15-11-2012, 05:13 PM
Biosensors Based on Biological Nanostructures
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Introduction
The term biomaterials is attributed to the materials employed to medical applications, such as
ceramic implants and biopolymer scaffolds, as well as a variety of composites (Hauser e
Zhang, 2010). In recent decades, researchers of distinct subjects have gathered efforts in
developing new biomaterials for applications in various branches of medicine. With the
advent of molecular biology and biotechnology, and knowing that many of these
biomaterials are not specific for medical applications, studies have been directed to directed
towards to biological and biomimetic materials preparation biological and biomimetic
materials (Sanchez, Arribart et al., 2005; He, Duan et al., 2008; Aizenberg e Fratzl, 2009).
In this new class of materials, the peptide compounds appear as promising candidates to
building blocks due to their easy preparation and physical and chemical stability (Cheng, Zhu
et al., 2007). Thus, we can propose different peptide sequences and from their selforganization
to obtain structures with different geometries (spherical, cylindrical, conical)
and even nanotubes and/or nanofibers (Hirata, Fujimura et al., 2007) are obtained.
Peptide nanomaterials form supramolecular structures which are interconnected by
intermolecular interactions such as van der Waals forces, electrostatic, hydrophobic and
hydrogen bonds, among others (Cheng, Zhu et al., 2007; Colombo, Soto et al., 2007). Due to
these characteristics, crystal engineering of supramolecular architectures has rapidly
expanded in recent years, mainly due to the possibility of intermolecular interactions,
structural diversity and potential applications (Sanchez, Arribart et al., 2005; Cheng, Zhu et
al., 2007; He, Duan et al., 2008; Aizenberg e Fratzl, 2009). This structural variety is possible
due to the planning and construction of supramolecular architectures, as promising building
blocks that allow the design of functional molecular materials that will display some sort of
ownership of technological interest (Sanchez, Arribart et al., 2005; Cheng, Zhu et al., 2007;
He, Duan et al., 2008; Aizenberg e Fratzl, 2009).
Peptide-based nanostructures
The formation of tubular peptide nanostructures has been performed using several different
peptide sequences, such as heptapeptide CH3CO-Lys-Leu-Val-Phe-Phe-Ala-Glu-NH2, (Lu,
Jacob et al., 2003) and dipeptides +NH3-Phg-Phg-COO- (Reches e Gazit, 2004) and +NH3-Phe-
Trp-COO- (Reches e Gazit, 2003). The first peptide nanotubes were obtained by M.R.
Ghadiri and co-workers from cyclic compounds (Ghadiri, Granja et al., 1993). The L-amino
acids are the most used building blocks. However, D-amino acids can also self-assemble to
form nanofibers similar to those obtained from L-amino acids (Poteau e Trinquier, 2005).
The properties of peptides can be modified through changes in the sequence of amino acid
residues used in their preparation, providing a highly relevant factor in building these new
systems (Poteau e Trinquier, 2005). Such changes were reported in a study by varying the Damino
acids (D-Alanine, D-Leucine and D-phenylanine) to obtain different peptide
nanotubes (De Santis, Morosetti et al., 2007). It was observed that by employing enantiomers
(D, L) the possibility of obtaining different supramolecular systems arises, with possible
changes in their structural and electronic properties (De Santis, Morosetti et al., 2007).
One of the most commonly used peptides in synthesis of nanotubes is +NH3-Phe-Phe-COO-
.These nanotubes exhibit several unique properties such as high uniformity along the entire
length of the tube, biocompatibility, stability against various solvents and thermal stability.
In this sense, there are several studies that investigate the structural control of the nanotubes
by changing variables such as temperature, solvent and pH (Adler-Abramovich, Reches et
al., 2006).The +NH3-Phe-Phe-COO- nanotubes maintain their morphology up to 200º C, and
total degradation or loss of tubular morphology occurs between 200 and 300º C (Ryu e Park,
2010). The thermal stability has been attributed to π-stacking interactions among aromatic
residues that mediate the formation of structures (Reches e Gazit, 2003). The investigation of
stability in different organic solvents shows that the nanotubes do not suffer morphological
changes after treatment in ethanol, methanol, 2-propanol, acetone and acetonitrile (Adler-
Abramovich, Reches et al., 2006).
Preparation methods of peptide nanostructures
Obtaining nanostructures in liquid phase
The liquid phase method for obtaining nanostructures is divided in two steps. To obtain a
nanostructure based on (+NH3-Phe-Phe-COO-), for example, the first step is the dissolution of the compound in an organic solvent (1,1,1,3,3,3-hexafluoro-2-propanol, HFP) at a
concentration of 100mg mL-1. In the second step, nanostructures are obtained in a
spontaneous process, after the dilution in water to achieve 2mg mL-1 of concentration. By
this protocol, +NH3-Phe-Phe-COO- self-assemble as nanotubes of 80 to 300 nm thick.
The self-assembling mechanism in which nanotubes are produced is not yet fully
understood. However, the most acceptable explanation suggests that the π-π stacking
interactions and hydrogen bonds between aromatic rings are responsible for the material
nano-organization (Reches e Gazit, 2003).
Nanostructure preparation in solid-vapor phase
Peptide nanostructures have been prepared by self-assembly oriented in the solid-vapor
phase method, which consists of using two solvents, one to solubilize the peptide and
another one to encourage the nanostructure assemble. Based on the bottom-up strategy, the
first step consists on the formation of a peptide film onto substrate surface (usually silicon),
with posterior evaporation of the solvent in the absence of humidity. In this case, the
peptide film is referred to as the solid phase. The next step consists of keeping the solid film
in a vapor solvent atmosphere, the commonly called vapor phase. Parameters like
temperature, vapor pressure, concentration of solid film and exposure time of the film to
vapor solvent govern the nanostructure formation.
Ryu et al. described this methodology as the one to obtain 1D nanostructures (Ryu e Park,
2008b; a). The authors studied the influence of temperature and water activity of a solution
containing metallic salts in the nanostructures formation and they reported that
nanostructures are formed at high water activity, while for activity values lower than 0.3, no
nanostructures were obtained. Also, it was observed that nanostructures were only achieved
at a working temperature of 100 to 150 °C. Fig. 1 shows the experimental schematic process
to prepare nanowires or nanotubes in solid-vapor phase.
Obtaining nanostructures for physical vapor deposition
Recently, +NH3-Phe-Phe-COO- nanotubes were obtained vertically oriented, employing the
physical vapor deposition technique (Fig. 3) (Adler-Abramovich, Aronov et al., 2009). Size
and quantity of peptide nanotubes were controlled through deposition parameters
adjustment such as time, solvent of preparation, temperature and distance between
substrates. The peptide nanotubes formation using this technique became possible because
of the low molecular weight and high volatility of precursor species. In a typical synthesis,
the +NH3-Phe-Phe-COO- is heated at 220°C in a vacuum chamber containing a clean
substrate, heated at 80°C, that is located at the top of the chamber. The nanotubes formed
exhibit length of hundreds of micrometers and diameters of 50 to 300nm, with morphologies
similar to those from the liquid phase. This method has been employed in the fabrication of
electronic devices, such as capacitors, but it can be used in the modification of electrodes for
electrochemical uses.
Electrospinning
The electrospinning technique is a technology that uses a high tension electric field (5-50kV)
and low currents (0.5-1μA) to obtain 1D nanostructures. In this process a fluid material is
accelerated and drawn trough an electric field producing structures with reduced diameters.
In the work of Singh et al. (Singh, Bittner et al., 2008) +NH3-Phe-Phe-COO- nanotubes were
prepared from solution in HFP. Then, this solution was diluted in water to 2.9 mmol L-1 of
concentration and sonicated for 1 hour. Variations in the obtaining parameters of the
nanostructures, like electric field, concentration, and flow injection speed on silicon wafer
were investigated and their influence on the nanostructure formation was reported.