ARTICLE pubs.acs.org/Langmuir
Protein Denaturation Detected by Time-of-Flight Secondary Ion Mass Spectrometry Manuela S. Killian, Heike M. Krebs, and Patrik Schmuki* Department of Materials Science and Engineering 4, Chair for Surface Science and Corrosion, Friedrich-Alexander-University of Erlangen-Nuremberg, Martensstr.7, 91058 Erlangen, Germany
bS Supporting Information ABSTRACT: In the present work we investigate the denaturation of a functional protein, horseradish peroxidase (HRP), under various experimental conditions using time-of-flight secondary ion mass spectrometry. HRP was immobilized on TiO2, and the samples were stored under different conditions. The activity of the enzyme was assessed colorimetrically and compared to ToF-SIMS spectra. We show that denaturation of the protein can be monitored using the ToF-SIMS signal of the disulfide bonds, which is related to the tertiary structure of the protein. As disulfide bonds appear in a vast range of proteins, the present findings may be of wide significance; i.e., a tool is provided that can allow the investigation of the presence of an active protein structure by a comparably simple surface analytical method.
’ INTRODUCTION As interest in the application of protein chips, biosensors, and biochemically functionalized implant materials grows, the processing and analysis of protein-modified surfaces is becoming increasingly important. A variety of schemes have been employed to optimize the orientation and activity of immobilized proteins,1 including attachment via charge-driven orientation,24 physical adsorption through hydrophobic interactions,57 and bioactive ligands.829 In many cases, it is particularly important to determine whether an immobilized protein indeed retains its activity during and after the attachment to the surface. Activity assays often are based on enzyme linked immunosorbent assays (ELISA)8,16,30,31 or fluorophore-labeling.13,32 A surface analytical method such as X-ray photoelectron spectroscopy (XPS) can be used to obtain the coverage and thickness of protein coatings and to identify and distinguish pure protein films. However, XPS is, for example, not specific enough to distinguish different proteins adsorbed from protein mixtures. Also, conformational changes are not easily observable, as the information depth of the technique is larger than the average diameter of a protein.33 Time-of-flight secondary ion mass spectrometry (ToF-SIMS) provides information about chemical structures within the protein and consequently may be a promising tool for surface protein analysis. The fragment pattern generated by amino acids allows for confirmation of the attachment of proteins on the surface and it also is basically possible to distinguish specific proteins deposited from mixtures.9,3442 Furthermore, the information depth is shallow enough to observe only the amino acids located at the proteinair interface. r 2011 American Chemical Society
The exposed amino acids are related to the protein orientation, allowing one to track changes in the latter.33,42 However, it has to be considered that changes in the amino acid ratios for even selectively attached proteins may contain ambiguous information due to some degree of randomness in the orientation and thus may only give indirect clues on the presence of an active protein. In the present work, we use horseradish peroxidase (HRP) to study the feasibility of correlating protein activity to secondary ion mass spectral information. The activity of HRP can be assessed easily, by a comparably simple color reaction. HRP was shown to retain its activity upon immobilization on TiO2 or TiO2 nanotubes via silane linker chemistry and subsequent release by UV-illumination.43,44 Here we investigate the influence of distinct storage conditions on the activity of this enzyme. We use the enzyme immobilized on TiO2 and compare colorimetric activity assays to the corresponding ToF-SIMS spectra.
’ EXPERIMENTAL SECTION All chemicals were purchased from chemical suppliers and used without further purification. All organic solvents were water-free grade. TiO2 Layer Formation. Titanium foils [99.6% purity, Advent Ltd., 0.1 mm thickness, (1.5 1.5) cm2 pieces] were ultrasonically cleaned in ethanol and deionized (DI) water. The samples were anodized in a three-electrode cell, with a Pt gauze counter electrode and an Ag/AgCl (1 M KCl) reference electrode, at 20 V versus Ag/AgCl for 20 min, to obtain a 50 nm thick compact oxide film of TiO2. The specimens were completely immersed in the electrolyte upon anodizing. Anodization Received: November 10, 2010 Published: May 31, 2011 7510
dx.doi.org/10.1021/la200704s | Langmuir 2011, 27, 7510–7515
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ARTICLE
Figure 1. (a) ABTS activity assay of HRP-coated TiO2 stored at different temperatures. Absorption values are relative to the pure ABTS solution; lines are guide to the eye only. (b) XPS ratio of N 1s to Ti 2p of freshly prepared HRP-coated TiO2 and samples stored at elevated temperatures. was carried out using a high-voltage potentiostat (Jaissle IMP 88-200 PC), connected to a digital multimeter (Keithley 2000) interfaced to a computer. One molar H2SO4 (Merck) was used as electrolyte solution. For nanotubular samples, the Ti-foil was anodized at 20 V for 120 min, and as electrolyte a 1:1 mixture of H2O and glycerol containing 0.27 M NH4F was used.45 Samples were rinsed thoroughly and soaked in water overnight. Nanotubular TiO2 was only used in the colorimetric activity assay and to cross-validate the findings from compact TiO2 by control samples. The colorimetric activity assay in return was crossvalidated with compact TiO2 samples. Surface Modification and HRP Attachment. Grafting of horseradish peroxidase (HRP) was achieved by silanization of TiO2 with 3-aminopropyltriethoxysilane (APTES) and using an ascorbic acid linker for attachment of HRP. The samples were refluxed in a 10 mM APTES solution in toluene (Aldrich, 99.8% purity) for 24 h at 70 °C.44,46 According to the method reported by Tiller and co-workers,19 the APTES-modified samples were steeped in a concentrated solution of ascorbic acid in dimethyl sulfoxide (DMSO, Aldrich, purity >99.9%) for 30 min. In order to oxidize the ascorbic acid, the samples were dried for at least 2.5 h in air. Afterward, HRP (400 unit/mL) was attached from aqueous solution by immersion of the titanium samples for 24 h at 4 °C. In between all steps and after the last step the samples were thoroughly rinsed (for APTES, acetone rinse, ethanol rinse, ethanol ultrasonic treatment; for ascorbic acid, H2O rinse; for HRP, H2O rinse) in order to remove loosely bound products. Samples were stored in glass vessels tightly wrapped with Al-foil either at 4 or 70 °C under atmospheric conditions in the dark. Activity Assay. Samples were placed in a beaker containing 0.75 mL of PBS solution, 0.3% H2O2, and 0.05 M 2,20 -azinobis(3ethylbenzothiazoline-6 sulfonic acid) diammonium salt (ABTS). H2O2 is added as a substrate for the enzyme and subsequently oxidizes the ABTS molecule, detectable by the intense green color of the stable ABTS• radical.43,44,47,48 The intensity of the absorption of the differently stored samples was measured at λ = 747 nm on a UVvis spectrometer (Lambda Bio XLS) after 120 min incubation time at room temperature. Reference measurements of the unmodified TiO2 in ABTS solution as well as the pretreatment steps or the pure solution did not lead to an increase in the ABTS• radical absorbance. In order to decrease the reaction time for the ABTS assay by increasing the surface area (for flat surfaces the absorption was less than half as intense in the same time interval), TiO2 nanotubes (100 nm diameter, 1 μm length)45 were also employed. All experiments were repeated several times, to ensure reproducibility. Surface Analysis. Positive and negative static SIMS measurements were performed on a ToF.SIMS 5 spectrometer (ION-TOF, M€unster, Germany) on at least three different spots on each sample. The samples were bombarded with a pulsed 25 keV Bi3þ liquid-metal ion beam. Spectra were recorded in the high mass resolution mode (m/Δm > 8000
at 29Si). The beam was electrodynamically bunched down to
