 microm for BTI above 0.9. This means that the systems with (-zeta(a)zeta(b)d) < 120 show neglectable electrostatic bio-adhesion, and would have a considerable probability of forming stable expanded beds in a biomass suspension under the particular experimental conditions.     

An ISFET biosensor for the monitoring of maltose-induced conformational changes in MBP

Here we describe an ion sensitive field effect transistor (ISFET) biosensor, which was designed to monitor directly the surface charge of structurally altered maltose binding protein (MBP) upon stimulation with maltose. This study is the first report of the application of a FET biosensor to the monitoring of conformationally changed proteins. Consequently, a significant drop in current on the basis of the charge-dependent capacitance measurement has been clearly observed in response to maltose, but not for the glucose control, thereby indicating that the substrate-specific conformational properties of the target protein could be successfully monitored using the ISFET. Collectively, our results clearly suggest that ISFET provide a high fidelity system for the detection of maltose-induced structural alterations in MBP.     

Surface Charge Density of Hetero-Fused Plant Protoplasts: an Electrophoretic Study

Electrophoretic mobilities of hetero-fused plant protoplasts,which were obtained by electrofusion of barley mesophyll cellprotoplasts and Rauwolfia serpentina cultured cell protoplasts,and those of the unfused parent protoplasts were measured invarious media of different pH values. At pH 5.2, the zeta potentialof the fused protoplasts was intermediate between those of thebarley and R. serpentina protoplasts and the average surfacecharge density of the fused protoplasts was closer to that ofR. serpentina than to that of barley. The distribution of thesurface charge density of fused protoplast obtained at pH 5.2is discussed in terms of the surface charge densities and thesizes of parent protoplasts. These results revealed that thesurface charge density of fused protoplasts was determined bythe surface charge densities and the ratio of the surface areasof the respective parent protoplasts. (Received December 28, 1989; Accepted August 10, 1990)     

Detection of DNA and proteins using amorphous silicon ion-sensitive thin-film field effect transistors

Amorphous silicon-based ion-sensitive field-effect transistors (a-Si:H ISFETs) are used for the label-free detection of biological molecules. The covalent immobilization of DNA, followed by DNA hybridization, and of the surface adsorption of oligonucleotides and proteins were detected electronically by the a-Si:H ISFET. The ISFET measurements are performed with an external Ag/AgCl microreference electrode immersed in 100mM phosphate buffer electrolyte with pH 7.0. Threshold voltage shifts in the transfer curve of the ISFETs are observed resulting from successive steps of surface chemical functionalization, covalent DNA attachment to the functionalized surface, surface blocking, and hybridization with a complementary target. The surface sensitivity achieved for DNA oligonucleotides is of the order of 1pmol/cm(2). Point-of-zero charge estimations were made for the functionalized surfaces and for the device surface after DNA immobilization and hybridization. The results show a correlation between the changes in the point-of-zero charge and the shift observed in the threshold voltage of the devices. Electronic detection of adsorbed proteins and DNA is also achieved by monitoring the shifts of the threshold voltage of the ISFETs, with a sensitivity of approximately 50nM.     

An acid-labile block copolymer of PDMAEMA and PEG as potential carrier for intelligent gene delivery systems

Intelligent gene delivery systems based on physiologically triggered reversible shielding technology have evinced enormous interest due to their potential in vivo applications. In the present work, an acid-labile block copolymer consisting of poly(ethylene glycol) and poly(2-(dimethylamino)ethyl methacrylate) segments connected through a cyclic ortho ester linkage (PEG- a-PDMAEMA) was synthesized by atom transfer radical polymerization of DMAEMA using a PEG macroinitiator with an acid-cleavable end group. PEG- a-PDMAEMA condensed with plasmid DNA formed polyplex nanoparticles with an acid-triggered reversible PEG shield. The pH-dependent shielding/deshielding effect of PEG chains on the polyplex particles were evaluated by zeta potential and size measurements. At pH 7.4, polyplexes generated from PEG- a-PDMAEMA exhibited smaller particle size, lower surface charge, reduced interaction with erythrocytes, and less cytotoxicity compared to PDMAEMA-derived polyplexes. At pH 5.0, zeta potential of polyplexes formed from PEG- a-PDMAEMA increased, leveled up after 2 h of incubation and gradual aggregation occurred in the presence of bovine serum albumin (BSA). In contrast, the stably shielded polyplexes formed by DNA and an acid-stable block copolymer, PEG- b-PDMAEMA, did not change in size and zeta potential in 6 h. In vitro transfection efficiency of the acid-labile copolymer greatly increased after 6 h incubation at pH 5.0, approaching the same level of PDMAEMA, whereas there was only slight increase in efficiency for the stable copolymer, PEG- b-PDMAEMA.     

Biothermodynamic analysis of BSA adsorption to alum gel using isothermal titration calorimetry

In this study, we used ITC (isothermal titration calorimetry) to quantitatively investigate the impacts of temperature and protein concentration on adsorption behavior on a solid surface, using BSA (bovine serum albumin) as a model protein, and alum (aluminum hydroxide) gel as an adsorbent. The zeta potential measurement for alum gel (0.25 mV at pH 9.3) revealed that its surface charge was not strong enough for electrostatic interaction. ITC analysis showed that the BSA-alum gel interaction was entropy-driven, suggesting that during adsorption, water molecules were expelled from the hydration layers of the alum gel and BSA. Therefore, the major mechanism for the BSA-alum gel interaction was hydrophobic interaction rather than electrostatic interaction. This biothermodynamic approach can be helpful not only to identify interaction mechanisms, but also to explore the optimum conditions for protein-adsorbent interactions.     

pH-sensitive vesicles containing a lipidic beta-amino acid with two hydrophobic chains

The lipidic beta-amino acid 2-(aminomethyl)-2-pentadecylheptadecanoic acid (1) was synthesized via the alkylation of the C(alpha)-atom of fully protected beta-alanine. Mixed large unilamellar vesicles with a diameter between 100 and 200 nm containing POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and 1 at a molar ratio of 9 : 1 were prepared and found to have a surface charge which is dependent on pH. At slightly acidic pH, the vesicles were positively charged, and at alkaline pH negatively charged. Dynamic light scattering, zeta potential, and cryo-transmission electron-microscopy measurements indicated that the mixed vesicles fused at pH 4-5 with negatively charged mixed vesicles composed of POPC and POPG (9.8 : 1, molar ratio), POPG being 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)].     

A fully electronic sensor for the measurement of cDNA hybridization kinetics

Ion sensitive field effect transistors (ISFET) are candidates for a new generation of fully electrical DNA sensors. To this purpose, we have modified ISFET sensors by adsorbing on their Si(3)N(4) surface poly-L-lysine and single (as well as double) stranded DNA. Once coupled to an accurate model of the oppositely charged layers adsorbed on the surface, the proposed sensor allows quantitatively evaluating the adsorbed molecules densities, as well as estimating DNA hybridization kinetics.     

Hydrophobic and electrostatic cell surface properties of Cryptosporidium parvum.

Microbial adhesion to hydrocarbons and microelectrophoresis were investigated in order to characterize the surface properties of Cryptosporidium parvum. Oocysts exhibited low removal rates by octane (only 20% on average), suggesting that the Cryptosporidium sp. does not demonstrate marked hydrophobic properties. A zeta potential close to -25 mV at pH 6 to 6.5 in deionized water was observed for the parasite. Measurements of hydrophobicity and zeta potential were performed as a function of pH and ionic strength or conductivity. Hydrophobicity maxima were observed at extreme pH values, with 40% of adhesion of oocysts to octane. It also appeared that ionic strength (estimated by conductivity) could influence the hydrophobic properties of oocysts. Cryptosporidium oocysts showed a pH-dependent surface charge, with zeta potentials becoming less negative as pH was reduced, starting at -35 mV for alkaline pH and reaching 0 at isoelectric points for pH 2.5. On the other hand, variation of surface charge with respect to conductivity of the suspension tested in this work was quite small. The knowledge of hydrophobic properties and surface charge of the parasite provides information useful in, for example, the choice of various flocculation treatments, membrane filters, and cleaning agents in connection with oocyst recovery.     

Temperature- and ionic strength-induced conformational changes in the lipid head group region of liposomes as suggested by zeta potential data.

Neutral liposomes composed of DMPC (dimyristoylphosphatidylcholine), DPPC (dipalmitoylphosphatidylcholine) or DSPC (distearoylphosphatidylcholine) are found to exhibit non-zero zeta potentials in an electric field even when they are dispersed in solution at pH 7.4. A model for the orientation of lipid head groups is proposed to explain the observed non-zero zeta potentials. The dependence of the zeta potential on temperature and ionic strength is analyzed via this model to obtain the information on the direction of the lipid head group in the liposome surface region. The direction of the lipid head group is found to be sensitive to the temperature and the ionic strength of the medium. At low ionic strengths, the phosphatidyl groups are located at the outer portion of the head group region. At constant temperature, as the ionic strength increases, the choline group approaches the outer region of the bilayer surface while the phosphatidyl group hides behind the surface. At the phase transition temperature of the lipid, the phosphatidyl group lies in the outer-most region of the surface and the choline group is in the inner-most region.     

New technology for the detection of pH

The measurement of pH is the most widely employed test for (bio-)chemical lab. Since the first use of glass electrode to detect pH, new techniques and methods have broaden the scope of pH detection. Metal/metal oxide, ion sensitive field-effect transistors (ISFET), fibre-optical techniques, nanotechniques, and conducting polymer techniques have been extensively developed. This review covers the various methods for pH detection. New development trends were discussed.     

Molecular charge contact biosensing based on the interaction of biologically modified magnetic beads with an ion-sensitive field effect transistor

In this article, we report a novel method of biomolecular recognition based on the molecular charge contact (MCC). As one of the MCC biosensing method, the interaction between DNA-coated magnetic beads and a silicon-based semiconductor, an ion-sensitive field effect transistor (ISFET) could be detected for DNA molecular recognition events using the principle of the field effect, which enables detecting ionic or molecular charges. After DNA-coated magnetic beads had been introduced and brought in contact with the gate surface by a magnet, the threshold voltage of the ISFET was shifted in the positive direction by immobilization, hybridization and extension reaction of DNA molecules on magnetic beads. This positive shift was based on the increase in negative charges of the phosphate groups in them. Then, the ISFET device could be reused a couple of dozen times continuously and cost-effectively because the oligonucleotide probes were tethered to the magnetic beads, but this was not done directly on the gate surface of the ISFET. Moreover, the MCC biosensing method enabled discrimination of a single nucleotide polymorphism. By creating an interaction of magnetic beads with the semiconductor, we can expect enhancement of the reaction efficiency in a solution and reuse of the device by separating the reaction field from the sensing substrate.     

Surface immobilization of protein via biosilification catalyzed by silicatein fused to glutathione S-transferase (GST)

Silicatein from Suberites domuncula was known to catalyze silica deposition in vitro under near neutral pH and ambient temperature conditions. In this study, we employed GST–glutathione (GSH) interaction system to increase the production of silicatein and develop an efficient protein immobilization method. Recombinant silicatein fused with GST (GST-SIL) was produced in E. coli and the GST-SIL protein was employed on GSH-coated glass plate. GST-SIL bound surface or matrix can catalyze the formation of silica layer in the presence of tetraethyl orthosilicate as a substrate at an ambient temperature and neutral pH. During silicatein-mediated silicification, green fluorescent protein (GFP) or horseradish peroxidase (HRP) can be efficiently immobilized on the silica surface. Immobilized GFP or HRP retained their activity and were released gradually. This biocompatible silica coating technique can be employed to prepare biomolecule-immobilized surfaces or matrixes, which are useful for the development of biocatalytic, diagnostic and biosensing system, or tissue culture scaffolds.     

An AFM determination of the effects on surface roughness caused by cleaning of fused silica and glass substrates in the process of optical biosensor preparation

The covalent attachment of organic films and of biological molecules to fused silica and glass substrates is important for many applications. For applications such as biosensor development, it is desired that the immobilised molecules be assembled in a uniform layer on the surface so as to provide for reproducibility and speed of surface interactions. For optimal derivatisation the surface must be appropriately cleaned to remove contamination, to create surface attachment sites such as hydroxyl groups, and to control surface roughness. The irregularity of the surface can be significant in defining the integrity and density of immobilised films. Numerous cleaning methods exist for fused silica and glass substrates and these include gas plasmas, and combinations of acids, bases and organic solvents that are allowed to react at varying temperatures. For many years, we have used a well established method based on a combination of washing with basic peroxide followed by acidic peroxide to clean and hydroxylate the surface of fused silica and glass substrates before oligonucleotide immobilisation. Atomic force microscopy (AFM) has been used to evaluate the effect of cleaning on surface roughness for various fused silica and glass samples. The results indicate that surface roughness remains substantial after use of this common cleaning routine, and can provide a surface area that is more than 10% but less than 30% larger than anticipated from geometric considerations of a planar surface.     

Tracking of the kinetic stability of 2 types of total nutrient admixtures containing different lipid emulsions

The physical stability of 2 types of total nutrient admixtures was studied as a function of storage time and temperature. One of them contained only structured triglycerides and the other exclusively long-chain triglycerides as lipid components. To evaluate the possible changes in the kinetic stability of the emulsions and in the surface characteristics of the droplets during storage, particle size analysis, zeta potential, and dynamic surface tension measurements were performed. To follow any chemical decomposition processes that occurred during storage, the pH of the emulsions was also monitored. The mean droplet size of emulsions prepared with lipids containing exclusively long-chain triglycerides showed a remarkable increase after 4 days of storage, in contrast with that of the mixtures containing structured lipids. A combination of size distribution, zeta potential, and dynamic surface tension measurements proved to be useful for an adequate tracking of the kinetic stability of total nutrient admixtures. Structured triglycerides not only provide advantageous metabolic effects but improve the physical stability of total parenteral nutrition admixtures.