Polymyxin B-induced phase separation and acyl chain interdigitation in phosphatidylcholine/phosphatidylglycerol mixtures.

Monolayers, fluorescence polarization, differential scanning calorimetry and X-ray diffraction experiments have been carried out to examine the effect of the polypeptide antibiotic polymyxin B on the phase behaviour of dipalmitoylphosphatidylglycerol (DPPG) either pure or mixed with dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC). It is shown that in both phosphatidylglycerol alone and phosphatidylglycerol/phosphatidylcholine mixtures, polymyxin B can induce either phase separation between lipid domains of various compositions or interdigitation of the acyl chains in the solid state, without segregation of the two lipids. Phase separation was observed by fluorescence and differential scanning calorimetry after addition of the antibiotic to vesicles composed of mixtures of DMPC and DPPG in conditions where polymyxin B did not saturate phosphatidylglycerol (DPPG to polymyxin B molar ratio, Ri, higher than 15). Phase separation was also observed in mixed monolayers of DPPC and of the 5:1 DPPG/polymyxin B complex, at high surface pressure. Acyl chain interdigitation was observed by X-ray diffraction in both 5:1 DPPG/polymyxin B mixtures and preformed 5:5:1 DMPC/DPPG/polymyxin B mixture, in which the antibiotic saturates phosphatidylglycerol (Ri 5). In both cases, raising the temperature gave rise to a complex double-peaked phase transition by differential scanning calorimetry, from the interdigitating phase to a normal L alpha lamellar phase. As it is known that polymyxin B does not interact with phosphatidylcholine, the data presented show that, when phosphatidylcholine and phosphatidylglycerol are mixed together, a phase perturbation such as acyl chain interdigitation, which normally affects only phosphatidylglycerol, is also felt by phosphatidylcholine.

Immunomagnetic concentration of antigens and detection based on a scanning force microscopic immunoassay.

Scanning Force Microscopy has already been shown to be a convenient and rapid method for sensitive antigen detection and quantification. Here, we describe different improvement steps brought to a TSH Scanning Force Microscopic ImmunoAssay (SFMIA), each of them aiming to solve a previous limitation of the solid phase test format and leading to a significant sensitivity enhancement. First, superparamagnetic nanoparticles conjugated to monoclonal anti alphaTSH antibodies were used for the specific TSH capture step. Their magnetic properties allowed easy separation of the complexes obtained from relatively large reaction volumes by application of a High Gradient Magnetic Field System. As a consequence, complex formation could proceed in a stirred solution, which greatly enhances binding rates compared to previous 'static' conditions of solid-phase reactions. It was established that, despite their small size, magnetic complexes could be moved over short distances by a NdFeB magnet magnetic field. This property was exploited to overcome diffusion barrier and boundary layer constraints and to drive magnetic complexes through the liquid, towards anti-betaTSH antibodies immobilized on silica wafers. Finally, we significantly increased the complex number/surface area by a stepwise reduction of the biospecific solid phase area. The proposed steps permitted a 3-fold improvement in the TSH SFMIA dynamic range. Moreover, as little as 0.02 pg/ml (0.1 nIU/ml or 0.8 amol/ml) of TSH could be detected using 1 ml sample volumes. This is over 100 times more sensitive than the current performance of commercialized automated systems.

Under buffer SFM observation of immunospecies adsorbed on a cyano grafted silicon substrate.

Scanning force microscopy (SFM) in contact mode and in liquid medium has been employed to study immunospecies layers adsorbed on a silicon wafer. The silicon wafer has been grafted with a cyanosilane monolayer in order to create a surface with strong adhesive properties which prevent proteins being swept by the scan of the SFM tip. The force curves reveal that the adhesive force has been increased by a factor six without roughness modification (< 1 nm). After the incubation of the surface in a monoclonal antibody (mouse anti-human alpha-fetoprotein IgG) solution, SFM surface images suggest an homogeneous layer composed by ellipsoidal objects (40-60 nm in diameter, 6-13 nm in height). The substrate was moreover incubated in an antigenic solution (human alpha-fetoprotein): SFM images reveal that proteins have been added onto the antibody layer.

A dumbbell-like complex formation for DNA target assay.

A simple and highly sensitive test for the detection of nucleic acid targets is described. It is based upon complex formation between a small-diameter magnetic particle and a larger and nonmagnetic particle through a hybridization reaction, what we have called a dumbbell-like complex. During the different steps, nonreacting nonmagnetic conjugates were eliminated by magnetic separation. At the end of the process, dumbbell complex number was estimated by counting under a microscope. Compared to the already described two-particle tests, our model was able to reach higher sensitivities, with a threshold typically in the amol/mL range (10(6) copies of HIV DNA/mL) without the need for complex instrumentation or genomic amplification reactions.

Thyroid stimulating hormone assays based on the detection of gold conjugates by scanning force microscopy.

Low bulk concentrations of thyroid stimulating hormone (TSH) were detected by scanning force microscopy (SFM) using gold-labeled conjugates. Anti-TSH antibodies were covalently bound onto amino-modified silicon oxide wafers. Surface modification was examined by contact-angle measurements, ellipsometry, X-ray photoelectron spectroscopy, and SFM. Antibodies were found to form a monolayer of prone molecules with an average surface density of 5000 IgG/mum2. TSH molecules were then allowed to bind to immobilized antibodies. The immunological reaction was quantified by SFM using gold-labeled species. Two scanned force microscopic immunoassays (SFMIA) were compared: first, a competitive test which used gold-labeled TSH molecules mixed with free TSH antigens was performed . Afterward, a sandwich assay was carried out, using gold-labeled anti-TSH antibodies. This latter method was found to be far more sensitive than competitive SFMIA. Gold conjugates were also found to be of great use to quantify antigens in large volumes by a sandwich test: a sensitivity threshold as low as 0.015 ng of TSH/ml (0.075 UI/ml or 6 x 10(-13) M) was estimated.

A study of the structure and dynamics of complexes between polymyxin B and phosphatidylglycerol in monolayers by fluorescence.

Interactions between the antibiotic polymyxin B and monolayers of dipalmitoylglycerophosphoglycerol have been reinvestigated through a study of the structure and dynamics of the complexes by means of an interface fluorimeter of our fabrication. A fluorescence technique has been developed where the use of linearly polarized incident beams gives the simultaneous determination of the orientation and the lateral diffusion rate of a fluorescent probe inserted in the film. The present investigation was carried out with 12-(9-anthroyloxy)-stearic acid, a fluorescent compound which forms non-fluorescent photodimers upon illumination. Orientation of the probe was studied by computing the ratio of the two dimerization constants KD and the ratio of the fluorescence intensities obtained with crossed linearly polarized incident lights. The lateral diffusion rate of the probe was obtained by measuring fluorescence recovery after photobleaching (photodimerization) of the probe. Control experiments, carried out with dimyristoylglycerophosphocholine, a lipid which does not interact with polymyxin B, show that the antibiotic does not significantly modify the behaviour of the probe. Both in terms of orientation and dynamics, with respect to dipalmitoylglycerophosphoglycerol, when the antibiotic is present in the subphase (1 microM, saturating conditions), data indicate that the lipid remains in a liquid-expanded state. This is true even at a high surface pressure (pi approximately equal to 37 mN X m-1), above the apparent 'transition' which can be observed at 30-35 mN X m-1 on its compression isotherm. Computation of the contribution of polymyxin B to the film expansion to the conclusion that this 'transition' would be a structural transition between two models of interaction: one, below the 'transition', where the polypeptide ring penetrates between the film-forming lipid molecules and another one, above the 'transition', were the antibiotic is adsorbed at the lipid-water interface with only its hydrocarbon chain penetrating the film.

Determination of the Main Forces Driving DNA Oligonucleotide Adsorption onto Aminated Silica Wafers

The adsorption of a 20-base long 5'-amino-linked oligodeoxyribonucleotide (ODN) onto aminopropylsilane-modified silica wafers has been investigated. At first, silanized surfaces were characterized by contact angle measurements, X-ray photoelectron spectroscopy (XPS), ellipsometry, and atomic force microscopy (AFM). Adsorbed amount of oligonucleotides was estimated by radioactive counting or colorimetric hybridization reaction. The first technique was useful for direct counting, while colorimetric detection, implying an hybridization reaction between adsorbed ODN and its complementary sequence, provided information about its accessibility on the wafer. With the purpose of determining the driving forces of the ODN adsorption onto these surfaces, conditions such as initial ODN concentration, pH, and ionic strength have been examined. The adsorption process could be described as a Langmuir reversible adsorption type. Surface charge contribution has been investigated by raising pH values from 4 to 10.8. Electrostatic interactions between the negatively charged ODN and the aminated groups on the silica wafers were found to play a major role in the adsorption process. Moreover, a drastic influence of the ionic strength on the ODN adsorbed amount was evidenced. Copyright 1997 Academic Press. Copyright 1997Academic Press