Biopolymers for Biosensors: Polypeptide Nanotubes for Optical

Chapter 22

Biopolymers for Biosensors: Polypeptide Nanotubes for Optical Biosensing 1

1

1

Hatice Duran , King Hang Aaron Lau , Anke Lübbert , Ulrich Jonas , Martin Steinhart , and Wolfgang Knoll Downloaded by COLUMBIA UNIV on August 5, 2012 | http://pubs.acs.org Publication Date: March 28, 2008 | doi: 10.1021/bk-2008-0977.ch022

1

2

1

1

Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany Max Planck Institute of Microstructure Physics, Weinberg 2, 06120 Halle, Germany 2

In this work, N-carboxy anhydride (NCA) monomer molecules were condensed on the pore walls of an initiator-coated nanoporous alumina template, leading to polypeptide (poly(gamma-benzyl-L-glutamate), PBLG) film formation. Three different ways were followed for peptide nanotube formation: NCA polymerization in solution, in melt and polymerization from surface-initiated vapor deposition. While the NCA monomer was polymerized within the pores, the wall thickness of the resulting polypeptide was tuned by changing the polymerization time. This polypeptide-coated alumina membrane will be used as planar optical waveguide to monitor both the changes in the refractive index andfluorescentsignals of the composite membrane through specific binding of a bioanalyte. We monitored for the first time the in-situ formation of an initiator layer (3-Aminopropyltriethoxysilane, APTE) inside the pores of an alumina membrane via optical wave guide spectroscopy. Attachment of initiator molecule effectively changed the dielectric constants of the interfaces, resulting in detectable shifts of the waveguide modes. We have previously demonstrated that unmodified nanoporous alumina waveguide sensor having a 10 times higher sensitivity than surface plasmon spectroscopy (SPR). The sensitivity may be further increased if the pores are coated with PBLG polypeptides, which has many functional sites on each polypeptide chain. © 2008 American Chemical Society In Polymers for Biomedical Applications; Mahapatro, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

371

Downloaded by COLUMBIA UNIV on August 5, 2012 | http://pubs.acs.org Publication Date: March 28, 2008 | doi: 10.1021/bk-2008-0977.ch022

372 Nowadays, carbon nanotubes (CNTs) are very attractive substances as potential building blocks for high technology applications. Many of these applications require both uniform dispersion of CNTs in solution and further functionalization of their inner or outer wall in order to have a useful signal which is also a prerequisite for the use in biosensor devices. A major concern about carbon nanotubes for biological applications is their potential toxicity unless they are functionalized or coated with a biocompatible agent (1). For example, research over the past several years has shown that, when inhaled, carbon nanotubes can accumulate in the lungs and cause inflammation. Polymeric nanotubes, on the other hand, can be potential candidates to overcome such severe obstacles. They are also regarded as promising building blocks for a multitude of applications. Polypeptides are polymers of exceptional interest because of the close relationship they bear to proteins. Synthetic polypeptides offer a unique opportunity for exploring the interrelation of these fundamental properties, which determine protein structure and function (2)\ their flexibility in functionality and their molecular-recognition properties distinguish them from other materials. The ability of peptide-based nanotubes to target specific cells makes them attractive for biomedical applications (e.g. smart drug-delivery). This type of nanotubes can be inserted into cell membranes and can mimic the ion channels in membranes (3). Furthermore, inner surfaces can be designed that have properties distinct from the outer surfaces. For example, one can fabricate them with hydrophobic outer surfaces and hydrophilic inner surfaces. Since polypeptide nanotubes are non-toxic to cells and they are biocompatible, this makes them even more valuable for future in vivo experiments in biosensor and biomoleculer filters applications. Peptide nanotubes could be formed via selfassembly by stacking cyclic peptide monomers (4). Self-assembled peptide nanostructures can be organized into nanowires, nanotubes, or nanoparticles via their molecular recognition function (4). Peptide rings are stacked through backbone-backbone hydrogen bonds between neighboring amide groups to form peptide nanotubes. Most recently, dendritic peptide monomers have been used to form polypeptide nanotubes (5) via hydrogen bonding. One of the disadvantages of nanotube formation using cyclic peptide monomers is that in order to produce larger-diameter peptide nanotubes, the ring diameters of the cyclic peptide monomers need to be larger. However, many cyclic monomers can only be synthesized between 10 to 20A in diameter to maintain a stable cyclic structure, and nanotubes need to be bundled to obtain larger nanotube diameters (6). In order to achieve a wide range of peptide-nanotube sizes, it may be more advantageous to fabricate them via template-directed synthesis. Martin et al. (7, 8) have pioneered template-assisted fabrication of hybrid nanostructures by combinjng the tools of synthetic chemistry and material science. They grew organic materials within the pores of anodic alumina templates, which are characterized by regularly arranged parallel pores with uniform pore depth

In Polymers for Biomedical Applications; Mahapatro, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.


373 (9, 10). Using this approach, one can now produce polymeric nanotubes in confined geometries for applications in biosensor technology. In the present work, poly(y-benzyl-Z-glutamate) (PBLG) polypeptide nanotubes have been fabricated for optical biosensor applications. This technique is based on the synthesis of nanostructures within the pores of a nanoporous membrane (7, 11). PBLG (12) is a polymer with non-linear optical properties. This polymer is attractive not only because of its large intrinsic dipole moments and birefringence but also because its ester side chains may be easily modified. So far, the tethering of polypeptides onto flat substrates (13) has been studied extensively. However, this concept still presents a challenge due to the low surface area coverage, resulting in a smaller amount of functional or reactive sites. Our technique overcomes these drawbacks. Its advantages include reproducibility, flexibility regarding the aspect ratios of the nanotubes, largescale production, and sharp distributions of length and diameter. Furthermore, the nanoporous structure of the alumina membrane results in a very high internal surface area. Consequently, we increased the effective surface density of functional peptide groups. This is particularly important since the sensitivity and the precise quantification of analyte molecules depend on the number of active biofiinctional groups. However, a problem still to be solved is the recycling of the alumina templates. Functionalities can be enriched in nanotubes through well-established chemical or non-covalent attachment of functional molecules (14) to the peptide nanotubes. This feature enables decoration of the inside and outside walls of peptide nanotubes with different functionalities, which provides the flexibility to further control the nanotube structure and behavior at the molecular level. The hollow structure of nanotubes offers functionalisation and/or templating various molecules inside the nanotubes. These nanotubular polypeptides can be further designed to have distinct functional groups (-COOH or -NH ) depending on the analyte of interest. Furthermore, debenzylated polypeptide nanotubes act as polyelectrolytes and might be useful for monitoring changes in pH for in vivo cell studies. The polypeptide nanotubes embedded in the robust alumina template form a mechanically stable biosensor membrane so that aggregation and mechanical deformation of the nanotubes are prevented. In this way, polypeptide probes have a sufficiently long lifetime and during this period it is easy to functionalize and calibrate them. 2

Experimental Materials and Templates The N-carboxy anhydride (NCA) monomer of PBLG was synthesized by phosgenation of L-glutamic acid (Merck Biosciences) with stoichiometric


374 quantities of triphosgene (Sigma-Aldrich) in dry ethyl acetate (Fluka) (15). Then, the monomer was purified two times by recrystallization. 'H-NMR spectra were recorded on a Bruker Dpx250 spectrometer, using the residual proton resonance signal of the deuterated dichloro methane (CD C1 ) as the internal standard. H NMR (CD C1 250 MHz): 5 (ppm): 2.17 (m, p-CH ), 2.52 (m, y-CH ), 4.32 (t, C-H), 5.14 (s, -CH -benzylic), 6.31 (s, N-H), 7.41 (s, Ar-H) 3-Aminopropyltriethoxysilane (APTE) (Sigma-Aldrich) was used as initiator. The initiator was dried and distilled prior to usage for better polymerization. For substrate directed growth of PBLG, it is immobilized in the alumina pores by liquid phase silanization as described below. For the polymerization of NCA monomer in a solvent for PBLG powder: In a dry box, NCA was dissolved in DMF (150-200mg NCA per ml solvent) and placed inside a syringe attached with a Rotilator-Syringe filter (pore diameter 5mn) on it. Then, transferred in a 25mL Schlenk tube from the top which was sealed with a Teflon stopcock. An aliquot of APTE was rapidly added via syringe to the flask, while continuously stirring the monomer solution. A stirrbar was added and the flask was sealed, removed form the dry box, and stirred in a thermostated bath at 25°C for 5 days. Polymer was isolated by addition of the reaction mixture as small drops to a distilled water flask (the amount of water is X10 that of the solution) causing precipitation of the polymer. The polymer was then dissolved in THF and reprecipitated by addition to water. The white precipated PBLG wasfilteredand dried in vacuo overnight to give a white solid. *H NMR (DMSO, 500 MHz at 323K): 8 (ppm): 2.06 (m, P-CH ), 2.28 and 2.48 (m, y-CH ), 3.95 (t, C-H), 5.03 (s, -CH -benzylic), 7.26 (s, Ar-H), 8.25 (s, N-H). C NMR (DMSO, 500 MHz at 323K): 8 (ppm): ™ ™ 'P-CH ), 29.50 (yCH ), 57.38 (-CH -benzylic), 64.86 (C-H), 127.17, 128.18, 135.48 (Ar-H), 170.88, 174.32 (C=0). The number average molecular weight (83,200 g/mol with a degree of polymerization of 380 and an average chain length of 57nm) and polydispersity index (1.73) were measured with GPC in DMF against standard PS. Silicon substrates (Si-Mat, double side polished) were cleaned by immersing them in a mixture of N H (8 mL, 25%), H 0 (8 mL, 35%) and Millipore water (100 mL) at 80-85 °C, for 20-25 min. Afterwards, the substrates were rinsed with copious amount of Millipore water and dried under N . Self-ordered porous alumina templates (pore diameter ranges from 25nm to 380nm; pore depth isfrom5um to lOOum) were prepared according to protocols reported elsewhere (16, 17). 2

2

!

2

2

2

2


2

2

2

2

,3

2

2

2

3

2

2

2


375 Deposition of Thin Alumina Films on LaSFN9 Substrates for Optical Waveguide Studies Glass substrates (LaSFN9, Hellma Optik, Halle) were cut into 25 mm x 25 mm pieces and sonicated in 2% Hellmanex solution (Hellma Optik). Before aluminum deposition, 3 nm Cr followed by 45 nm Au were deposited as an optical coupling layer by thermal evaporation, both at a rate of 0.1 nm/s and at -lxlO" mbar (BOC Edwards Auto300). A l (500-1000 nm) was deposited by DC magnetron sputtering (BOC Edwards Auto500) in an Ar plasma, at a rate of 1 nm/s/A using the following conditions: 0.7 A (300 W) or 1.4 A (600 W), process pressure at 3xlO" mbar, base pressure at

Biopolymers for Biosensors: Polypeptide Nanotubes for Optical

Recommend Documents