Antibodies (also known as immunoglobulins - Ig [1]) are gamma globulin proteins found in blood or other bodily fluids of vertebrates. They are used by the immune system to identify and neutralize bacteria and viruses. Five different antibody isotypes are known in mammals, which act differently and induce the appropriate immune response for each different type of foreign object they encounter [2]. They are known as IgA, IgD, IgE, IgG and IgM. Even if the general structure of all Ig’s is very similar, a small region at the protein tip is changing, allowing millions of antibodies with slightly different tip structures to exist. Each of these variants can bind to a different target, known as an antigen [3]. This huge diversity of antibodies allows the immune system to recognize an equally wide diversity of antigens. The unique part of the antigen recognized by an antibody binds with their antibody in a highly specific interaction that allows antibodies to identify and bind only their unique antigen. Antibodies can neutralize targets by binding to a part of an infectious pathogen [4].

IgG antibodies are large molecules of ~150 kDa composed of 4 peptide chains: 2 identical heavy chains of ~50 kDa and 2 identical light chains of ~25 kDa. The 2 heavy chains are linked to each other and to a light chain each by disulfide bonds. The resulting tetramer has 2 identical halves which form the Y-like shape. Each end of the fork contains an identical antigen binding site. IgG is largely involved in the secondary immune response. The presence of specific IgG corresponds to maturation of the antibody response. IgG is the only isotype that can pass through the human placenta, providing protection to the fetus in utero. IgG can bind to many kinds of pathogens, e.g. viruses, bacteria, fungi, and protects the body against them by agglutination and immobilization.

The proteins can offer important advantages such as molecular recognition, self-assembly or genetic manipulation [5]. There is a permanent need for flexible immobilization methods able to attach specific biomolecules to relevant material surfaces. Laser-assisted methods exhibit some advantages over other techniques: the accurate control of the layer thickness and/or expulsed material, absence of contamination, uniform distribution of material over quite large areas and rather easy synthesis of multistructures. Matrix Assisted Pulsed Laser Evaporation (MAPLE) process was particularly developed to provide a soft laser transfer of organic and/or biologic materials [6].

We recently confirmed the sheltered transfer and immobilization of rabbit anti-human antiserum IgG by MAPLE [7]. The targets consisted of 0.2–2 mg/mL IgG blended or not with lipid (L-a-phosphatidylcholine dipalmitoyl) dissolved in distilled water-based saline buffer. The optical microscopy, AFM, SEM, and FTIR evidenced an optimal protection regime of protein transfer for the incident laser fluence of 0.5 J/cm². Spectrofluorimetry and fluorescence microscopy were selected to verify the biosensor transduction principle due to their high sensitivity for detecting low amounts of antigen (IgG). Protein immobilization to the substrate was demonstrated after immersion in the donkey anti-rabbit secondary antibody solution. The IgG transfer and immobilization onto substrates were improved by addition of lipid to MAPLE solutions.

The immobilization of proteins in thin films or nanostructures is a real defy and of great interest for miniaturized biosensors based on the direct reaction antibody - antigen. Two well-known methods for protein immobilization are electrostatic layer-by-layer (LbL) [8,9] and Langmuir–Blodgett (LB) [10] processes, acting complementary for different types of materials. The surface functionalization [11], complex biological interactions biotin - avidin [12] and proteins A or G [13,14] chemical interactions [15-18] and physical adsorption [19] were tested to optimize the protein immobilization which depends on composition, reactivity, wettability, and roughness of the surface [20,21].

Based on Ref. [7], we advance the suggestion of using MAPLE immobilized IgG films as immuno-sensors for the detection of specific antigens in research or clinical studies. To this prospective, the observed possibility to control the sensor surface morphology by the content of salts and lipid in MAPLE solutions confers a large avenue for finding the best compromise between the IgG content and surface condition over sensing capabilities. This is an essential step in developing personalized and miniaturized biosensors. Specific masks or photomasks [22] in MAPLE experiments can be used to synthesize well controlled microsized samples for microarray chip use. An interesting alternative that avoids the use of expensive photolithographic masks and presents higher integration scale than microspotting or ink-jet printing is the laser direct write (LDW) technique [23,24].

Exopolysaccharides (EPSs) are high-molecular-weight polymers composed of sugar residues and secreted by microorganisms in the surrounding environment to serve as a protective barrier against external stress. They have many prospective applications due to their unique physicochemical and rheological properties, biocompatibility and biodegradability [25]. Extremophilic microorganisms provide non-pathogenic products, suitable for uses in food industry, pharmacy and cosmetics as emulsifiers, stabilizers, gel agents, coagulants, thickeners and suspending agents [26]. One EPS extremophile producer is the halophilic Halomonas sp. AAD6 bacteria, which secrets high level of levan, a long linear homopolymer of β(2-6) linked fructose residues [27]. Levan is a water soluble, strongly adhesive and film-forming biopolymer, presenting low viscosity, high solubility in oil, compatibility with salts and surfactants, stability to heat, acid and alkali media, high holding capacity for water and chemicals, and good biocompatibility. It has many potential uses as emulsifier, stabilizer and thickener, encapsulating agent, osmoregulator and cryoprotector in food, cosmetics, or pharmaceutical industries. In medicine, levan is used as plasma substitute, prolongator of drug activity, antitumor and antihyperlipidemic agent [28]. Levan by Halomonas sp. was reported as a good candidate for development of nanocarrier systems for peptide and protein drug delivery [29]. Large decrease of this polymer fabrication costs has been attained by optimization of fermentation conditions in large-scale levan production by Halomonas sp. bioreactor cultures [30].

Levan was found to dissolve more easily in acidic solutions than in alkaline ones (Table 1). Besides HCl, the polymer was found to dissolve quickly in 1-3% acetic acid and 1% Lactic acid solutions. Levan did not dissolve in ethanol, methanol and acetone. Particle size affects the solubility and fine powdered Levan dissolved 10 fold quicker than its dried fiber form. A precipitate was formed when 5% (w/v) NaOH or 20% (w/v) Na₂SO₄ were added to 0.5% (w/v) aqueous Levan solutions.

Table 1: Solubility of Levan produced by Halomonas sp.

 

Levan coatings could have great commercial potential for specific applications. Currently, films of polymers with desired shape and area are obtained by solvent casting [31] or thermal processing [32]. Presently, drug tablets are coated with a thick film (hundreds of microns) of polymer and polysaccharide, with plasticizers and pigments added [33]. The thicker the film, the higher the risks of poor adhesion, cracking or easy peeling [34], making impossible to control the film dissolution.

Films at nanometric scale would reduce the cost of production and increase the specific surface area. For drug release and delivery, thin coatings of desired thickness would be attractive to control the rate of dissolution in the gastrointestinal tract since some drugs are absorbed better at different points in the digestive system. Nanostructured layers could boost the potential of biopolymer surface for applications as nanocarriers or drug delivery. Moreover, uniform distribution on different collectors allows for a wide range of new uses, especially in biology and medicine.

In our group, MAPLE was for the first time extended to obtain nanostructured thin films of levan and oxidized levan [35]. Solutions of pure levan in dimethyl sulfoxide were frozen in liquid nitrogen to obtain solid cryogenic pellets. The transfer of pure levan and oxidized levan by MAPLE was achieved without any addition of plasticizers or pigments. The deposition of levan was unapproachable by any other laser or other techniques. The coatings preserved the bulk composition, presented a compact structure, good adhesion to substrate and a uniform, homogenous nanostructured surface. They exhibited high specific surface areas fully compatible with potential use in biology or medicine. The biocompatible behavior of the synthesized nanostructures was confirmed by cell viability and proliferation studies.

 

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