Formation of Highly Stable Self-Assembled Alkyl Phosphonic Acid

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Formation of Highly Stable Self-assembled Alkyl Phosphonic Acid Monolayers for the Functionalization of Titanium Surfaces and Protein Patterning Xuemingyue Han, Sun Xiangyu, Tao He, and Shuqing Sun Langmuir, Just Accepted Manuscript • DOI: 10.1021/la504644q • Publication Date (Web): 05 Dec 2014 Downloaded from http://pubs.acs.org on December 12, 2014

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Formation of Highly Stable Self-assembled Alkyl Phosphonic Acid Monolayers for the Functionalization of Titanium Surfaces and Protein Patterning ABSTRACT

A protocol for the preparation of improved phosphonate monolayers on a titanium (Ti) substrate is presented. Zirconium ions were used to enhance the bonding between the phosphonate headgroup and the pretreated Ti surface. Contact angle and X-ray photoelectron spectroscopy were used to characterize self-assembled monolayers (SAMs) of alkylphosphonic acid that formed spontaneously on Zr-mediated Ti (Zr/Ti) surfaces. The surfaces that were treated with an aqueous solution of zirconium oxychloride showed significantly enhanced stability in buffer compared to those formed directly on the native oxidized Ti. A bifunctional molecule, 10-mercaptodecanyl phosphonic acid (MDPA), was also used to form SAMs on Zr/Ti surfaces using an identical method; which enabled us to regulate the surface functionality through the terminal functional group. Protein patterning on the surface was carried out using UV irradiation through a mask to selectively degrade regions of the MDPA molecules. The surface was then backfilled with a protein-resistant molecule in the exposed regions followed by selective immobilization of proteins to the unexposed areas using a heterobifunctional linker molecule. The presented 1 ACS Paragon Plus Environment

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strategy significantly improved the stability of the phosphonate SAMs on oxidized Ti surfaces, which provided an ideal approach foundation for biomolecular immobilization and patterning onto the Ti surfaces. Thus, this method provided a versatile platform to activate the surfaces of biomedical Ti implants.

INTRODUCTION

Titanium and its alloys are widely used as implant materials for biomedical applications because of their good biocompatibility, excellent chemical stability and convenient mechanical properties.1, 2 To better facilitate the direct connections between Ti and bones, many approaches to improve the interactions between cells and implants by tailoring the implants’ surface properties have been studied.3 These approaches include hydroxyapatite coating,4 sputter coating,5 sol-gel deposition,6 pulse laser deposition7 and mineral deposition from supersaturated salt solutions.8 The long-term success of an implant depends largely on the stability of the biocompatible coating on the implant surfaces. Detachment of the coating from the Ti surface may result in the eventual clinical failure of the implant.9 Therefore, the main problem that remains with all of these biocompatible coatings is their weak adhesion strength at the Ti surface-coat interface.

To resolve this issue, some studies explored chemical modification of the Ti implant using self-assembled monolayers (SAMs) of silane,10, 11 phosphonate12 or phosphonic acid3, 13, 14 based molecules. SAMs provide an ideal method for surface modification. In SAMs, the adsorbates anchor to surfaces by covalent bonding of the 2 ACS Paragon Plus Environment

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head group and are densely packed by Van der Waals forces between neighboring alkyl chains. Through careful selection of the terminal group of the adsorbate molecule, we are able to create a new surface with defined functionalities. Cell adhesive peptide RGD,11 bone morphogenetic proteins (BMPs), or other biomolecules with physiological functions can then be attached to the surfaces.15 However, although phosphonic acid and phosphonate SAMs are more stable than silane monolayers under physiological condition16-18, they still hydrolyze in aqueous solution.19, 20

High quality phosphonate SAMs on Ti substrates have been prepared conveniently,21 and immobilization of a wide variety of biomolecules22, 23 and catalysts24 to these surfaces using zirconium-phosphonate interactions has also been realized. In such a system, oxygen atoms octahedrally coordinate with tetravalent Zr ions, and the three oxygen of each group is anchored to three Zr ions.25, 26 Zr-phosphonate films are known to be highly stable in both water and saline solutions.26 Furthermore, our previous work has demonstrated zirconium ion-mediated phosphonate SAM formation on hydroxylate glass.27 Furthermore, phosphonate SAMs on Ti are known to photodegrade under weak UV exposure mainly because of catalysis of native oxide Ti substrates.28, 29 This feature of Ti surfaces make controlling the patterning of SAMs on Ti possible using photolithography, a relatively simple, clean, and biocompatible technique compared to other patterning techniques.

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In this study, a protocol for the formation of more stable phosphonic acid SAMs on zirconated Ti (Zr/Ti), subsequent photopatterning of these SAMs, and creation of protein arrays were demonstrated. In this protocol, native oxides of Ti substrates were cleaned by sonication and alkaline solution treatment, and the surface was modified with Zr from aqueous solution. Then, SAMs were prepared by immersing the substrates in an ethanol solution of 10-mercaptodecanylphosphonic acid (MDPA). After being covered with a patterning mask, the SAMs were irradiated with UV light. The irradiated areas were photodegraded and then backfilled with a protein-resistant molecule bearing a phosphonic acid group, resulting in bioinert background regions. Biomolecules with fluorescent markers were subsequently selectively anchored to the masked areas, which could be directly observed by fluorescence microscopy. Scheme 1 illustrates the fabrication of MDPA SAMs on Ti substrates, photopatterning of the SAMs, backfilling with a protein-resistant molecule, and protein immobilization.


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Scheme 1. Fabrication procedure for the fluorescent protein arrays created by sulfhydryl terminated SAMs on zirconated Ti substrates. (a) alkaline pretreated titanium substrate; (b) modification with Zr ions; (c) preparation of the MDPA monolayer; (d) sulfhydryl terminated SAMs covered with a patterned mask are exposed to 254 nm UV light; (e) SAMs in exposed areas are degraded by UV irradiation; (f) the patterned surface is backfilled with the protein-resistant molecule, P-10EG; (g) bifunctional linker MPS molecules are selectively anchored to sulfhydryl groups in the unexposed areas; (h) a multistep biomolecule immobilization protocol is used to generate fluorescent patterns.

EXPERIMENTAL

Materials.

Glass microscope cover slides were obtained from SPI. Titanium slugs (99.995%), n-octadecylphosphonic acid [CH3(CH2)17PO(OH)2, 97%, ODPA], 1-octadecanethiol [CH3(CH2)17SH, 96%, ODT], and 3-maleimidopropionic acid N-hydroxysuccinimide ester (99%, MPS) were obtained from Alfa Aesar. AcroSeal® ethanol (99%) was obtained from Acros. Zirconium oxide chloride (ZrOCl2•8H2O, 99%) was obtained from Sinopharm. PEO 10 OH Terminated Phosphonic Acid (HO(CH2CH2O)10(CH2)3PO(OH)2, SP-1P-1-006, P-10EG) was obtained from Specific Polymers. EZ-Link® Amine-PEG2-biotin (NH2(CH2O)2(CH2)2NHCO(CH2)4-Biotin, NH2-biotin) was obtained from Thermo Scientific. Electron microscope copper grids (hexagonal, 400 mesh) were obtained 5 ACS Paragon Plus Environment

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from Agar Scientific. Streptavidin, mouse IgG/Biotin (IgG/biotin), and goat anti-mouse IgG/Alexa Fluor 488 (αIgG/488) were obtained from Biosynthesis. Other solvents and reagents were reagent grade chemicals and obtained from Beijing Chemical Works. All reagents used in this work were utilized once available and without further treatment unless otherwise mentioned. Ultra-pure water of 18 MΩ cm at 25℃ was obtained from Millipore-ELIX water purification system. MDPA [HS(CH2)10PO(OH)2] was synthesized according to a previous method.27

SAM formation and microcontact printing (µCP) of alkyl phosphonic acid on Zr/Ti.

Glass microscope cover slides were treated with RCA1 solution of NH3• H2O/H2O2/H2O (1:1:5) and subsequent RCA2 solution of HCl/H2O2/H2O (1:1:6) at 80°C for 1 h. Slides were rinsed with water after each cleaning step and then dried with nitrogen. A 50 nm Ti film was deposited onto the clean glass slides at a rate of 0.05 nm s-1 by electron beam evaporation and stored in air. Before further use, Ti substrates were sonicated and rinsed with acetone, ethanol, and water and were immersed in a mixed solution of H2O/H2O2/NH4OH (200:4:1) at 70°C for 3 min. The hydrophilic surfaces were immersed in a 5 mM aqueous solution of ZrOCl2 and kept at 60°C for 2 days. After Zr-deposition treatment, the substrates were rinsed and sonicated with water for 5 min and dried with nitrogen. SAMs of ODPA and MDPA were formed by immersing the Zr/Ti substrates in 1 mM ethanol solution at room temperature for 1-3 days. For µCP of ODPA ink on the Zr/Ti substrates, the 6 ACS Paragon Plus Environment

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poly(dimethyl)siloxane (PDMS) stamp was prepared according to previous methods.30 Briefly, the stamp was first fabricated by pouring the mixture of prepolymer and curing agent (Dow Corning, Sylgard 184) onto a silicon-based master with patterns prepared by UV photolithography. Then, the stamp was fluorinated. The plate was heated to 80°C for 6 hours. Before use, the stamp was rinsed and sonicated in hexane for half an hour and then immersed in ethanol for 2 hours. The stamp surface was drop-casted with the ethanol ink of 1 mM ODPA and incubated for 3 min. Next, excess ink on the stamp was blown away with a stream of nitrogen. Inked stamp was pressed on the Zr/Ti substrate for 3 min. The patterned substrate was thoroughly rinsed with ethanol and dried under a stream of nitrogen.

Surface photodegradation and patterning.

The photodegradation of SAMs on Ti substrates was carried out using an integrative electronic UV lamp with a 254 nm central wavelength at 220 V/15 W obtained from Cnlight. The surface received a light intensity of ~1.3 mW/cm2 at a distance of approximately 15 cm from the lamp. The patterning of SAMs was performed by UV exposure for 2 h through hexagonal 400 mesh electron microscope grids.

Activation of the patterned SAMs with MPS.

The patterned SAMs were dipped in an ethanol solution of P-10EG to backfill the degraded regions. The substrates were then immersed in a dry DMF solution of 5 mg/mL MPS under nitrogen at room temperature overnight. After removal of the solution, substrates were rinsed with DMF, and dried with nitrogen.31, 32 The DMF 7 ACS Paragon Plus Environment

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used here was dried over calcium hydride, refluxed for over 5 h and then distilled under reduced pressure.

Protein Immobilization.

The procedure developed for fluorescent protein patterning is outlined in Scheme 1. All of the following immobilization procedures were conducted in a humidity chamber at room temperature, and the PBS solutions for incubation contained 30% glycerol to prevent rapid evaporation of the drops.33 After the activation step, the patterned substrate was covered with 50 µg/mL of NH2-biotin in PBS overnight, thoroughly rinsed with PBS and water, and dried under a stream of nitrogen. For blocking non-specific adsorption, a PBS solution of 1% BSA was dropped on the sample to cover the whole surface for 1 h. The sample was rinsed thoroughly with PBS and water, followed by addition of a PBS solution of 50 µg/mL streptavidin to cover the whole surface for 1-h incubation. The sample was washed again with PBS and water, followed by incubation with 50 µg/mL IgG/biotin (with 250 µg/mL BSA) for 1 h. Subsequently, the substrate was rinsed and shifted to a dark environment before adding 50 µg/mL αIgG/488 (with 250 µg/mL BSA) onto the sample. The sample was incubated for 45 min, rinsed with PBS and water, and dried with nitrogen.

Analysis techniques.

Contact angles (CA) were measured by DSA100 Drop Shape Analysis System, KRÜSS. A minimum of 10 values were obtained for each sample. X-ray photoelectron spectroscopy (XPS) data were obtained using an ESCALAB 250Xi 8 ACS Paragon Plus Environment

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instrument, Thermo Scientific, having a monochromatized Al K α X-ray source. XPS high-resolution spectra of C 1s, Zr 3d, S 2p and P 2p were obtained from a circular area of 500 µm in diameter with 20 eV pass energy. In the survey spectra, the pass energy is 100 eV, and the C 1s peak position was adjusted to 284.8 eV. The Atomic Force Microscopy (AFM) scans were conducted in contact mode by a VEECO Dimension 3100 equipment in the atmosphere. Fluorescent images were captured by DMI 6000B scanning confocal microscope, Leica. Alexa Fluor 488-labeled protein patterns were observed with λex=450-490 nm (λem=515 nm).

RESULTS AND DISCUSSION

Formation of phosphonate SAMs on Zr-modified Ti specimens.

In general, zirconium-phosphonate systems are built by alternating biphosphonate and Zr ion layers on pretreated phosphorylated surfaces using the LB technique26, 34 or other covalent modification methods.21, 35 In the present study, we demonstrated that Zr4+ could be anchored to the native hydroxylated oxide surface of Ti, and alkylphosphonic acids can spontaneously assemble on the Zr/Ti surface to produce SAMs with improved stability that are resistant to hydrolysis caused by immersing them in water or saline solution.

To characterize the alkylphosphonic acid SAMs formed on the Zr/Ti surfaces, CA and XPS tests were conducted. Cleaned Ti substrates directly immersed in the ethanol solution of ODPA without the alkaline solution and Zr treatments (ODPA on Ti sample) and Zr/Ti substrates immersed first in an ethanol solution of ODT (ODT 9 ACS Paragon Plus Environment

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sample) and then transferred to an ODPA solution (ODT-ODPA sample) were used as control samples. Additionally, patterns of ODPA SAMs were generated using µCP from an ethanol solution on the Zr/Ti substrates and characterized by AFM.

The static water CA of the Ti substrate is approximate 35°. After removal from the aqueous Zr solution, the surface is rather hydrophilic. However, after formation of the well-defined ODPA SAM, the CA value reaches 110±1°, which is slightly higher than that of ODPA on the Ti sample (108±1°). These values are consistent with that of ODPA SAMs formed on other metal oxide surfaces, indicating successful formation of high quality ODPA SAMs on Zr/Ti substrates.36, 37 Conversely, the ODT control sample yields a static CA of 45±1°. When compared to the value of 110±1° for ODPA SAMs on Zr/Ti, the CA of 45±1° indicates that the interaction between sulfhydryl group and the Zr/Ti substrates is rather weak, and the changes in CA are probably due to the physical adsorption of molecules on the Zr/Ti substrate that exhibit very high surface energy. Furthermore, if the ODT control sample is then immersed in ODPA solution, the CA value will increase to 110±1°. This result indicates that the previous ODT immersion step does not affect the formation of ODPA SAM on the Zr/Ti substrate.

To further verify the CA values, the control samples of ODT and ODT-ODPA adsorbed on Zr/Ti substrates were characterized by XPS, as indicated in Fig. 1. The XPS spectra of the ODT control sample show a very weak sulfur signal in S 2p region that could not be detected after ODPA immersion on the ODT-ODPA sample. It is 10 ACS Paragon Plus Environment

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also clear that the phosphorus signal is absent from the ODT sample as expected, whereas an obvious peak was detected on the ODT-ODPA sample, Fig. 1, P 2p region. Additionally, the intensity of C 1s peak of the ODT-ODPA sample is distinctly stronger than that of the ODT sample, Fig. 1 C 1s region. These results are consistent with the CA data, indicating that the anchoring of the ODPA molecules on the Zr/Ti substrate is overwhelmingly more stable than that of ODT molecule.

Figure 1. High-resolution XPS spectra of C 1s, S 2p, and P 2p regions of the zirconated Ti substrates immersed in ODT solution (ODT), and then transferred to an ODPA solution (ODT-ODPA).

µCP was utilized to transfer ODPA molecules onto the Zr/Ti substrate. After incubation in ethanol solution of ODPA for 3 min, the PDMS stamp was blow dried with N2 and brought into contact with the Zr/Ti substrate for 3 min. The substrate was rinsed with ethanol completely, subsequently dried and investigated by AFM in contact mode. From Fig. 2 we can see that after µCP, ODPA molecules are transferred to the substrate and formed a dot arrays. Fig. 2 (a) shows a typical height image of a µCP sample. It is clear that the round regions printed with ODPA are higher compared to that of the zirconium-modified surroundings. In addition, the 11 ACS Paragon Plus Environment

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section analysis below Fig. 2 (a) shows that these round features are approaching 2 nm in height, which is consistent with the data obtained from the same SAM of phosphonic acid on mica.38 Thus, these results confirm that the printing procedure successfully transfer the ODPA molecules from the stamp to the Zr/Ti substrate. In the friction retrace image, Fig. 2 (b), the contacted regions show bright contrast while the substrate show dark contrast, which suggests that the interaction between the tip and ODPA monolayer is weaker than that of the surroundings. The obvious contrast between the interaction forces of the two regions may be due to a decline in the surface energy resulting from the ODPA assembly. The AFM images suggest that ODPA molecules are able to form a monolayer on the Zr/Ti substrate in a short time.

Figure 2. AFM contact mode images (30×30 µm2). (a) Height and (b) friction (retrace) images of the ODPA pattern and a corresponding height profile made by microcontact printing (µCP) onto zirconium-modified Ti substrate.

All of the results suggest that the ODPA SAMs were successfully formed on the Zr/Ti substrates. Although there may be some physical adsorption of ODT molecules on the Zr/Ti substrates immersed in the ODT ethanol solution, thiol molecules could be 12 ACS Paragon Plus Environment

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replaced by subsequent treatment with an organic phosphonic acid. Although some papers have reported the use of thiol or thiolate molecules for Ti modification,39 our data indicate that the interaction between the sulfhydryl group and the Zr/Ti substrate is rather weak. In summary, we speculate that the sulfhydryl group of the MDPA molecule will not hinder the phosphonic acid group’s ability to anchor to the Zr/Ti substrates.

The left part of Fig. 3 illustrates that MDPA SAMs have been generated on the Zr/Ti substrates, and each step of the procedure was characterized by XPS. In Fig. 3, the right part shows XPS spectra of Zr 3d, S 2p, and P 2p regions for (a) the hydroxylated Ti substrate, (b) the Zr/Ti substrate, and (c) the MDPA SAM on the Zr/Ti substrate. In addition, there is no chlorine signal in the XPS survey spectrum, and the peak position of Zr 3d is very close to that of bulk zirconium dioxide, suggesting that the Zr anchored onto the Ti substrate is probably in the form of zirconium oxide. Especially in the spectrum of the Zr 3d (c), the P 2s peak at 191.5 eV and Zr 3d peak appear to simultaneously add to the S 2p and P 2p signals, suggesting that the MDPA SAM has successfully formed on the Zr/Ti substrate. While the exact conformation of the ion layer is still unclear, it is thought to be made up of Ti-O-Zr bonds25 that mediate the well-ordered phosphonate mono- or multilayer formation40.


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Figure 3. Schematic drawing of the preparation procedure of MDPA SAM on zirconated a Ti substrate and the corresponding XPS spectra of Zr 3d, S 2p, and P 2p regions: (a) alkaline pretreated titanium substrate; (b) modification of Zr ions; (c) preparation of MDPA monolayer. The detailed peak positions are Zr (3d5/2 at 182.6 eV), S (2p at 163.6 eV), and P (2s at 190.9 eV; 2p at 133.1 eV).

Investigation of SAM stability on Ti and Ti with Zr modification.

Ti is covered by a native oxide layer under ambient conditions. Unlike the strong and stable interaction between alkylphosphonic acids and ZrO2,41 the SAM of phosphonic acid formed on untreated Ti substrate is weak and can be removed by rinsing.42 To verify the stability of the SAMs formed on the Zr/Ti and native oxide Ti, both were immersed in water or in a 10 mM PBS (pH 7.4) solution and incubated for 2 weeks. 14 ACS Paragon Plus Environment

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In previous studies,19, 20 SAMs with hydrophobic terminal groups that were kept in water or a saline solution exhibited decreasing CA values because of disorder and/or desorption of the adsorbate molecules. Our CA experiments also indicate that some fraction of the ODPA molecules desorbed from the native oxides of Ti substrate after immersion in water and saline solution for a long time. As shown in Fig. 4 (a), ODPA SAMs that assembled on Ti substrates directly are stable at ambient atmosphere, which is an advantage over the traditional thiol SAMs on gold.43 In the case of ODPA SAMs on Ti both in water and in PBS, there is a significant drop in CA from ~110° to ~90° and ~70°, respectively. These results clearly indicate that there are the ODPA SAM formed on Ti substrate is damaged or desorbed during the immersion period. However, for ODPA SAM on the Zr/Ti, the CA values were constant after immersion in PBS for up to 2 weeks, as shown in Fig. 4 (b). These results suggest the ODPA SAM formed on the Zr/Ti is much more stable under physiological conditions.


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Figure 4. Contact angle data collected during immersion of ODPA SAMs on native oxides of Ti (a), and zirconated Ti substrates (b) in water and PBS solution for up to 15 days.

To fully explore the stability of SAMs, ODPA SAMs on Ti or Zr/Ti were characterized by XPS before and after immersion in aqueous solution. Tab.1 provides the elemental concentration data of ODPA SAMs formed on Ti or Zr/Ti substrates before and after immersion in water and PBS for 2 weeks. For SAMs on Ti, obvious decreases in elemental P and C concentrations were observed after immersion in both water and PBS for 2 weeks. Over the same time period, elemental Ti and O concentrations increased, indicating that a considerable part of the adsorbates


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detached from the Ti substrates. However, for ODPA SAMs on the Zr/Ti substrates, there was no obvious difference in any elemental concentration after immersion in both water and PBS solution. These observations confirmed that the phosphonate SAMs are highly insoluble on Zr/Ti substrates in water and saline solutions.

Table 1. Atomic concentrations (%) of ODPA SAMs immersed in water and in PBS solution for 2 weeks, determined by XPS. C 1s

O 1s

Ti 2p

P 2p

SAM on Tia

47.8±0.6

41.2±0.2

8.9±0.6

2.1±0.5

Immersion in water

40.4±1.2

45.9±1.1

12.1±0.9

1.6±0.8

Immersion in PBS

35.7±0.7

48.9±0.9

14.2±1.2

1.2±0.1

on 50.8±0.3

34.1±0.8

9.4±0.5

2.5±0.4

3.2±0.5

SAM Zr/Tib

Zr 3d

Immersion in water

50.5±0.8

34.6±1.0

9.4±0.8

2.4±0.3

3.1±0.4

Immersion in PBS

50.1±1.9

35.2±1.5

9.3±1.0

2.3±0.2

3.1±0.3

a

ODPA SAM on native oxides of titanium substrate. b ODPA SAM on zirconated titanium substrate.

According to the data in Tab.1, the extent of desorption of the ODPA SAMs were estimated by Eq.(1),

Desorption extent =

: :

:

Eq.(1) 17

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in which “0” and “t” refer to the conditions before and after the 2 week incubations, respectively. Using the decreased substrate screening caused by removal of the adsorbates, we calculated that 44.1% and 64.2% of ODPA molecules were desorbed after immersion in water and PBS, respectively, for 2 weeks. However, in the case of SAMs on the Zr/Ti, the extent of desorption was minimal even after 2 weeks of immersion in PBS. An analogous trend was found for the ratio of C 1s to Ti 2p. These results indicate that the amount of adsorbates detached from the Ti substrates in water are lower compared to those in PBS, which is in excellent agreement with previously reported data.43 In addition, these data are also consistent with our CA measurements, suggesting that the ODPA SAMs on the Zr/Ti substrates are extremely stable in aqueous solution, while the SAMs on native oxide Ti surfaces are not.

Photodegradation and photopatterning of phosphonate SAMs on Zr/Ti.

Photodegradation of SAMs of ODPA on Zr/Ti was conducted by exposure to 254 nm UV light. For comparison, photodegradation was also performed for ODPA SAMs on native oxides of Ti. As shown in Fig. 5, there is a sharp decline in the CA of ODPA SAM on Ti, in agreement with the work of other authors.29 Although the decline rate was slightly lower than that of the SAMs on Ti, a similar trend was observed in the CA values of SAMs on Zr/Ti. The mechanism of photodegradation of phosphonate SAMs on titanium oxides is discussed elsewhere.29, 44 Here we speculate that the Zr4+ layer that exists between the adsorbates and the Ti substrate slightly reduces the photocatalytic effect of the Ti substrates. After 150 min, the CA value decreased to 18 ACS Paragon Plus Environment

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lower than 10°, indicating that photodegradation was complete for ODPA SAMs on both of the substrates. This photocatalytic property makes the Zr/Ti and the native oxides of Ti ideal surfaces for photopatterning by UV light.

Figure 5. Contact angle data of ODPA SAMs on native oxides of Ti and zirconated Ti substrates during UV exposure.

Fig. 6 (a) illustrates the XPS spectra in the C 1s region of MDPA SAM before and after exposure to UV light for 2 h. After photodegradation, the C 1s peak area diminished and exhibited asymmetry, which could be attributed to the generation of an oxide compound on the surface. However, a substrate without SAMs would tend to adsorb adventitious oxycarbide contaminants from the atmosphere due to its high surface energy.45 At the same time, as seen from Fig. 6 (b), the peak for the thiol sulfur of the MDPA SAM is mainly at 163.5 eV with some weak peaks at higher binding energy, which could be attributed to the unavoidable oxidized form of sulfur. However, after UV irradiation, the peak for sulfur is completely shifted to 169 eV with no signal at the previous position, and the peak area dramatically decreased. These results indicate not only thorough photooxidation but also photocleavage of the 19 ACS Paragon Plus Environment

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sulfhydryl groups occur during the UV exposure. This observation is in contrast to our previous study,27 wherein the quantitative conversion of a sulfhydryl to an oxidized form occurs with the same type of SAM on a glass substrate after even 6 h UV exposure. These results suggest that the sulfhydryl groups are oxidized thoroughly. Furthermore, some alkyl chains and terminal groups in SAM molecules are photodegraded mainly by breaking the P-C bond during UV exposure, leaving only the phosphonate groups on the surface. The peak area in the P 2p region remained comparatively unchanged and even increased slightly after the UV exposure, as illustrated in Fig. 6 (c). This observation may be caused by the degradation of the carbon backbones of the MDPA molecules, which may attenuate the phosphorus signal slightly giving rise to a slight increase in the peak area of P 2p. In addition to the peak area, the spectrum was fitted with two components. After UV irradiation, the strength of the peak with higher binding energy was relatively increased, which might be attributed to some oxidized state of phosphorus (i.e., phosphate instead of phosphonate).46 Further, XPS was utilized to investigate the photopatterning of the MDPA SAM covered by a mask, which was the first step in the fabrication of protein arrays (see Scheme1). The XPS detection range covers several feature dimensions of the mask. As shown in S 2p region for the patterned MDPA SAM, Fig. 6 (d), doublet peaks appear approximately 163.5, 166.4, and 168.3 eV representing the sulfhydryl groups, a bivalent form of sulfur, and a tetravalent form of sulfur, respectively. This observation, especially the relatively strong peaks at 163.5 eV, indicates that some sulfhydryl groups are protected by the mask, which means the covered areas retain the 20 ACS Paragon Plus Environment

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ability to couple to amino-functionalized biomolecules using the bifunctional linker MPS.

Figure 6. Illustration of C 1s (a), S 2p (b), and P 2p (c) XPS spectra of MDPA monolayers on zirconated Ti substrates before and after irradiated by 254 nm UV light for 2 h. Additionally, the S 2p spectra of patterned MDPA monolayer using a mask during UV exposure is shown in (d). The fitted peak positions are as follows: (a) C 1s at 284.8, 186.1 eV before exposure; 284.8, 286.3, 287.5, 288.8 eV after exposure; (b) S 2p at 163.5 and 164.7, 166.4 and 167.6, 168.3 and 169.5 eV before exposure; 168.4, 169.6 eV after exposure; (c) P 2p at 133.1, 134.9 eV before exposure; 133.3,


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134.4 eV after exposure; (d) S 2p at 163.6 and 164.8, 166.4 and 167.6, 168.2 and 169.4 eV.

Backfilling with a protein-resistant molecule, P-10EG.

To generate well-defined protein patterns by photolithography, the exposed regions were backfilled with P-10EG, a protein resistant molecule.47 The success of backfilling the photodegraded regions with other adsorbates has been shown elsewhere.29, 46 In the previous study, photooxidized regions were backfilled by immersing the specimens in a second phosphonic acid solution.29 In our case, the thoroughly oxidized ODPA SAMs were immersed into a solution of ODPA for 24 h, and a CA of 108.2±1.3° was recorded, indicating an ideal recovery of the monolayer. The XPS spectra in the C 1s region for MDPA SAM after UV exposure for 2 h are illustrated in Fig. 6 (a). While after backfilling with P-10EG molecules, the XPS in the C 1s region, Fig. 7 (a), shows mainly two peaks: one at 284.8 eV, attributed to the methylene of the alkyl chain, and another at 286.4 eV, corresponding to the units of the oligo(ethylene glycol). These results are indicative of successful backfilling of the UV exposed areas with P-10EG molecules.


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Figure 7. (a) C 1s XPS spectrum of MDPA SAM on zirconated Ti substrate backfilled with P-10EG after UV exposure for 2 h; the fitted peak positions are: 284.8, 286.4, 287.4, 288.8 eV. (b) N 1s XPS spectra of (і) P-10EG assembled on zirconated Ti, (іі) surface (і) after exposure to BSA, (ііі) UV irradiated MDPA SAM, (іv) surface (ііі) after exposure to BSA, (v) UV irradiated MDPA SAM and backfilled with P-10EG, (vі) surface (v) after exposure to BSA; the peak position is at 400.1 eV.

Adsorption of proteins was compared on the following three surfaces: P-10EG assembled on Zr/Ti, MDPA SAM exposed to UV for 2 h, and backfilled with P-10EG after UV exposure. As shown in Fig. 7 (b), no nonspecific attachment of proteins was observed on the SAM of P-10EG (i), while the MDPA SAM exposed to UV (ііі)


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shows significant increases in N 1s signal following exposure to BSA (іv). Conversely, for the sample backfilled with P-10EG after UV exposure (v), the nitrogen signal (vі) was much weaker than that in curve (іv). It is clear that P-10EG assembled on zirconated Ti substrates resists the adsorption of protein from solution. However, backfilling with this molecule to a UV irradiated area is not as effective as the primary SAM in blocking non-specific protein adsorption, which might be due to the defects in the backfilled SAM. Covalent attachment of protein by MPS and fluorescent protein patterning.

Immobilization of biomolecules containing amino group on the patterned SAM was conducted using MPS molecules that can link amino and sulfhydryl groups. The terminal sulfhydryl groups of MDPA SAMs exposed to UV light could no longer anchor MPS molecules.27 Thus, we hypothesized that MPS, and subsequently, proteins, would only anchor to the regions protected by the mask during the UV irradiation process. Meanwhile, MDPA molecules within the exposed regions would be degraded and replaced by the protein resistant molecule P-10EG to create a bioinert background.

A fluorescent protein binding assay was conducted to demonstrate the versatility of this method for constructing protein patterns, and a fluorescence microscope was used to record the protein adsorption behavior on the patterned SAMs. However, Ti substrates can quench fluorescence signals, and, though this quenching would suppress background fluorescence, it could still complicate the microscopy.33 To 24 ACS Paragon Plus Environment

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reduce the effects of quenching, a multistep method for fabrication of fluorescent protein pattern was designed to increase the distance between the labelled protein and the Ti substrate, as shown in Fig. 8 (a). The optical image of the mask covered the SAMs under UV exposure as shown in Fig. 8 (b). The patterned SAM surfaces were backfilled with P-10EG and treated with MPS solution permitting the immobilization of biomolecules in the masked regions while the irradiated regions remained protein-resistant. After sequential attachment of the biomolecules, observation by a confocal fluorescence microscope clearly indicates selective immobilization of the fluorescent protein within the covered regions, as illustrated in Fig. 8 (c). In this case, bright color duplicates the covered regions, indicating specific protein immobilization. The exposed areas are dark due to the protein resistance properties of P-10EG. In addition, a line profile of fluorescence intensity of the pattern shows clear periodicity, indicating the successful patterning of protein using the presented strategy.

Figure 8. (a) Schematic drawing of the multistep protein immobilization protocol. (b) Optical image of the mask covered the SAMs under UV exposure. (c) Fluorescence 25 ACS Paragon Plus Environment

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image and corresponding intensity profile of the NH2-biotin immobilized onto regions protected by the mask under UV irradiation using a bifunctional linker, streptavidin, mouse IgG/biotin (IgG/biotin), and subsequently by goat anti-mouse IgG/Alexa Fluor 488 (αIgG/488).

CONCLUSIONS In this study, we demonstrated the formation of stable phosphonic acid SAMs on Ti substrates mediated by Zr ions. Using photolithography, this type of SAM enabled the fabrication of surfaces functionalized with patterns of fluorescent proteins. Zirconium ions, used here as a reinforcing modification, can strongly interface between the native oxides of the Ti substrate and the organic phosphonic acid. CA and XPS data revealed near perfect stability of the ODPA SAMs formed on the Zr/Ti substrates when subjected to water and PBS immersion. MDPA molecules with sulfhydryl terminal group were synthesized and used to form ordered and stable monolayers with a sulfhydryl surface on the Zr/Ti substrate. Exposure of the MDPA SAMs to UV light in air leads to both photooxidation and photodegradation of the adsorbate molecules. Moreover, backfilling of the irradiated SAMs with glycol-terminated P-10EG molecule proved to be an effective way to build a bioinert background that could resist nonspecific protein adsorption. Photopatterning of the SAM surfaces was performed using a mask under UV exposure, and the photodamaged regions were backfilled with P-10EG molecules. Thus, the as-prepared surface could be used to establish fluorescent protein patterns through the heterobifunctional cross-linker MPS or guide biomolecules to immobilization in specific regions. The present study has 26 ACS Paragon Plus Environment

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therefore offered a method for improving the stability of phosphonate SAMs on titanium surfaces and verified the feasibility of biomolecule immobilization to bioactivate the surfaces of titanium implants. AUTHOR INFORMATION Corresponding Author * Tel.: +86-(0)755-26036026; Fax: +86-(0)755-26036026. E-mail addresses: [email protected]; [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENTS The work was supported by the National Nature Science Foundation of China (grant nos. 21273126). TH also thanks the National Research Fund for Fundamental Key Projects No. 973 (2011CB933200) and the Hundred-Talent Program of the Chinese Academy of Sciences.

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Scheme 1. Fabrication procedure for the fluorescent protein arrays created by sulfhydryl terminated SAMs on zirconated Ti substrates. (a) alkaline pretreated titanium substrate; (b) modification with Zr ions; (c) preparation of the MDPA monolayer; (d) sulfhydryl terminated SAMs covered with a patterned mask are exposed to 254 nm UV light; (e) SAMs in exposed areas are degraded by UV irradiation; (f) the patterned surface is backfilled with the protein-resistant molecule, P-10EG; (g) bifunctional linker MPS molecules are selectively anchored to sulfhydryl groups in the unexposed areas; (h) a multistep biomolecule immobilization protocol is used to generate fluorescent patterns. 196x117mm (300 x 300 DPI)

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Formation of Highly Stable Self-Assembled Alkyl Phosphonic Acid

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