Plastic Antibodies - Designing Receptors for the Next Generation of Biosensors - page 105

at low and high pH and at high temperature [ 14 - 16 ]. All these factors may alter
their recognition properties, thus shortening their shelf-life [ 17 , 18 ]. Finally,
biomolecules may be difficult to immobilize on suitable supports for use in assays
and sensors [ 15 , 19 ], which is an extremely important feature for developing
diagnostic devices. For all these reasons, other affinity tools, such as engineered
binding proteins, aptamers, and molecularly imprinted polymers (MIPs), have
gained interest as potential antibodies substitutes. Their attractive features include
enhanced stability, efficient selection and screening procedures, and cost-effective
production methods. In the following sections we provide a brief overview of
engineered binding proteins, aptamers, and MIP nanoparticles as affinity tools for
different applications such as diagnostics, therapeutics and drug delivery, as well as
separation and catalysis.
1.1 Engineered Binding Proteins
As already stated above, the recognition ability of antibodies relies on a limited
variable region structurally embedded in a more conserved framework. In the same
way, proteins capable of binding to a certain target might be selected from a random
library, characterized by a constant structural peptide framework and randomized
variable binding regions. However, a large amount of peptide derivatives have to be
generated and screened to successfully design and engineer a binding protein
scaffold. This can be achieved by performing high-throughput screening based on
molecular display technology, which establishes a physical link between phenotype
and genotype. The most commonly used display technology is phage display in
which genes encoding proteins of interest are fused to a gene that encodes a phage
coat protein. In this way, phage particles can be made to display peptides of interest
on their surface [ 20 , 21 ]. Escherichia coli ( E. coli ) cells are infected with the
members of the phage library to produce many copies of each of the library
members displaying the variant proteins. This library is screened against the
immobilized target molecule and the phages with appropriate specificity and
affinity are separated and collected in a process known as biopanning. The collected
high-affinity phages are used to re-infect E. coli cells and the process is repeated
iteratively (usually three to five rounds) using more stringent washing steps.
Eventually, monoclonal phages are selected, and the high-affinity protein scaffolds
identified by sequencing the DNA of the corresponding phage [ 6 ]. Scaffolds that
have good stability are required to have a sufficiently long shelf-life, which is
important from a commercial point of view. Among the successful examples of
engineered proteins there are fibronectin type III domain, which has a certain
degree of similarity with the structure of an immunoglobulin G variable domain,
and designed ankyrin repeat proteins (DARPins) [ 22 - 24 ]. However, developing
scaffolds for a certain application is not easy; in particular due to the unpredictable,
costly, and time-consuming nature of the screening procedure, hence the commer-
cialization of these products is still at early stage [ 6 ].