P.
Cao
a,
C. Q.
Yuan
*a,
C. Y.
Ma
a,
Y.
Yang
b,
X. Q.
Bai
a,
X. J.
Wang
a,
X. Y.
Ren
a,
H.
Xie
c and
X. P.
Yan
a
aSchool of Energy and Power Engineering, Wuhan University of Technology, Wuhan 430063, P. R. China. E-mail: ycq@whut.edu.cn
bSchool of Medicine, Keele University, Keele, Staffordshire ST5 5BG, UK
cSchool of Chemistry, Chemical and Life Sciences, Wuhan University of Technology, Wuhan 430070, P. R. China
First published on 4th September 2015
Biofouling on metal surfaces is one of the main reasons for increased ship drag. Many methods have already been used to reduce or remove it with moderate success. In this study, a synthetic peptide has been utilized to react with 304 stainless steel aiming to generate a bioorganic stainless steel using a facile technique. After the reaction, white matter was found on the surface of the treated stainless steel via SEM, whilst the nontreated stainless steel had none. Elemental analysis confirmed that excessive N existed on the surface of the treated samples using an integrated SEM-EDS instrument, implying the presence of peptides binding on the surface of the bioorganic stainless steel. The FTIR spectra showed amide A and II peaks on the surface of the bioorganic stainless steel suggesting that either the peptides grafted onto the steel surface or the polypeptide composition accumulated on the steel samples. XPS analysis of the treated steel demonstrated that there was nitrogen bonding on the surface and it was a chemical bond via a previously unreported chemical interaction. The treated steel has a markedly increased contact angle (water contact angle of 65.7 ± 4.7° for nontreated steel in comparison to treated, 96.4 ± 2.1°), which supported the observation of the wettability change of the surface, i.e. the decrease of the surface energy value after peptide treatment. The changes of the surface parameters (such as, Sa, Sq, Ssk and Sku) of the treated steel by surface analysis were observed.
Experimental results show that homarine and its aqueous extract can inhibit the growth of the diatoms effectively, and prevent barnacle larvae and marine benthic diatoms from attaching to the surface of ships. Application of anti-adhesion compounds could lead to the development of hull coatings.8,9 The research indicated that the drag reduction via antifouling and the alteration of surface energy of the material surface are closely related. Fouling will become difficult or defaced desorption becomes very easy when the surface energy of the material is low or ultra-low, in turn, achieving the effect of drag reduction.10
In recent years, a new concept of bioorganic stainless steel has been proposed11 in which a synthetic peptide is made to react with the metallic material surface. It was reported that such materials have a lower surface energy and are difficult to attach to by fouling organisms. Biological peptides, such as silver bonded peptides, palladium bonded peptides, and platinum bonded peptides,12 can react with metals to generate new materials. The iron oxide bonded peptide is the first polypeptide connecting a biological peptide to a metal. It is one of the synthetic peptides in the peptide library.13–15 Stainless steel is a very common metallic material, and is widely used in various industries. A new bioorganic metallic material was obtained by the reaction between the pili of microorganisms and steel.16–19 Wong et al. obtained a material with low surface energy by reacting a polypeptide with stainless steel.20 The reaction activity of stainless steel with peptides can be increased via dopamine addition.21 Later, another scholar studied the factors affecting the binding capacity to stainless steel.19 Davis et al. proved that the peptide–steel reaction led to a formal or semi-formal organic–metal covalent bond formation because stainless steel shared electrons with the dithiocycloheptane of the peptide.11 Some peptides with a linear chain do not contain a disulfide ring, but an indole group of L-tryptophan that has a cyclic chain structure, and may share electrons with metal to generate a new material. L-Histidine can be used as a protein tag. The imidazole group located in the residues of histidine acts as an electron donor and can form a coordinate bond by reacting with metal ions, which are immobilized on a matrix material. This group is likely to produce specific chelation with a metal ion (Ni2+, Cu2+, or Co2+, etc.) which is fixed on the chromatography filler.22
Previous studies on bioorganic stainless steel focused more on the identification of favourable peptides using the phage display technique or the identification of binding domains. However, the functional and surface property changes of these bioorganic stainless steels have not been investigated or reported in detail. This study fabricated one new bioorganic stainless steel, aiming to reveal the alterations of the surface parameters and functions after reaction with a specifically selected peptide. The multiple characterization assays used in this study confirm that the bioorganic stainless steel has modified surface properties.
Grade 304 stainless steel discs (ϕ 10 × 1 mm, with constituent elements as follows: C: ≤0.08%; Si: ≤1.00%; Cr: 18.00–20.00%; Mn: ≤2.00%; Ni: 8.00–11.00%; P: ≤0.035%; S: ≤0.030% and negligible N)23 were annealed at 1040 °C for 1 hour. One surface of the discs was polished using sandpaper of five increasing grit sizes (Eagle Inc, Korean): 120#, 240#, 400#, 600# and 1200# and an aqueous slurry of 0.05 μm colloidal silica. The polished samples were washed using dish washing detergent and distilled water, and then immersed in 95% (v/v) ethanol for 20 minutes on a shaker with a slow shaking rate. Then these samples were washed with distilled water, immersed in acetone for 5 min, and rinsed with distilled water. They were placed into 12-well cell culture plates, and covered by 4 ml phosphate buffered saline (PBS, pH 7.4) containing 10 μg ml−1 peptide for the peptide reaction. The reaction plates were placed on a shaker, at a rate of 100 rpm and were incubated at room temperature for 1 hour. After the reaction, the samples were washed more than 6 times with distilled water until the peptide was not detected in the wash liquor using a spectrophotometer and dried in the drying chamber. The treated samples are denoted as BioS corresponding to the reaction products of the BioP.
A Phenom ProX SEM equipped with an EDS (Phenom, Germany) was used to conduct the elemental analysis of the sample surface. The instrument uses quad backscattered electron detectors, which can give information on the composition and morphology of the samples. The image acquisition device contains four image capture functions. The memory sample position function allows the selection of the best location automatically. Images were obtained by choosing the “full mode” under 25000 magnification conditions, and the distribution of the elements at those points were obtained via tipping some points in the samples surfaces. To confirm the presence of a specific element, the certainty values were set to greater than 90%.
X-ray Photoelectron Spectroscopy (XPS) (AXIS-ULTRA DLD-600W, Shimadzu-Kratos, Japan) was used to examine the electronic state of the elements of the sample surfaces. The base pressure in the analytical chamber was lower than 7 × 10−8. Monochromatic Al Kα radiation source was used at a power of 450 W. The analysis spot was 300 × 700 μm. The resolution of the instrument was 0.48 eV for Ag 3d5/2 peak. The scan step was 0.05 eV. XPS spectra were generated using XPS software equipped within the instrument.
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However, after the reaction with the BioP, all of the stainless steel surfaces had a layer of white substance visible from the SEM images, whilst the original sample surface did not, as shown in Fig. 3(b). The layer was ascribed to the bonded peptide on the 304 stainless steel.
A strong peak that appeared in the nontreated sample spectrum (Fig. 4(a)) in the 1100–1150 cm−1 region was the Si–O–Si vibration.26,27 The BioS spectra showed that the surfaces of stainless steel treated with the BioP exhibited some changes. Compared with the nontreated sample, a broad peak occurred in the infrared spectra of the peptides with treated sample surfaces in the 3300–3500 cm−1 region, which proved the presence of amide A. There were 2 obvious and small peaks in the vicinity of 1610–1370 cm−1 in the BioS spectra. The result indicated that the peptides have aromatic CC stretching vibration. Since peptides contain peptide bonds, i.e. CO
NR(H), the peaks appearing around 3000 cm−1 corresponded to ν(C–H) (including saturated or unsaturated); the peak at 1680 cm−1 to ν(C
O) of the carbonyl group; 1450 cm−1, 1380 cm−1 to an alkyl group; N–H bending of amide II was found at 1490–1620 cm−1; 1229–1301 cm−1 was C–N stretching and NH bending. All were indicators of the presence of amide groups.
Using the EDS detector, the surface spectroscopic analysis showed that the nontreated sample mainly contained iron, chromium and nickel, the concentrations of which were 72.4%, 18.2% and 7.7%, respectively; and the chromium and nickel content ratio was about 18:
8, a ratio in line with the proportion of the chromium and nickel content in 304 stainless steel (the formula is 0Cr18Ni9). Table 1 shows the summary of the chemical composition of the steel samples.
Element | Nontreated | BioS |
---|---|---|
Fe (%) | 72.4 ± 0.2 | 71.8 ± 0.1 |
Cr (%) | 18.2 ± 0.1 | 17.9 ± 0.3 |
Ni (%) | 7.7 ± 0.2 | 7.4 ± 0.2 |
Si (%) | 1.6 ± 0.1 | 2.2 ± 0.3 |
N (%) | 0.02 ± 0.01 | 0.8 ± 0.1 |
The surfaces of the samples treated with the peptide also contained iron, chromium and nickel. The content ratio was essentially the same as the nontreated stainless steel. However, the surfaces of the treated samples also contained N and Si. The element Si may come from residue chemicals after using the silicon containing sandpaper and silica slurry. Excessive nitrogen was detected only on the surfaces of the samples treated with the peptide compared to the nontreated specimen (max 0.08%). The content of N was about 0.9% by weight, which supported the idea that the N element-containing substance was formed on the sample surface after treatment with the polypeptide.
The water contact angle of the nontreated sample surface was around 65.7 ± 4.7°, and the contact angle of the BioS was 96.4 ± 2.1°. The glycerol contact angle of the original sample was 53.5 ± 4.7°, and the contact angle of the BioS was 83.5 ± 1.2°. The value of the contact angle increased significantly after being treated with the peptides.
The surface energies of the samples were calculated via Owens–Wendt–Rabel–Kaelble eqn (1) and the parameters are shown in Table 2. The surface energy value of the nontreated, and BioS samples were 41.3, and 25.0 mN m−1 respectively which demonstrated that the surface energy of 304 stainless steel decreased by reacting with the peptides.
Liquid | γ L | γ LWL | γ L + | γ L − |
---|---|---|---|---|
Water | 71.8 | 21.8 | 25.5 | 25.5 |
Glycerol | 64.0 | 34.0 | 3.92 | 57.4 |
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Number | S a (μm) | S q (μm) | S sk | S ku |
---|---|---|---|---|
Nontreated | 0.945 ± 0.053 | 1.496 ± 0.007 | 4.077 ± 0.236 | 37.9 ± 2.5 |
BioS | 1.187 ± 0.004 | 1.679 ± 0.056 | 0.720 ± 0.016 | 14.8 ± 1.8 |
SEM analysis is a convenient and powerful technique to study surface topographic changes, which has been used to study stainless steel surfaces modified by peptides.11,21 In combination with EDS analysis, we not only observed the new substance on the treated steel surfaces, but also detected their composition using nitrogen as the marker for the peptide grafting degree. All peptides have unique peptide bonds, i.e. CONR(H). The reaction extent of stainless steel and peptides can be reflected by the N content.11,21 EDS analysis is an accurate technique to determine the composition of the surface substance of the sample. In the treated steel sample surfaces, Fe, Cr and Ni component proportions have been detected. Their concentrations were same as the component ratio of the original stainless steel. The element nitrogen on the surface proved that the stainless steel surface treated with the peptide was comprised of a novel nitrogen-containing substance, and the content is relatively stable (Table 1). The new steel sample generated by the reaction between the peptide and stainless steel can be classed as bioorganic stainless steel.
ATR-FTIR has been used to study the surfaces treated with the peptide.31,32 Peptide bonds, carbon–hydrogen bonds, and carbon–oxygen bonds, etc. were found on the surface of the treated samples in our study (Fig. 4). There were 2 obvious and some small valleys in the vicinity of 1610–1370 cm−1 in the BioS spectra. The result indicated that the peptide has aromatic CC stretching vibration. N–H bending of amide II was found at 1490–1620 cm−1; 1229–1301 cm−1 is C–N stretching and NH bending.33 These spectra suggested that the occurrence of a reaction between steel and the peptide generated a substance containing organic ingredients which may be obtained by the joining of the peptide with the free electrons of stainless steel. The treated steel material exhibited new spectral peaks which did not belong to the original steel material. Hence, the peptide has been physically and chemically bonded to the steel surfaces through the active groups and elements on the steel. The FITR and XPS spectra of the treated sample supported the EDS results. However, the specific reaction mechanisms are complex and will be studied in subsequent experiments.
The contact angle is determined by the surface morphology and chemical substances.34 The generation of a new substance on the sample surface, is the main cause that led to the changes in the surface parameters. According to Young’s equation,35 the surface energy of the samples reduced when the contact angles were increased which is consistent with the calculation results. Thus, contact angle changes in samples can be used to analyse changes in the surface energy of samples qualitatively. As can be seen from Fig. 7, the value of the contact angle increased significantly (p < 0.001) in comparison to the nontreated sample. Thus, the hydrophobic properties of the steel surfaces increased. However, the amount of improvement in the contact angle or surface energy was not big, and further investigations to generate a new generation of antifouling materials are required. The increase in the sample contact angles could be partially due to the surface topographic change after peptide bonding. The changes in the contact angle of the steel sample indicated that the reaction with the BioP can considerably influence the steel surface.
Surface morphology has a greater effect on the contact angle compared to the chemical composition.34 In this study, it was found that the sample surface became smoother after treating with the peptide (Table 3). The changes of the surface roughness parameters were mainly caused by the new substance generated on the surface of the sample. The steel sample contained numerous grain boundaries, and the BioS has been shown to bind with higher adhesive force to grain boundaries compared to regions within grains.36 The majority of the reactions probably happened in the trough of the surface, and the complex structure of the new substance on the surface led to the smoother surface after the BioS bonded with stainless steel.
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