Titania Binding Peptides as Templates in the Biomimetic Synthesis of

Subscriber access provided by Mount Allison University | Libraries and Archives

Article

Titania Binding Peptides as Templates in the Biomimetic Synthesis of Stable Titania Nanosols: insight into the role of buffers in peptide mediated mineralization Valeria Puddu, Joseph M Slocik, Rajesh R. Naik, and Carole C. Perry Langmuir, Just Accepted Manuscript • DOI: 10.1021/la401777x • Publication Date (Web): 01 Jul 2013 Downloaded from http://pubs.acs.org on July 4, 2013

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Titania Binding Peptides as Templates in the Biomimetic Synthesis of Stable Titania Nanosols: insight into the role of buffers in peptide mediated mineralization. Valeria Puddu1, Joseph M. Slocik2, Rajesh R. Naik2, Carole C. Perry1*. 1

Interdisciplinary Biomedical Research Centre, School of Science and Technology, Nottingham

Trent University, Clifton Lane, Nottingham NG11 8NS, United Kingdom 2

Materials and Manufacturing Directorate, Air Force Research Laboratory, Wright-Patterson Air

Force Base, Ohio 45433, United States KEYWORDS: Titania, peptides, bio-inspired synthesis, capping agent.

ABSTRACT: In this contribution we report the unusual behavior of two peptides (Ti-1 (QPYLFATDSLIK) and Ti-2 (GHTHYHAVRTQT)) with high affinity for titania that efficiently promote titania mineralization from an aqueous titanium bisammonium lactatodihydroxide (TiBALDH) solution, yielding small (ca. 4 nm) titania nanoparticles. As a result, we were able to produce for the first time using a biomimetic approach; highly stable sub-10-nm titania sols. Both sequences show a high titania mineralization activity per unit peptide concentration and a

ACS Paragon Plus Environment

1

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 38

capacity to control particle size and stabilize nanoparticles through specific surface interactions. We also show that phosphate ions disrupt the controlled particle formation and stabilization achieved in the presence of the two peptides. The products obtained from phosphate buffered solutions are titanium containing materials (not pure oxide) with poor morphological control similar to those previously reported by others. Our results provide important insights into understanding the mechanism of titania mineralization in a range of different aqueous media (water, Tris and Phosphate buffer).

INTRODUCTION. The capacity of (bio)molecules to guide and control the formation of (bio)minerals by specifically tailoring their properties and functions is commonly used in Nature.1 Synthetic approaches that mimic mineralization in living systems at neutral pH and room temperature present a promising and sustainable alternative to conventional synthetic methods for the preparation of inorganic materials.2-5 Nanostructured titania (TiO2) is a very attractive semiconductor material due to its optical and electrochemical properties and finds widespread application as a white pigment, catalyst support, as a photo catalyst, in self-cleaning coatings and in solar energy conversion devices.6 As the specific technological properties of TiO2 are defined by its physical properties (e.g.: particle size, crystallinity and morphology) synthetic routes that allow for a fine control of such properties are sought after. At the industrial scale, multimillion tons per year7 of powdered titania is produced by flame oxidation of purified titanium tetrachloride (TiCl4) at high temperatures (1500-2000 K).8 Other traditional synthetic routes to colloidal TiO2 involve hydrolysis and condensation reactions under extreme pH or non–aqueous media are needed to control reaction rates and subsequently the size and shape of the final product.9-11 Such synthetic paths lead to poorly


2

Page 3 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

crystalline crude products that require harsh conditions of temperature or pressure to be converted to crystalline phases with the required technological properties.6 The addition of amino acids to traditional condensing systems12,13 has been used to modify phase distribution, size, shape and surface roughness of titania. In this scenario, the appeal and potential of a bio-inspired approach is clear in that, under benign conditions of temperature and pH material formation could be controlled using (bio)molecules as mineralization mediators. Biomimetic synthesis approaches reported in the literature use titanium bisammonium lactatodihydroxide (TiBALDH), a water stable titanium complex as precursor.14-19 The most remarkable example of such an approach to titania synthesis is that of Kröger and colleagues that used silaffins, proteins involved in the biomineralization of silica in diatoms, to induce mineralization of rutile particles characterized by a complex hierarchical architecture.14 The complexity of the silaffin molecular structure and cooperative effects between the protein domains was considered responsible for the specific rutile crystallization and for the level of hierarchical organization achieved.14 Other biomimetic syntheses starting from TiBALDH at ca. neutral pH in the presence of smaller, less structurally complex biomolecules such as peptides17, 19 and amino acids,12,13 or bioinspired polymers like polyamines15,16 also yield titania-based materials. However, addition of these simpler (bio) molecules was found to provide no significant morphological and structural control over the final material. The products formed were highly aggregated materials with particle size in the range from 500 nm to several microns in diameter. Furthermore, (bio)molecules, and/or other ions present in the mineralization system (e.g. phosphate ions), are often trapped within the mineral phase during precipitation, thereby forming inorganic-organic composite materials13,15,20 rather than the pure oxide. This lack of control over morphology and


3

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 38

composition represents a serious drawback compared to the traditional synthesis method which can readily lead to pure colloidal nanoparticles as small as 2 nm.21 A major challenge in the improvement of biomimetic synthetic strategies for titania is understanding the mechanism of (bio)mineralization. Since several (bio) molecules capable of precipitating silica are also able to precipitate titania, a mechanism for titania formation has been rationalized by comparison with that of silica formation. For polyamines16 and peptides,17,19 a general correlation between TiO2 mineralization activity and the number of net positive charges per molecule has been observed. Peptide affinity to a TiO2 surface is a critical but not sufficient factor to guarantee titania mineralization as local structure and peptide conformation have been recently shown to be important in defining precipitation activity and reaction kinetics.22 Also, although buffer chemistry and buffer concentration can influence both the mineralization activity of the biomolecules themselves and the morphology of precipitates formed, the role played by ions present in buffered solutions (such as phosphate or citrate) has not been widely considered in the process of biomolecule mediated titania formation. To date, biomolecules with the ability to promote titania mineralization and efficiently control particle growth at physiological conditions have not yet been reported.22 In this contribution we report the unusual behavior of two peptides with high affinity for titania that not only efficiently promote titania mineralization from an aqueous TiBALDH solution, but also effectively control the morphology and size of the precipitates. As a result, we were able to produce for the first time using a biomimetic approach; a highly stable titania sol made of ca. 4 nm nanoparticles. We investigated the role played by these peptides at different stages of the mineralization process obtaining experimental evidence for peptide-mineral precursor interaction in solution during the early stages of mineral formation. We also show that controlled particle


4

Page 5 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

formation and stabilization achieved in the presence of the two peptides is disrupted by the presence of phosphate ions in the mineralization medium, providing important insights into understanding the mechanism of titania mineralization in phosphate containing media as reported by others.15-17,19

EXPERIMENTAL Titania binding peptides were identified by biopanning using a Ph.D.-12 phage display library (New England Biolabs) on a titania powder target from Sigma Aldrich (CAS#26,849-6) following the standard protocol for biopanning. Sequences Ti-2 and R5 were synthesized in house by microwave assisted solid state synthesis using an automated

peptide synthesizer

(CEM), and characterized by HPLC (purity >90%) and Mass spectroscopy. Sequence Ti-1 was obtained from Pepceutical (purity >85%). For titania mineralization 125 µL of 1M TiBALDH was added to 2.375 ml of a 0.4 mg/ml solution of titania binding peptide in Tris buffer (100 mM, pH 7.4), phosphate buffer (150mM, pH 7.4) or distilled water (pH adjusted to 7.4 with NaOH 0.01 M). Mineralization kinetic profiles were obtained by adding aliquots (10 or 20 µL) taken at regular time intervals from the mineralization mixture to 280 µL of 5 mM Tiron in acetate buffer at pH 5. The adsorption at 380 nm was measured using a Tecan plate reader after equilibration for one hour. The concentration of TiIV in solution was calculated by comparison with a calibration curve. Blank and control experiments were carried out in absence of peptide and in the presence of R5, respectively, at the same conditions reported above. Other experiments where the amount of peptide, buffer or TiBALDH concentration were altered in a systematic


5

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 38

manner whilst keeping all other reagent concentrations constant were also performed in triplicate. All kinetic profiles were fitted with first and second order exponential decay equations. The first order exponential equation was: 0

Eq. 1

Where: y0 represents the TiBaldH % at the endpoint, A is the amplitude and k is the rate constant. The second order exponential equation was: 0 1 2

Eq. 2

The second order exponential decay is compatible with a double step mechanism where k 1 and k

2

represent the rate constants of the fast and slow process, respectively; A1 and A2 represent

the corresponding amplitudes; and y0 represents TiBaldH % at the endpoint. The parameters in the above equations were estimated by nonlinear regression analysis using Origin software and the F-test was used to establish the best fitting model for the experimental data. Samples were dialysed, lyophilized and characterized by Dynamic Light Scattering using a NanoS Zetasizer (Malvern Instrument). TEM images were acquired on a Phillips CM200 transmission electron microscope operating at 200 kV using a single tilt holder. Electron diffraction patterns were obtained in diffraction mode and adjusted for eucentric height at a distance of 360 mm, beam spot size of 7, and 0.5 sec exposure time. Diffraction patterns were referenced to a 10 nm gold nanoparticle sample (Ted Pella) at same eucentric height and parameters. Samples were prepared by depositing 10 µL of peptide-titania solution onto a 3 mm copper grid coated with carbon film (Ted Pella) with grids from Quantifoil (R 1.2/1.3) being used to collect the HRTEM images.


6

Page 7 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

CD spectra were recorded on freshly prepared samples on a Jasco J-815 CD spectrometer using 0.5mm path length quartz cuvettes. For samples in H2O and PBS, peptide solutions were 0.5mg/ml, for samples in the TiBALDH experiment, peptide solutions were 2.48mg/ml and TiBALDH dilution was 1:202. QCM analysis of peptide binding was performed using a Q-sense E4 QCM-D quartz crystal microbalance with flow modules. QCM sensors coated with 100 nm titanium (Q-sense, QSX310) were cleaned by UZ/ozone treatment (Novascan PSD Pro Series Digital UV/Ozone system) for 10 minutes, immersion in a 2% SDS solution for 30 minutes, thorough rinsing with deionized water, N2 drying, and another UZ/ozone treatment for 10 minutes. After cleaning, sensors were mounted in QCM flow modules, while Ti-1 and Ti-2 peptides (10 µg/mL) were flowed across sensors at 0.17 mL/min at 23°C. The 3rd overtone frequency was measured and used to calculate the adsorbed mass of peptide using the Sauerbrey equation.

RESULTS AND DISCUSSION. A combinatorial method was used to identify two novel sequences: Ti-1 (QPYLFATDSLIK) and Ti-2 (GHTHYHAVRTQT) by screening a phage display library (Ph.D.-12, New England Biolabs) against a commercial titania powder sample. The amino acid composition of Ti-1 and Ti-2 (Table 1) is enriched in basic (K, H) and polar (Y, S, T) residues. These characteristics are consistent with those of previously reported titania binding peptides isolated by phage display.19,22 We studied Ti-1 and Ti-2 mineralization activity from a TiBALDH solution and compared it with that of peptide R5 (SSKKSGSYSGSKGKRRIL), the repeat unit of silaffin, which has previously been shown to induce the mineralization of titania.14,16 Mineralization reactions were


7

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 38

first studied at pH 7.4 in water (non-buffered system) and Tris buffer (TB) (buffered systems), from a 50mM TiBALDH solution exposed to peptide concentrations of 0.4 mg/ml (0.28 mM for Ti-1 and Ti-2 and 0.20 mM for R5).

Table 1. Amino acid composition and properties of the peptides identified by phage display against a titania substrate.

Peptide Sequence

Peptide composition acidic

polar

basic

pI*

Net charge @pH 7.4

Ti-1

QPYLFATDSLIK

1

3

1

6.2

0

Ti-2

GHTHYHAVRTQT

0

4

4

9.4

1.3

The kinetics of TiIV depletion was monitored for 90 minutes using a miniaturized colorimetric assay in a 96-well plate, based on the formation of a yellow complex between TiIV and Tiron.23 After 90 minutes reaction, both water and TB solutions were clear with no visible precipitate. The reaction profiles (Figure 1, A and B) show that, as expected, in the absence of peptides precursor solutions were hydrolytically stable in both water and TB. Also, DLS on the 50 mM TiBALDH solutions prior to addition of peptide showed a flat baseline, in contrast with what has recently been reported for more concentrated precursor solutions.24 The presence of peptide R5 did not induce significant precursor disappearance while binding peptides Ti-1 and Ti-2 were found to induce a rapid depletion of TiIV from both the buffered (TB) and the non-buffered (water) solutions. Ti-1 and Ti-2 show a slight difference in the total mineralization activity after 90 minutes, with a total 25% and 30% precursor depletion respectively (Figure 1 A and B). In vitro mineralization systems commonly use phosphate (or phosphate/citrate) buffers.15-17,19 To


8

Page 9 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

enable comparison with current literature on the biomimetic formation of titania, we also studied the kinetics of titania mineralization in phosphate buffer (PB). In the presence of phosphate ions a white precipitate was visible within minutes after TiBALDH was added to the peptide solutions.

Figure 1. Kinetics of TiIV disappearance from a 50 mM TiBALDH solution in A) H2O; B) Tris buffer 150 mM; and C) Phosphate buffer 150 mM (all solutions at pH 7.4). Symbols indicate: (○) blank,

(•)Ti-1

(QPYLFATDSLIK);

(∆)

Ti-2

(GHTHYHAVRTQT);

and

()

R5

(SSKKSGSYSGSKGKRRIL). Additives are present at a concentration of 0.20 mM (R5) and 0.28 mM (Ti-1 and Ti-2).

Remarkably, at our experimental conditions, phosphate buffer alone (blank) is sufficient to initiate the condensation of titania from the TiBALDH solution. Binding peptides Ti-1 and Ti-2 were found to induce precursor depletion at comparable rates and efficiency reaching a maximum conversion of 45% after 90 minutes (Figure 1C); while sequence R5 was able to convert 60% of the initial precursor after 90 minutes. The presence of phosphate buffer has already been shown to significantly improve the mineralization activity of peptides such as R5,


9

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 38

although it should be noted that µm sized Titanium phosphate precipitates are generated instead of pure oxide nanoparticles.15

All kinetic profiles were fitted by a first and a second order exponential decay, respectively (see Eq. 1 and 2 in the Experimental section). An exemplar fitting for Ti-1 in water is reported in the Supplementary Information (Figure S1). The experimental data were much better described by the second order exponential equation than the first order equation (over 99% confidence using the F-test). This is in agreement with data reported in the literature when spermidine and spermine were used as catalysts in the mineralization of titania.16 The analysis of the kinetic profiles for mineralization in water, TB and PB show that for a given peptide, the profiles and kinetic constants, show different activity towards precursor depletion in the presence of phosphate ions, Table 2.

Table 2. Values of k1obs and k2obs and correlation coefficients obtained from the two-exponential decay fitting of kinetic data (as defined in Eq. 2) relative to TiBALDH conversion in different mineralization systems (PB, Tris and water) and in the presence of different peptides. Mineralization in water and Tris buffer occurs only in the presence of binding peptides Ti-1 and Ti-2. A - symbol is used to represent no mineralization.

PB Blank R5 Ti-1 Ti-2 TB Blank

A1

k1obs s-1

A2

k2obs s-1

R2

18.78 29.32 25.21 27.62

2.0788 6.1554 26.532 0.9669

15.12 27.29 18.22 29.30

0.0256 0.0335 0.1364 0.0227

0.96 0.96 0.99 0.95

-

-

-


10

Page 11 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

R5 Ti-1 Ti-2 H2O Blank R5 Ti-1 Ti-2

17.60 19.98

1.5891 0.1952

18.02 16.55

1.2803 0.2352

14.13 11.43

0.0590 0.0346

0.98 0.99

13.26 6.61

0.0457 0.0341

0.99 0.99

A second order exponential decay is generally considered compatible with a double step mechanism,25 where k1obs and k2obs represent the corresponding rates. From the values of the rate constants we identify a fast process (described by k1obs) and a slow process (described by k2obs) of comparable amplitudes. Generally, rate constant values in the presence of phosphate ions show a tenfold higher value compared to the other mineralization systems. Peptides Ti-1 and Ti-2 give similar rate constants in water and in Tris buffer. Ti1 shows faster kinetics than Ti-2 for both fast and slow processes, however while the rates obtained for the slow process are of comparable scale, the difference in the fast process is of the order of one order of magnitude. The faster rate might correspond to a sequence dependent process, we suggest that the first step could be the complexation of TiBaldH with the peptide (see discussion of CD data); while the slower rate (similar for the two sequences) might correspond to the nucleation and growth process. More kinetic data is needed to understand the nature of the two processes identified, and the contribution of each reaction component to the overall mechanism of reaction. The minerals obtained after 2 hours reaction were collected and characterized, revealing striking differences depending on the presence or not of ions from the buffer solutions, especially in the case of the phosphate mineralizing system. DLS analysis showed that Ti-1 yielded a


11

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 38

monodisperse population of particles with hydrodynamic diameter of ca.4 nm in water and Tris buffer (Figure 2A). Peptide Ti-2 behave identically both in water and in Tris buffer (Figure S2).

Figure 2. Exemplar particle size distribution by DLS analysis at 2, 18 and 72 hours of reaction of titania nanoparticles obtained in the presence of Ti-1 in: water (A), and Phosphate buffer (B) Insets in A shows the high stability of the titania sol obtained with Ti-1 (and Ti-2) over time. Inset in B shows the evolution of the mean particle size with time for Ti-1 (and Ti-2) in the presence of PB. (C) Hydrodynamic diameter measured by DLS of particles before and 24 hours after the addition of more peptide or titania precursor in the water system. The particle size of the materials was confirmed by TEM (Figure 3-a, b): it can be noticed that upon drying nanoparticles synthesized in water were still monodisperse while those prepared in the presence of Tris buffer aggregate, probably due to a salt bridging effect via the particle’s double layer. The elemental composition was analyzed by EDX yielding strong signals for Ti and O alone (Figure 3-f). XRD analysis did not yield clear diffraction peaks, indicating that the majority of the material is amorphous. However, high resolution TEM allowed the identification of lattice fringes (d=0.353 nm) on vast areas of the samples, revealing the presence of both anatase and monoclinic TiO2(β) crystals. The electron diffraction patterns show relatively broad diffraction rings,

yielding d spacings compatible with the presence of anatase (d=0.353


12

Page 13 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

matching the (101) plane and 0.176 nm matching the (105) plane); and of monoclinic TiO2(β) (d=0.353 and 0.205 nm matching the (110) and the (601) plane, respectively).19

Figure 3. Morphology, composition and crystallinity analysed by TEM/EDX of materials obtained from TiBALDH solutions at pH 7.4 in the presence of: a) Ti-1 in H2O, inset shows higher magnification image; b) Ti-2 in Tris-buffer, inset shows higher magnification image; c) Ti-1 in phosphate buffer, EDX in inset shows the presence of P in the material. High resolution TEM image and electron diffraction (insets) of particles obtained in the presence of: d) Ti-1 in water; e) Ti-2 in Tris buffer, showing lattice fringe spacing; f) exemplar EDX for samples obtained in the absence of phosphate ions.

ATR-IR of titania nanoparticles obtained in water (or TB) in the presence of Ti-1 or Ti-2 shows a very weak signal in the amide band region, indicating the presence of a small amount of


13

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 38

peptide in the precipitated solid, estimated to be ca. 5%wt by TGA (Figure 4). The titania nanoparticles formed are characterized by high stability towards growth and aggregation over time as particle size and distribution do not change over 72 hours (Figure 2A-inset). It should also be noted that the stability of the nanoparticles in solution was maintained even after a period of 18 months, showing only a small degree of aggregation, as assessed by DLS (Figure S3). Further addition of either peptide or precursor to the obtained sols, did not promote further particle growth (Figure 2C). The mineralization reaction in the presence of peptides in phosphate buffer yielded materials with very different characteristics to those obtained from water or TB. In phosphate buffer, polydisperse materials of particle size distribution ranging from 10 to 100 nm after two hours of reaction were obtained (a representative example is reported in Figure 2B). The evolution of size with reaction time up to 72 hours, shows an increase of the bigger size population and consequently of the average particle size of the materials (Figure 2B). TEM microscopy on dialyzed and lyophilized samples after two hours reaction, shows spherical particles of 150-300 nm diameter (Figure 3-c). The appearance of larger particles as measured by DLS for the same reaction time is probably due to the formation of aggregates in solution. Materials obtained in the presence of a phosphate buffer also differ in composition: EDX analysis shows the presence of Ti, O, and P in the samples (inset in Figure 3-c) indicating that phosphate ions are entrapped in the solid during mineralization. This was confirmed by the presence of the characteristic P-O vibration in the ATR-IR spectrum26 (Figure 4). ATR spectra of materials obtained from phosphate buffered solutions in the presence of peptides also show a distinct amide I band centered at 1641 cm-1, and a broad feature at 1400 cm-1 indicating the presence of peptide in the solid. The peptide content,


14

Page 15 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

quantified by TGA, varied between 40 and 60% depending on the peptide used for mineralization (Figure 4). The presence of phosphate ions in the reaction medium, results in lack of control over the morphology, composition and stability of the minerals obtained. The characteristics of the minerals obtained from PB are in agreement with those reported by others for similar mineralization systems containing phosphate ions.7,9,20

Figure 4. TGA (A) and ATR-FT-IR (B) of mineralized materials obtained in the presence of peptide Ti-1 compared with pure peptide and commercial TiO2, show a small amount of peptide present in the material prepared from H2O compared to that prepared from PB. In order to understand which reaction parameters affect mineralization activity, and to gain further insight on the role of phosphate ions in titania formation, we investigated TiBALDH


15

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 38

conversion as a function of reagent concentrations, in both water and PB, using Ti-1 as a model peptide. In PB, precipitation activity is limited by the availability of TiBALDH; by the concentration of phosphate ions and was found to be independent of peptide concentration over the concentration range (0.08-1mM) studied (Figure 5). These data confirm the important role of phosphate ions in mineral formation conducted in the presence of (bio)molecules and is consistent with previous studies reporting the requirement of phosphate ions for precipitation18 or to increase mineral yields.17 In contrast to the situation when phosphate is present, for titania mineralization in water, exposure to Ti-1 (0.28 mM solution), results in an increase in mineralization activity with increasing precursor concentration, up to 50 mM (Figure 5).

Figure 5. Titania precursor conversion dependence on: (A) initial peptide concentration in a 50 mM TiBALDH solution, (B) initial precursor concentration using 0.28 mM peptide and (C) concentration of phosphate buffer in a 50 mM TiBALDH solution using 0.28 mM peptide. All experiments at pH 7.4. For a 50 mM TiBALDH solution the maximum mineralization (and plateau) is achieved at 0.28 mM peptide in line with previous studies that report a sequence specific mineralization yield, dependent on the amount of peptide present in solution.13 The existence of a sequence


16

Page 17 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

dependent correlation between (bio)molecule concentration and mineralization yield, may explain why peptide R5 does not show a significant activity towards titania mineralization in water or TB under our experimental conditions, but is active towards titania mineralization when used at a concentration >3mM.11 In water, Ti-1 mineralization activity is limited by both reagents, up to a certain precursor/peptide ratio, indicating a different role for the peptide and a different overall reaction mechanism than that observed in the presence of phosphate ions. It is worth noting that the mineralization activity per unit concentration of peptide for Ti-1, Ti-2 exceed those previously reported in the literature for titania mineralizing peptides.10,13 As an example, the mineralization activity per unit concentration of Ti-1 and Ti-2 in water (or TB) is 15 times higher than that of R5 (in water) as reported by Sewell and Wright.17 In this context, the observed mineralization activity of peptides Ti-1 and Ti-2, at concentrations lower than 0.28 mM is remarkable when compared with the biomolecule concentration (at least ten-fold higher) generally used in biomimetic mineralization studies.10,11,13 By comparison with the mechanism of silicification in vivo and in vitro, it has been proposed that TiIV can be complexed by basic groups in the (bio) molecules which then catalyze hydrolysis and promote condensation of neighboring Ti complexes.16 The formation of a precursor-peptide complex is compatible with the two step-mechanism identified by our kinetic data analysis. In order to gain more insight into the specific role of the peptide and to collect direct evidence to support such interactions, we studied the changes in the CD spectra of equimolar solutions of the peptides in the presence of the titania precursor TiBALDH in water. Both peptides have a random coil conformation in solution (Figure 6). In the presence of TiBALDH, although the peptide structure is not modified, other spectral changes are observed. As examples, in the presence of TiBALDH, the spectrum of Ti-1 shows a red shift of the negative peak as well as an


17

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 38

enhancement of the peak arising from TiBALDH (Figure 6A); while in the case of Ti-2, a decrease in peak intensities is observed (Figure 6B). These spectral changes clearly indicate an interaction between the peptide and the titanium precursor at the very early stages of the mineralization process, that appears to be more complex for Ti-1 than for Ti-2.

Figure 6. A) CD spectra of Ti-1 in water and Ti-1 in water in the presence of TiBALDH. B) CD spectra of Ti-2 in water and Ti-2 in water in the presence of TiBALDH. The evidence that Ti1 interacts with TiBALDH more strongly than Ti2 correlates with the difference in rate constant k1obs observed, where Ti-1 shows values tenfold bigger than Ti2


18

Page 19 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(Table 2) and supports our assumption that the fastest of the two processes could be the complexation between the TiBALDH and the peptide present in the mineralization system. Taking into consideration both the kinetic profiles and the material characterization information, binding sequences Ti-1 and Ti-2 appear to play a dual role in titania mineralization. First, they catalyze the nucleation of titania at neutral pH, and secondly they control the mineral size and growth by stabilizing the forming nanoparticles. It is also clear that the presence of phosphate ions in the mineralization systems has a detrimental effect over such a stabilization mechanism. We propose a mechanism, for mineralization shown in Figure 8 that accounts for the role of the peptides at several distinct stages during particle formation and also accounts for the role of phosphate ions. According to the proposed model, hydrolysis/condensation reactions at ca. neutral pH are triggered by interaction between cationic amino acids in the peptides and the anionic hydrolytically stable TiBALDH complex. Both peptides contain Lysine and/or Histidine that can locally act as acid-basic catalysts, thereby initiating hydrolysis of the lactate groups and allowing complexation of TiIV. Condensation can occur between neighboring complexes in the XY plane or it can occur in the axial direction for two complexes in close proximity.26 The first titania nuclei formed grow following the same peptide mediated mechanism at the nuclei surface.


19

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 38

Figure 7. Binding of Ti-1 and Ti-2 to an hydrated Ti crystal measured by QCM. Arrows indicate peptide and water injection (washing) in the system. We attribute the ability of Ti-1 and Ti-2 to make ca. 4 nm nanoparticles to a capping mechanism. The low amount of peptide present in the material and the inactivity of the particle’s surface toward growth in the presence of additional reagents are compatible with the formation of a peptide monolayer on the titania nanoparticles surface.28 As a comparative example, peptides have been used as capping agents or stabilizers in the preparation of functional gold colloidal nanoparticles, by exploiting specific binding interactions between chemical functional groups and the mineral’s surface.29 Sequences Ti-1 and Ti-2 high affinity for titania was confirmed by QCM-D analysis. The adsorption of synthetic peptides Ti-1 and Ti-2 at pH 7.4 (PBS) on a titanium sensor covered by a thin layer of amorphous and non-stoichiometric TiO230 was monitored (Figure 7). Both sequences show high affinity for the titania surface, but the variation of adsorbed mass as a function of time reveal different adsorption kinetics for the two peptides. Ti-1 shows lower affinity and a more gradual adsorption than Ti-2. For both peptides


20

Page 21 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

no adsorption saturation is observed indicating the formation of peptide multilayers on the sensor’s surface. In order to explain the observed differences in reactivity the surface chemistry of titania needs to be considered. Prior studies suggest the hydroxyl groups on the titania surface to be the recognition sites for peptide binding.31 Since the hydroxyl groups are amphoteric in nature forming Ti-OH2+ and Ti-O-, both positively and negatively charged functional groups can electrostatically bind to the titania surface, presumably via interaction through the ordered water layers at the interface.31 Ti-1 presents oppositely charged groups (aspartic acid and lysine) that could bind in synergy on both types of recognition sites, in the same way as proposed by Sano and Shiba.20,30 Ti-2 presents four basic amino acids that could electrostatically interact with the negatively charged sites on the titania surface. However, of the four basic amino acids present in Ti-2, only Arginine carries a net positive charge at pH 7.4 while the Histidines are mainly non protonated. Additional binding comes through the enrichment of both peptides in polar residues containing hydroxyl groups, which can also favor binding via the cumulative effect of multiple H bond interactions.32 The higher affinity of Ti-2 could suggest that the actual interfacial interaction involves predominantly the Ti-O- groups and the positively charged side chains, at least for this peptide sequence which is in accord with the pzc values for similarly passivated TiO2 layers,32 where Ti-O- groups are thought to be the dominant charged group at pH 7.4. The difference in affinity could also result from an intrinsically more favorable molecular conformation of Ti-2 that may allow for a better spatial orientation of the cationic and polar groups during the binding event. We suggest that the high affinity and specific binding to the titania surface demonstrated by Ti-1 and Ti-2, is the driving force for the capping mechanism responsible for particle growth interruption by the blocking of the condensation reaction at the titania surface. When the primary particle reaches 4-5 nm we propose that the interaction


21

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 38

between the TiO2 surface and the peptides becomes dominant. At this point the peptides bind to the nanoparticles, effectively stabilizing them and inhibiting any further growth.

A model

accounting for phosphate induced assembly similar to that encountered in polyamine27,34 and dendrimer35 directed silica formation, has been used to explain the role of phosphate in titania mineralisation.27 Further, in a recent study it has been suggested that Phosphate ions stabilize a linear peptide conformation, thus providing a rigid template for mineral condensation.17 Our study shows that the presence of phosphate in the mineralization medium disrupts the peptide controlled growth mechanism.

Figure 8. Proposed mechanism of formation of peptide capped titania. The specific surface recognition properties of Ti-1 and Ti-2 are responsible for capping resulting in the formation of stable titania sols. The presence of phosphate in the reaction media results in the co-precipitation of peptide and phosphate ions in the mineral phase, effectively disrupting the surface recognition and specific interactions.


22

Page 23 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Condensation continues beyond the formation of stable primary particles with further growth and agglomeration occurring as well as co-precipitation of the (bio) molecules and phosphate present. The formation of composite material and presence of phosphate in the mineral phase may be responsible for the deficiency of surface recognition and specific interface interaction with the peptides and the consequent absence of an effective capping mechanism. Further, the kinetic profiles also suggest a possible catalytic role of phosphate ions in mineralization that may be involved in the growth control disruption observed in this study. More detailed studies are needed to validate this possibility.

CONCLUSION In conclusion, we have reported on two titania binding peptides with remarkable titania mineralization activity that can be used to produce highly stable, monodispersed titania nanoparticles of ca 4 nm size. To the best of our knowledge, this is the first report of the controlled preparation of small titania nanoparticles (sub-10-nm) using a biomimetic approach. We propose that the biomolecules’ recognition and affinity for titania are central in the capping mechanism at the basis of small particle formation; mechanism that is disrupted by the presence of phosphate ions in the mineralization system. The preliminary results reported herein open up the possibility of exploiting peptides selective binding properties for the preparation of peptide capped nanoparticles with potential applications in translocation studies and drug delivery. A detailed investigation of the interactions at the titania-peptide interface responsible for surface recognition and binding resulting in nanoparticle stabilization and the correlation between binding properties and capping ability of different sequences is currently in progress and will be the subject of a future publication. In particular, more work is needed to understand the reaction


23

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 38

kinetics in detail together with the precise contribution that the different components play in the mineral formation mechanism.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

ACKNOWLEDGMENTS The authors thank the American AFOSR (Grant: FA9550-10-1-0024) for their funding of this study. REFERENCES (1) Mann, S. Biomineralization, Oxford University Press, Oxford, New York, 2001. (2) Dickerson, M. B.; Sandage, K. H.; Naik, R. R. Protein- and Peptide-directed Syntheses of Inorganic Materials. Chem. Rev. 2008, 108, 4935-78. (3) Liang, M. K.; Deschaume, O.; Patwardhan, S. V.; Perry, C. C. Direct Evidence of ZnO Morphology Modification via the Selective Adsorption of ZnO-binding Peptides. J. Mater. Chem. 2011, 21, 80-89. (4) Wang, Y.; Chen, L. Q.; Li, Y. F.; Zhao, X. J.; Peng, L.; Huang, C. Z. A One-pot Strategy for Biomimetic Synthesis and Self-assembly of Gold Nanoparticles. Nanotechnology 2010, 305601-305631.


24

Page 25 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(5) Belton, D. J.; Patwardhan, S. V.; Annenkov, V. V.; Danilovtseva, E. N.; Perry, C. C. From Biosilicification to Tailored Materials : Optimizing Hydrophobic Domains and Resistance to Protonation of Polyamines. PNAS 2008 , 105, 16, 5963-5968. (6) Chen, X.; Mao, S. S.; Titanium Dioxide Nanomaterials: Synthesis, Properties, Modifications, and Applications. Chem. Rev. 2007, 107, 7, 2891-2959. (7) West, R.H.; Celnik, M. S.; Inderwildi, O. R.; Kraft, M.; O. Beran, G. J.; Green, W.H. Toward a Comprehensive Model of the Synthesis of TiO2 Particles from TiCl4 Ind. Eng. Chem. Res. 2007, 46, 6147-6156 (8) Deberry, J. C.; Robinson, M.; Pomponi, M. D.; Beach, A. J.; Xiong, Y.; Akhtar, K. Controlled Vapor Phase Oxidation of Titanium Tetrachloride to Manufacture Titanium Dioxide. U.S. Patent 6,387,347, May 14, 2002. (9) Buonsanti, R.; Grillo, V.; Carlino, E.; Giannini, C.; Kipp, T.; Cingolani, R.; Cozzoli, D. Nonhydrolitic Synthesis of High Quality Anisotropically Shaped Brookite TiO2 Nanocrystals. J. Am. Chem. Soc. 2008, 130, 11223-11233. (10) Li, X. L.; Peng, Q.; Yi, J. X.; Wang, X.; Li, Y. D. Near monodisperse TiO2 Nanoparticles and Nanorods. Chem. Eur. J. 2006, 12, 2383-2391. (11) Aboulaich, A.; Boury, B.; Mutin, P. H. Reactive and Organosoluble Anatase Nanoparticles by a Surfactant-Free Nonhydrolytic Synthesis. Chem. Mater. 2010, 22, 4519-4521. (12) Kyoshi, K.; Sugimoto, T. Shape Control of Anatase TiO2 Nanoparticles by Amino Acids in a Gel–sol System. Chem. Comm. 2004, 1584-1585.


25

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 38

(13) Durupthy, O.; Bill, J.; Aldinger, F. Bioinspired Synthesis of Crystalline TiO2: Effect of Amino Acids on Nanoparticle Structure and Shape. Cryst. Growth Des. 2007, 7, 2696-2704. (14) Kroger, N.; Dickerson, M. B.; Ahmad, G.; Cai, Y.; Haluska, M. S.; Sandhage, K. H.; Poulsen, N.; Sheppard, V. C. Bioenabled Synthesis of Rutile (TiO2) at Ambient Temperature and Neutral pH. Angew. Chem. Int. Ed. 2006, 118, 7239-7243. (15) Cole, K. E.; Ortiz, A. N.; Schoonen, M. A.; Valentine, A. M. Peptide- and Long-Chain Polyamine- Induced Synthesis of Micro- and Nanostructured Titanium Phosphate and Protein Encapsulation. Chem. Mater. 2006, 18, 4592-4599. (16) Cole, K. E.; Valentine, A.M.

Spermidine and Spermine Catalyze the Formation of

Nanostructured Titanium Oxide. Biomacromolecules, 2007, 8, 1641-1647. (17) Sewell S. S.; Wright, D. W. Biomimetic Synthesis of Titanium Dioxide Utilizing the R5 Peptide Derived from Cylindrotheca fusiformis. Chem. Mater. 2006, 18, 3108-3113. (18) Kharlamplampieva, E.; Jung, C. M.; Veronika, K.; Tsukruk , V. V. Secondary Structure of Silaffin at Interfaces and Titania Formation. J. Mater. Chem., 2010, 20, 5242-5250. (19) Dickerson, M.B. ; Jones, S. E. ; Cai, Y. ; Ahmad, G. ; Naik, R. R. ; Kröger, N.; Sandhage, K. H. Identification and Design of Peptides for the Rapid, High Yield Formation of Nanoparticulate TiO2 from Aqueous Solutions at Room Temperature. Chem. Mater. 2008, 20, 1578-1584. (20) Sano, K.; Sasaki, H.; Shiba, K. Specificity and Biomineralization Activities of Ti-binding Peptide-1 (TBP-1). Langmuir 2005, 21, 3090-3095.


26

Page 27 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(21) Oskam, G.; Nellore, A.; Penn, R. L.; Searson, P. C. The Growth Kinetics of TiO2 Nanoparticles from Titanium (IV) Alkoxide at High Water/Titanium Ratio. J. Phys. Chem. B. 2003, 107, 1734-1738. (22) Choi, N.; Tan, L.; Jang, J.; Um, M.; Yoo, P. J.; Choe, W. The Interplay of Peptide Sequence and Local Structure in TiO2 Biomineralization. J. Inorg. Biochem. 2012, 115, 20-27. (23) Yoe, J. H.; Armstrong, A. R. Colorimetric Determination of Titanium with Disodium-1,2dihydroxybenzene- 3,5-disulfonate. Anal. Chem. 1947, 19, 100-102. (24) Seisenbaeva, G. A.; Daniel, G.; Nedelec, J. M.; Kessler, V. G. Solution Equilibrium behind the Room Temperature Synthesis of Nanocrystalline Titanium Dioxide. Nanoscale 2013, 5, 3330. (25) Scott, R. A ; Lukehart C. M. Applications of Physical Methods to Inorganic and Bioinorganic Chemistry. Wiley 2007. Pp 474. (26) Wang Q.; Zhong, L.; Sun, J.; Shen, J. A Facile Layer-by-Layer Adsorption and Reaction Method to the Preparation of Titanium Phosphate Ultrathin Films. Chem. Mater. 2005, 17, 35633569. (27) Kinsinger, N. M.; Wong, A.; Li, D.; Villalobos, F.; Kisailus, D. Nucleation and Crystal Growth of Nanocrystalline Anatase and Rutile Phase TiO2 from a Water-Soluble Precursor. Cryst. Growth Des. 2010, 10, 5254–5261. (28)The surface coverage per titania particle was estimated taking into account the organic content, particle size, and a van der-Waals peptide area of 2-3 nm2 (according to TGA,


27

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 38

DLS/TEM, and atomistic models, respectively). An approximate titania density of 4.63 g/cm3 was used. (29) Porta, F.; Speranza, G.; Krpetic, Z.; Dal Santo, V.; Francescato, P.; Scari, G. Gold Nanoparticles Capped by Peptides. Materials Science and Engineering B 2007, 140, 187-194. (30) Sano, K.; Shiba, K. A Hexapeptide Motif that Electrostatically Binds to the Surface of Titanium. J. Am. Chem. Soc. 2003, 125, 14234-14235. (31) Skelton, A. A.; Liang, T.; Walsh, T. R. Interplay of Sequence, Conformation, and Binding at the Peptide-Titania Interface as Mediated by Water. ACS Applied Materials & Interfaces 2009 17, 1482-1491. (32) Monti, S.; Walsh, T. R. Free Energy Calculations of the Adsorption of Amino-Acid Analogues at the Aqueous Titania Interface. J. Phys. Chem. C 2010, 114, 22197-22206. (33) Kosmulski, M. pH-dependent Surface Charging and Points of Zero Charge II. Update. J. Coll. Interface Sci. 2004, 275, 214-224. (34) Sumper, M.; Lorenz, S,; Brunner E. Biomimetic Control of Size in the PolyamineDirected Formation of Silica Nanospheres. Angew. Chem. Int. Ed. 2003, 42, 5192-5195. (35) Knecht, M. R.; Sewell, S. L.; Wright, D. W. Size Control of Dendrimer-Templated Silica. Langmuir 2005, 21, 2058.


28

Page 29 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

TOC graphic


29

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

83x63mm (150 x 150 DPI)


Page 30 of 38

Page 31 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

236x72mm (150 x 150 DPI)


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

230x69mm (150 x 150 DPI)


Page 32 of 38

Page 33 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

251x132mm (150 x 150 DPI)


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

124x181mm (150 x 150 DPI)


Page 34 of 38

Page 35 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

270x71mm (150 x 150 DPI)


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

81x128mm (150 x 150 DPI)


Page 36 of 38

Page 37 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

128x94mm (150 x 150 DPI)


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

252x122mm (150 x 150 DPI)


Page 38 of 38

Titania Binding Peptides as Templates in the Biomimetic Synthesis of

Recommend Documents