Proteins passive adsorption

Figure 2 Immobilized antigen ELISA format. Antigen is immobilized to a solid phase by passive adsorption. Following removal of unbound antigen, analyte (free H) and antigen (H-protein) compete for a fixed number of primary antibody (Y) binding sites. Unbound materials are removed (dotted line). Secondary antibody-enzyme conjugate (Y-E) is added to bind to primary antibody followed by another wash step. Substrate (A) for the enzyme is added to detect the bound enzyme. The amount of colored product ( ) detected is inversely proportional to the amount of analyte present...

One of the simplest methods of attaching biomolecules to hydrophobic polymeric particles is the use of passive adsorption. Some of the earliest examples related to the use of particles in immunoassays include the use of non-covalently adsorbed antibody or antigen onto latex microspheres. Protein adsorption onto hydrophobic particles takes place through strong interactions... 

The following protocol for passive adsorption is based on methods reported for use with hydrophobic polymeric particles, such as polystyrene latex beads or copolymers of the same. Other polymer particle types also may be used in this process, provided they have the necessary hydrophobic character to promote adsorption. For particular proteins, conditions may need to be optimized to take into consideration maximal protein stability and activity after adsorption. Some proteins may undergo extensive denaturation after immobilization onto hydrophobic surfaces therefore, covalent methods of coupling onto more hydrophilic particle surfaces may be a better choice for maintaining native protein structure and long-term stability. 

The membrane surface may become passivated by some solution components that are strongly adsorbed. This effect is often encountered in measurements on biological fluids containing proteins. These adsorption effects can sometimes be prevented by selecting a suitable compoation of the sample and standard solutions for example by adding trypsin and triethanolamine to dissolve proteins [108]. Passive electrodes can sometimes be reactivated by soaking in suitable solutions (for example pepsin in 0.1M HCl [68]) and in more serious cases the membrane must be replaced or a solid membrane be repolished. 

QDs have been shown to be quite stable to metabohc degradation (with the exception of problems associated with heavy metal leachants) [ 177,180]. QDs can be conjugated to the linker [181] (e.g., avidin, protein A or protein G, or a secondary antibody) by covalent binding, passive adsorption, multivalent chelation or by electrostatic interactions [182,183]. 

Usually, immobilization of antibodies can be performed either directly by passive adsorption, covalent coupling, or indirectly by using a solid surface pre-coated with protein A or G [25] or a secondary immunoglobulin (Ab2, anti-Abi) [26] for a favourable orientation of the antibody on the solid surfece with the binding sites towards the sample solution. This orientated immobilization of antibodies is important for the assay performance, taking into account the special characteristics of solid-phase immunoassays (see Section 9.3.4.4). 

The selectivity of immunosensors for steroid analytes is achieved with the use of appropriately selected monoclonal antibodies. The carbon working electrode provides a suitable surface for passive adsorption of proteins, and can therefore be tailored with an appropriate antibody, so that it will act as an immunoactive surface upon which an immunoaffinity assay can be performed an electrochemical signal can then be generated by monitoring the production of an electroactive species at the underlying electrode surface. We and other workers have found that to retain maximum monoclonal antibody activity, it is desirable to use a primary antibody (rabbit IgG), which serves both to capture (e.g., from a culture medium) and to orientate the mAb. Hence this approach... 

A key feature of the solid-phase ELISA is that antigens or antibodies can be attached to smfaces easily by passive adsorption. This proeess is commonly called coating. Most proteins adsorb to plastic smfaces, probably as a result of hydrophobie interactions between nonpolar protein substructures and the plastie matrix. The interactions are independent of the net charge of the protein, and thus each protein has a different binding constant. The hydrophobieity of the plastic/protein interaction can be e qjloited to increase binding since most of proteins hydrophilic residues are at the outside and most of the hydrophobic residues orientated towards the inside (1). 

Not only the components of the sensing layer are important but also the strategies of integration between them and with the primary transducer are of paramount importance. Passive adsorption is one of the simplest and most frequently used immobilization techniques, based mainly on weak noncovalent bindings. However, random protein orientation can lead to the obstruction of functional binding sites and can result in the loss of affinity or activity. Thus, covalent attachment of a protein layer on a chemically functionalized surface produces a more stable layer with correct orientation. This influences sensitivity and specificity of the immunoassay as density of immobilized protein can be better controlled and nonspecific adsorption may be decreased [155]. On the other hand, covalent procedures are usually longer and more tedious and are less justified when disposable surfaces can be used. 

Pinto et al. reported the PDMS surface functionalization for the development of an immune-sensor for salivary cortisol analysis, using three different antibodies immobilization methods immobilization by passive adsorption on pristine PDMS silaniza-tion of PDMS surface with (3-aminopropyl)-triethoxysilane (APTES) to generate amino groups and posterior covalent immobilization of antibodies on APTES-PDMS using crosslinker glutaraldehyde (GA) coating the PDMS surface with BSA to block nonspecific protein adsorption, and then covalent bond of the protein A via GA (Pinto etal.,2015). 

The qualitative thermodynamic explanation of the shielding effect produced by the bound neutral water-soluble polymers was summarized by Andrade et al. [2] who studied the interaction of blood with polyethylene oxide (PEO) attached to the surfaces of solids. According to their concept, one possible component of the passivity may be the low interfacial free energy (ysl) of water-soluble polymers and their gels. As estimated by Matsunaga and Ikada [3], it is 3.7 and 3.1 mJ/m2 for cellulose and polyvinylalcohol whereas 52.6 and 41.9 mJ/m2 for polyethylene and Nylon 11, respectively. Ikada et al. [4] also found that adsorption of serum albumin increases dramatically with the increase of interfacial free energy of the polymer contacting the protein solution. 

The avidin-biotin immobilization method maintains the biological activity of the receptor molecules. Besides the biocompatibility of the procedure, the surface geometry of these films provides high accessibility of the immobilized biomolecules. In addition, the avidin molecules form a passivation layer on the transducer surface that prevents nonspecific adsorption of proteins on the surface. In contrast with conventional grafting or affinity binding, this step-by-step approach can also be applied to the preparation of assemblies containing multilayers of biological molecules [42],... 

Despite the problems mentioned, the passive control of protein adsorption is excellently suited for short-term applications and many situations in a controlled environment ex vivo. 

Whitesides and coworkers describe the use of an elastomeric membrane to pattern proteins and cells on bacteriological polystyrene (PS), glass, and poly(dimethyl-siloxane) (PDMS) substrates [92], A patterned PDMS membrane was casted from lithographically structured photoresists and brought into close contact with the substrates (Fig. 6). When incubated with a solution of fibronectin (FN), adsorption of the cell-adhesion-mediating protein to the surface was restricted to the exposed areas. The membrane was peeled off and cells were seeded from a serum-free medium. Passivation to cell attachment of the untreated portions of the surface was achieved by adding 1% bovine serum albumin (BSA) to the cell-seeding medium, which... 

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