BA8-35 suffers from the disadvantage that high levels of non-specific binding can be observed
when the ligands to be tested are cationic or expose cationic patches. This has led to the development
of a biotinylated non-ionic APol (Ferrandez et al. 2014), whose use will be described in § 13.2.3.
In § 13.3, we will consider the use of alternative tags. Right now, we will turn to Study 13.2 and the
possibility of using tagged APols in multiplexing experiments, taking BA8-35 as a prototype.
13.2.2 Multiplexing
The development of protein arrays with micrometer features is of great interest because of their
potential applications in life science and biomedical diagnostics (Christman et al. 2006a; Wingren
and Borrebaeck 2007). Indeed, this technology can provide high-throughput screening of protein
functions and biomolecular interactions while requiring very small amounts of analytes and short
processing times (Bano et al. 2009). Further miniaturization of protein arrays to the nanometer scale
could further improve detection sensitivity and give access to specific biomolecular reactions that are
not accessible to micrometer-scale structures (Coyer et al. 2007). Arrays of proteins with micrometer
and/or submicrometer features have been fabricated using patterning techniques such as microcontact
printing (Coyer et al. 2007; Hoff et al. 2004); scanning near-field optical (Leggett 2007), dip-pen (Lee
et al. 2002), and photo- and ion beam lithography (Kannan et al. 2004; Christman et al. 2006a, b;
Zhang et al. 2007); atomic force microscopy (AFM) (Gu et al. 2004), and electrochemical-based
approaches (Hoover et al. 2007). However, only a handful of examples of anchoring simultaneously
multiple soluble proteins onto nanoarrays has been reported (Christman et al. 2006b), and, to date,
none of the techniques mentioned above has been applied to nanopatterning multiple purified MPs
(cf. Valiokas et al. 2006). Whereas there is a strong interest in the production of MP micro- and
nanoarrays for both basic and applied research, the insolubility of MPs in surfactant-free aqueous
solutions and their instability in the presence of detergents strongly complicate the fabrication of MP
biochips (Bieri et al. 1999; Fang et al. 2002; Hong et al. 2005; Majd and Mayer 2008). Techniques
involving drying steps and/or harsh conditions such as high temperatures and mechanical shear may
denature MPs (Fang et al. 2002). Furthermore, as noted in Chap. 2, MP stability often depends on the
retention of associated lipid molecules, which is difficult to ensure in the presence of detergents. This
requirement sets further constraints on the use of the solvents that are normally used to prevent
evaporation and keep the protein hydrated in scanning probe lithography techniques (Zheng et al.
2009). All of these problems can be circumvented or, at least, mitigated by the use of APols.
Study 13.2 describes a novel strategy to fabricate micro- and nanoarrays carrying multiple MPs,
based on the combined use of BA8-35 and of the selective functionalization of gold electrodes by
electropolymerization of a biotin-doped polypyrrole (PPy-biotin) film. The electrosynthesis of
conducting polymers allows the controlled and reproducible functionalization of electrodes at the
micro- and nanoscale (Cosnier et al. 1998). Pyrrole functionalized with different biotin groups has
been synthesized (Cosnier et al. 1998, 1999; Torres-Rodriguez et al. 1998; Yang et al. 1998; Darmanin
et al. 2012), and polymeric films have been prepared and used as a support for the immobilization of
biotin groups to electrode surfaces (Cosnier et al. 1998, 1999; Bidan et al. 2000). After incubation with
avidin, biotin-tagged biomolecules can be anchored to the free sites of avidin to construct biosensors
(Cosnier et al. 1998). As biotin is negatively charged under the conditions used for polymerization of
PPy, it can also be incorporated in the polymeric film when used as a doping anion. This method is
particularly appealing, as it does not require any chemical synthesis. In Study 13.2, it has been used to
establish a straightforward approach to MP multiplexing on gold micro- and nanoelectrodes. As
schematically illustrated in Fig. 13.5, the key to the approach is the selective electropolymerization
of conducting surfaces with a PPy film containing biotin, followed by the immobilization of avidin,
neutravidin, or SA and the binding of BA8-35-trapped MPs.
600
13 Amphipol-Mediated Immobilization of Membrane Proteins and Its Applications
when the ligands to be tested are cationic or expose cationic patches. This has led to the development
of a biotinylated non-ionic APol (Ferrandez et al. 2014), whose use will be described in § 13.2.3.
In § 13.3, we will consider the use of alternative tags. Right now, we will turn to Study 13.2 and the
possibility of using tagged APols in multiplexing experiments, taking BA8-35 as a prototype.
13.2.2 Multiplexing
The development of protein arrays with micrometer features is of great interest because of their
potential applications in life science and biomedical diagnostics (Christman et al. 2006a; Wingren
and Borrebaeck 2007). Indeed, this technology can provide high-throughput screening of protein
functions and biomolecular interactions while requiring very small amounts of analytes and short
processing times (Bano et al. 2009). Further miniaturization of protein arrays to the nanometer scale
could further improve detection sensitivity and give access to specific biomolecular reactions that are
not accessible to micrometer-scale structures (Coyer et al. 2007). Arrays of proteins with micrometer
and/or submicrometer features have been fabricated using patterning techniques such as microcontact
printing (Coyer et al. 2007; Hoff et al. 2004); scanning near-field optical (Leggett 2007), dip-pen (Lee
et al. 2002), and photo- and ion beam lithography (Kannan et al. 2004; Christman et al. 2006a, b;
Zhang et al. 2007); atomic force microscopy (AFM) (Gu et al. 2004), and electrochemical-based
approaches (Hoover et al. 2007). However, only a handful of examples of anchoring simultaneously
multiple soluble proteins onto nanoarrays has been reported (Christman et al. 2006b), and, to date,
none of the techniques mentioned above has been applied to nanopatterning multiple purified MPs
(cf. Valiokas et al. 2006). Whereas there is a strong interest in the production of MP micro- and
nanoarrays for both basic and applied research, the insolubility of MPs in surfactant-free aqueous
solutions and their instability in the presence of detergents strongly complicate the fabrication of MP
biochips (Bieri et al. 1999; Fang et al. 2002; Hong et al. 2005; Majd and Mayer 2008). Techniques
involving drying steps and/or harsh conditions such as high temperatures and mechanical shear may
denature MPs (Fang et al. 2002). Furthermore, as noted in Chap. 2, MP stability often depends on the
retention of associated lipid molecules, which is difficult to ensure in the presence of detergents. This
requirement sets further constraints on the use of the solvents that are normally used to prevent
evaporation and keep the protein hydrated in scanning probe lithography techniques (Zheng et al.
2009). All of these problems can be circumvented or, at least, mitigated by the use of APols.
Study 13.2 describes a novel strategy to fabricate micro- and nanoarrays carrying multiple MPs,
based on the combined use of BA8-35 and of the selective functionalization of gold electrodes by
electropolymerization of a biotin-doped polypyrrole (PPy-biotin) film. The electrosynthesis of
conducting polymers allows the controlled and reproducible functionalization of electrodes at the
micro- and nanoscale (Cosnier et al. 1998). Pyrrole functionalized with different biotin groups has
been synthesized (Cosnier et al. 1998, 1999; Torres-Rodriguez et al. 1998; Yang et al. 1998; Darmanin
et al. 2012), and polymeric films have been prepared and used as a support for the immobilization of
biotin groups to electrode surfaces (Cosnier et al. 1998, 1999; Bidan et al. 2000). After incubation with
avidin, biotin-tagged biomolecules can be anchored to the free sites of avidin to construct biosensors
(Cosnier et al. 1998). As biotin is negatively charged under the conditions used for polymerization of
PPy, it can also be incorporated in the polymeric film when used as a doping anion. This method is
particularly appealing, as it does not require any chemical synthesis. In Study 13.2, it has been used to
establish a straightforward approach to MP multiplexing on gold micro- and nanoelectrodes. As
schematically illustrated in Fig. 13.5, the key to the approach is the selective electropolymerization
of conducting surfaces with a PPy film containing biotin, followed by the immobilization of avidin,
neutravidin, or SA and the binding of BA8-35-trapped MPs.
600
13 Amphipol-Mediated Immobilization of Membrane Proteins and Its Applications