Timothy Dubrovsky

Langmuir-Blodgett films of immunoglobulines IgG. Ellipsometric study of the deposition process and of immunological activity

Abstract The structure of the immobilized Langmuir-Blodgett films of IgG and their immunological activity were studied by means of ellipsometry. The dependence of the film thickness on the surface pressure of deposition provides evidence of the tilting of molecules with an increase in the pressure. Below pressures of 30 mN m −1 the thickness of the film is approximately 4 nm which coincides with the smallest dimension of the IgG molecules. At pressures between 30 and 40 mN m −1 the thickness increases sharply achieving a value of about 10 nm which is equal to the largest molecular dimension. A further increase of pressure does not show in terms of growth of the thickness. This means that the films are transferred from the water-air interface in the form of a 2-D ordered monomolecular layer. The dependence of the immunological activity on the pressure of deposition was shown to have a descending pattern. Different mechanisms are proposed which explain the decrease of the immunological activity of the IgG molecules in the film with an increase of the surface density such as the blocking of the active sites and the decrease of the conformation mobility of the Fab fragments.

Immunological activity of IgG Langmuir films oriented by protein A sublayer

Abstract The immunological reaction ability of IgG Langmuir-Blodgett monolayers organized by means of a protein A sublayer has been studied by the gravimetric technique. In order to discriminate the effects of molecular orientation and to preserve the native structure of IgG molecules using the protein sublayer, the kinetics of specific and non-specific binding at an immunoglobulin monolayer deposited onto different substrates, such as a silanized aluminium surface and a surface covered by ovalbumin and protein A sublayers, are compared. It is shown that the prevailing effect is molecular orientation. A sublayer of protein A appeared not only to increase the specific sensitivity of the IgG monolayer but to decrease the non-specific binding as well. For this structure the sensitivity of a monolayer of rabbit anti-mouse IgG towards mouse IgG is 10 pM. The sensitivities in the case of a bare metal surface and ovalbumin sublayer are 10 and 100 times less, respectively. Protein A and ovalbumin sublayers are obtained by the Langmuir-Schaeffer technique. The formation of monolayers on the water-air interface and the deposition onto the solid substrate are studied by means of compression isotherms, gravimetric and ellipsometric techniques. It is shown that the solubility of protein A depends on the surface pressure increasing sharply after a certain value of the pressure. It is also shown that protein A can be deposited onto the substrate in the form of dense two-dimensional monolayers.

LB-based PAB immunosystem : activity of an immobilized urease monolayer

Abstract A new configuration of the biosensing system named potentiomeric alternating biosensor (PAB) is analyzed. The system is based on an Si/SiO 2 /Si 3 N 4 transducer and allows the monitoring of several biochemical events, such as immunoenzymatic reactions and cell metabolism. the utilization of PAB a immunosensor implies the possibility of using a modified Langmuir-Blodgett (LB) technique for the production of an antibody monolayer. In the first stage the activity of an immobilized LB monolayer of an enzyme (urease) is monitored in order to simulate the final step of the immunoenzymatic assay; subsequently, the enzymatic activity of an immobilized monolayer of antigen (MAb) after incubation with antibodies labelled with urease is monitored. Vacuum silanization with glycidoxypropyltrimethoxysilane (GOPTS) is utilized to ensure covalent binding of the protein LB monolayer to a glass substrate. The analysis with PAB of films thus prepared is preceded by characterization of the layer by means of nanogravimetric and spectrophotometric techniques, which yield useful information on the surface density and activity, respectively. Furthermore, the operative lifetime of the enzyme layer within a period comparable to the standard time of an immunoassay has been studied.

High-sensitivity biosensor based on LB technology and on nanogravimetry

Abstract Quartz-resonator nanobalances are utilized here as effective transducers capable of determining with high sensitivity the mass changes due to specific protein-protein, antigen-antibody and ligand-receptor binding or self-assembly of functional complexes. The resulting highly sensitive biosensor is based on two quartz resonators (one active and the second used as a reference) and on Langmuir-Blodgett (LB) protein monolayers. The electronics are composed of two separate blocks, one designed to acquire by a personal computer the data coming from the other card, a 24-bit digital counter directly connected to the two oscillators. In the case of immunosensor application, the active and reference oscillators are covered respectively by antibodies specific to a given antigen and by antibodies non-specific to the antigen, in order to disciminate the physical adsorption effects. Deposition of antibody monolayers is performed by the LB technique in a surface pressure range of 20–35 mN m−1 onto gluteraldehyde pre-treated quartz resonators. A thermal treatment of the antibody layer up to 150 °C results in the reorganization of the film, and significantly improves the sensitivity and the properties of the immunosensor.

Determination of orientation of the IgG molecules in immobilized Langmuir monolayers by means of binding with fragment specific anti-immimoglobulin antibodies

Abstract We report the technique for determining the orientation of the IgG molecules in Langmuir films immobilized on a solid substrate. This technique is based on measuring the binding of the secondary antibodies specifically recognising Fab and Fc fragments of immobilized IgG molecules. Specific binding of anti-Fab and anti-Fc rabbit anti-mouse (RAM) polyclonal antibodies to the film of mouse IgG shows strong dependence on surface deposition pressure of the film indicating changes of molecular orientation occurring in the film. At high deposition pressures the amount of anti-Fab specific RAM antibodies bound with the mouse IgG film is much greater than the anti-Fc specific RAM antibodies. This means that IgG molecules of the film are preferentially oriented with their Fab fragments outside. The ratio of molecules oriented in this way to the opposing molecules is estimated to be 3.125. The technique can be considered rather general and suitable for similar studies of any protein 2-dimensional arrays since the antibodies to different parts or fragments of any protein can be obtained.

Langmuir films of Fc binding receptors engineered from protein A and protein G as a sublayer for immunoglobulin orientation

Abstract The paper describes the production and immobilization of Langmuir monolayers of recombinant proteins A and G and the chimeric protein A G . The films were used for the immobilization of IgG molecules on the solid supports. Making use of the high affinity of the proteins A and G to the Fc fragments, the orientation technique provides the IgG position with the Fab fragments directed outward, capturing the antigen in the mobile phase. The films of binding receptors were characterized by ellipsometry and nanogravimetry and its IgG binding capacities were tested by fluorescent assays. The immunological activity of IgG films organized by means of receptor sublayers was studied by the microgravimetric technique. The sensitivity of the monolayer of rabbit anti-mouse IgG oriented with protein A sublayer towards mouse IgG is in the range of 10–100 pM.

Deposition, molecular organization and functional activity of IgG Langmuir films

Abstract IgG monolayers, formed at the air-water interface and covalently immobilized on solid supports, were studied by means of ellipsometry, time-resolved fluorometry and immunoassay techniques. Film molecular packing and immunological activity were shown to depend drastically on surface pressure.

Preparation and immobilization of Langmuir-Blodgett films of antibodies conjugated to enzymes for potentiometric sensor application

Abstract A method of surface modification of chips of potentiometric sensors by 3-glycidoxypropyltrimethoxysilane (GOPTS)1 in gaseous phase for covalent immobilization of antibody Langmuir-Blodgett films has been studied. Antibody monolayers are formed at the liquid-gas interface and transferred onto sensor chips at a surface pressure of 25 mN m−1. The immunological activity of immobilized RAM antibodies is determined on the basis of specific binding of enzyme-labelled antigen. The binding of HRP-labelled RAM antibodies to the immobilized antigen monolayer is estimated by using the conventional spectrophotometric method. The enzymatic and immunological activities of different components of the ‘sandwich’ scheme of the antigen detection have been characterized.

Langmuir-Blodgett film of glutathione S-transferase immobilised on silanized surfaces

Abstract In this work the enzymatic activity of an immobilised Langmuir-Blodgett film of glutathione S-transferase (GST) that catalyses the glutathione conjugation to the l-chloro-2,4-dinitrobenzene has been studied. Silicon wafers or glass slices were used as solid supports and their surfaces were modified by means of silanization with 3-glycidoxypropyltrimethoxysilane. This chemical modification of the surface allowed one to bound the enzyme to the support, avoiding detachment during the measurements. A Langmuir-Blodgett trough was used to form the GST film at the air-water interface and the Langmuir-Schaefer deposition technique was adopted to transfer the enzyme film onto the activated support. Nanogravimetry and spectrophotometry were performed to analyse the formed film in order to obtain data on surface density and functional activity respectively. The results underlined a reliability of the deposition and a maintenance of the enzymatic activity of GST after its deposition onto the solid support. The activity of GST film related to organization, thermal treatment and storage conditions was also studied. The investigation points out that the organisation of the LB film increased the specific activity of the enzyme film with respect to the spread one and that it was maximised when the GST was deposited onto an activated support. The enzymatic activity of GST was still maintained up to 423 K; the immobilised enzyme even showed activity 5 months after its deposition onto the activated substrate and only a small decrease in activity was observed.

Optimisation of IgG Langmuir film deposition for application as sensing elements

Covalently immobilised IgG Langmuir films were studied by means of ellipsometry, time resolved fluorimetry, CCD-based image analysis and immunoassay methods. The film structure and immunological activity were shown to depend drastically on the surface pressure of deposition. As previously shown, under a pressure below 30 mN/m the molecules are oriented such that their Fab-Fab-Fc plane is parallel to the surface, at a pressure above 40 mN/m the molecules take perpendicular position. Deposited under high pressure the molecules have preferential orientation with their Fab fragments directed outwards the support. Despite this favourable for antigen binding orientation achieved under high pressures, the immunological activity of the films towards a high-molecular weight antigen decreases with pressure, obviously due to the lack of conformational mobility necessary for the antibody to bind the antigen. Whenever the IgG Langmuir films were oriented by protein A sublayer, the specific binding ability of the IgG monolayer appears to increase and the non-specific binding to decrease, with an overall increase in sensitivity by at least 10 times.