May 2008
Published by the American Chemical Society
Volume 9, Number 5
Copyright 2008 by the American Chemical Society
Communications Zwitterionic Polymers Exhibiting High Resistance to Nonspecific Protein Adsorption from Human Serum and Plasma Jon Ladd, Zheng Zhang, Shengfu Chen, Jason C. Hower, and Shaoyi Jiang* Department of Chemical Engineering, University of Washington, Box 351750, Seattle, Washington 98195 Received November 26, 2007; Revised Manuscript Received January 21, 2008
This study examined six different polymer and self-assembled monolayer (SAM) surface modifications for their interactions with human serum and plasma. It was demonstrated that zwitterionic polymer surfaces are viable alternatives to more traditional surfaces based on poly(ethylene glycol) (PEG) as nonfouling surfaces. All polymer surfaces were formed using atom transfer radical polymerization (ATRP) and they showed an increased resistance to nonspecific protein adsorption compared to SAMs. This improvement is due to an increase in the surface packing density of nonfouling groups on the surface, as well as a steric repulsion from the flexible polymer brush surfaces. The zwitterionic polymer surface based on carboxybetaine methacrylate (CBMA) also incorporates functional groups for protein immobilization in the nonfouling background, making it a strong candidate for many applications such as in diagnostics and drug delivery.
Introduction Protein adsorption is thought to be the first step in the inflammatory response, leading the way for platelet adhesion and thrombosis.1–3 For medical implants and in vivo sensors, limiting thrombosis and material encapsulation is paramount, due to a decrease in performance associated with encapsulation. For in vitro sensors, thrombosis and fibrous encapsulation with avascular tissue typically do not occur because of the short duration of contact between the sensor surface and blood components. The nonspecific interactions of proteins with the sensor surface can, however, produce false positive or negative results and limit the accuracy and precision of such instruments. This creates the need for sensor surface modifications that can significantly resist the nonspecific adsorption of proteins from applications in human media or other complex media. The majority of previous studies characterizing surfaces for protein resistance have focused on simple protein solutions, typically fibrinogen from buffer.4–10 These studies demonstrate surfaces with resistance to fibrinogen or other single protein solutions but do not investigate the interactions of complex protein solutions, such as human plasma, with surface modifications. Other studies have investigated platelet adhesion11–13 or adsorp* To whom correspondence
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tion of individual proteins from plasma14–16 on surfaces with varying modifications. Few studies have considered total protein adsorption from complex matrices. Ma et. al prepared ethylene glycol-containing polymer brush surfaces and tested the adsorption from fetal bovine serum (FBS) on the surface. They saw an immeasurable adsorption response from both 10% and 100% FBS, indicating that the ethylene glycol-based polymer brush has low fouling characteristics.17 While the nonspecific adsorption from FBS is important for designing surfaces for cell culture, medical diagnostics and disease discovery applications typically rely on human serum or plasma as media. These two media pose significant challenges for their nonspecific adsorption on the surfaces of diagnostic devices or implanted materials. Because of the high frequency of use of serum and plasma samples in the medical industry, surfaces capable of resisting nonspecific adsorption from these media are needed. Benesch et. al tested ethylene glycol-based self-assembled monolayers (SAMs) with both a 67% human plasma solution and a 67% human serum solution. They found the amount of nonspecific adsorption on the surfaces depended on the number of ethylene glycol units in the alkyl thiol chains.18 As the number of ethylene glycol chains increased from 2 to 4 to 6, the nonspecific resistance from heparinized plasma decreased from ∼1500 pg/ mm2 to ∼900 pg/mm2 to ∼500 pg/mm2. However, the surfaces based on ethylene glycol that were investigated were not able
10.1021/bm701301s CCC: $40.75 2008 American Chemical Society Published on Web 04/01/2008
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to completely resist nonspecific adsorption from complex media. To develop a surface adequate for use in human media, with adsorption levels of 99% pure), seen in Figure 1, in a nitrogen-purged 1:1 water/methanol solution and BPY in nitrogen-purged methanol were transferred to the tube using a syringe. After reacting for 1 h, the SPR chip was removed and rinsed with ethanol and water. Samples were stored in DI water overnight. Nonspecific Protein Adsorption. Sample solutions were flowed for 10 min on a custom-built SPR sensor from the Institute of Photonics and Electronics, Academy Sciences (Prague, Czech Republic), described previously.29 Briefly, the SPR sensor is based on the Kretschmann geometry of the attenuated total reflection (ATR) method and wavelength modulation. For the SPR sensor used in the study, a 1 nm SPR wavelength shift at 750 to 751 nm represents a surface coverage of ∼150 pg/mm2 for proteins. Pooled human plasma and serum were purchased from BioChemed Services (Winchester, VA). X-ray Photoelectron Spectroscopy (XPS). Gold-coated silicon chips were used for XPS analysis. The procedure for SAM formation was identical to that for SPR chips. XPS analysis was done using a Surface Science Instruments S-probe equipped with a monochromated Al KR X-ray source. The energy of emitted electrons is measured with a hemispherical energy analyzer at pass energies ranging from 50 to 150 eV. All data were collected at 55° from the surface normal takeoff angle. A survey scan was used to determine the elemental composition present on the surface. Multiple spots were analyzed from a single sample and averaged. Data analysis software was from Service Physics, Inc. Ellipsometry. Ellipsometry was performed on a spectroscopic ellipsometer (Sentech SE-850, GmbH). Sample preparation was identical to XPS experiments. A bare gold-coated silicon chip was used as a reference. Five separate spots were measured at three different angles of incidence (50, 60, and 70°) in the visible region. The thicknesses of the studied films were determined using the Cauchy layer model with an assumed refractive index of 1.45.
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Figure 2. Sensorgrams showing the sensor response to a fibrinogen solution (1 mg/mL) on a SAM and polymer-modified surface containing ethylene glycol units. Fibrinogen adsorption on all of the studied materials was below measurable quantities.
Results and Discussion Polymers were formed using atom transfer radical polymerization (ATRP) of monomers illustrated in Figure 1. ω-Mercaptoundecyl bromoisobutyrate (Br-thiol) was synthesized as previously described30 and formed into a SAM on a gold surface. The ATRP process creates polymer brushes on the surface with control over the chain length and surface density of the polymer. The polymerization reaction was carried out in a mixture of water and methanol. Monomers were polymerized on a pure Br-thiol SAM surface in the presence of catalysts CuBr and bipyridine for 1 h. The time of reaction was sufficient to create a high density polymer but maintain a thickness suitable for use in a SPR sensor. The sensor used for analysis has a light beam set at a fixed angle of incidence on the surface. As the polymer thickness increases on the surface, the angle of incidence must be increased relative to the surface normal to provide data. The physical limitations of the setup dictate that polymer brushes that are over ∼60 nm thick will be outside of the detection capabilities of the SPR sensor. Pure SAM surfaces of OEG-terminated alkyl thiols or mixed SAMs of chargeterminated alkyl thiols were prepared overnight in ethanol. For surface modifications on gold, a SPR sensor is an ideal choice for studying protein adsorption, due to the dependence of the SPR phenomenon on a thin metallic film, typically gold. In this work, polymers based on zwitterionic materials are developed and characterized on gold for nonfouling properties in human serum and plasma using a SPR sensor. Fibrinogen, pooled human plasma in citrate phosphate dextrose (CPD), and pooled human serum were used to study the protein resistance of each surface. Fibrinogen, a component in blood typically thought to be important in the inflammatory response, is commonly used to evaluate the nonfouling characteristics of surfaces.4,5 Figure 2 shows typical sensor responses for fibrinogen adsorption on an OEG SAM and the poly OEGMA surfaces. The bulk refractive index change from the fibrinogen solution is responsible for the increase and equal decrease seen in these sensorgrams. The net responses from fibrinogen for the OEG SAM and the poly OEGMA surface were shown to be 3.9 ( 8.2 pg/mm2 and 0.3 ( 1.8 pg/mm2, respectively. All surfaces showed a similar resistance to fibrinogen adsorption. Therefore, if fibrinogen was used as the evaluation criterion, each of the studied surfaces would be considered nonfouling. Poly OEGMA was previously synthesized using ATRP by Ma, et al. To compare the nonfouling characteristics of the poly OEGMA produced via ATRP in this study with that by Ma, et
Figure 3. SPR sensor response for (A) nonspecific adsorption of 10% human serum in PBS and 100% human serum and (B) nonspecific adsorption of 10% human plasma in PBS and 100% human plasma. Surfaces include self-assembled monolayers of oligo(ethylene glycol) (OEG), mixed trimethylamine and sulfonic acid (TMA/SA), and mixed trimethylamine and carboxylic acid (TMA/CA), and ATRP-created polymers of oligo(ethylene glycol) methacrylate (OEGMA), sulfobetaine methacrylate (SBMA), and carboxybetaine methacrylate (CBMA). A full monolayer of protein on the surface is equivalent to ∼2700 pg/ mm2. Error bars represent the standard error of the mean.
al., the protein resistance of the poly OEGMA surface was tested using fetal bovine serum solutions at different concentrations. The nonspecific adsorption to fibrinogen, a 10% FBS solution and a 100% FBS solution were –3.2 ( 2.9 pg/mm2, –4.6 ( 4.6 pg/mm2, and –0.4 ( 2.8 pg/mm2, respectively, in the study by Ma et al. In this study, we found nonspecific adsorption to these different media to be 0.3 ( 1.8 pg/mm2, –3 ( 0.5 pg/mm2, and –4.2 ( 2.4 pg/mm2. The values obtained in this study, as with those published by Ma et al., are close to the detection limits of the instruments used in this work (∼3 pg/mm2) and in the work by Ma et al. (∼10 pg/mm2). Therefore, the ATRP reaction produces poly OEGMA surfaces with similar protein resistance characteristics as previous studies in the literature.17 Fibrinogen adsorption was used to test these surfaces for their resistance to a single protein from a buffer solution. Human serum and plasma, however, are components of human blood, comprised of complex mixtures of hundreds of proteins.31 Figure 3a,b show sensor responses to human serum and human plasma, respectively. The 10% serum and plasma solutions are often used for clinical medical diagnostics. The human serum used for this study has no added anticoagulant, while the human plasma has CPD added. Poly CBMA demonstrated the best nonspecific resistance to the test media. The poly SBMA showed nonspecific adsorptions in-line with the poly OEGMA. The zwitterionic polymer surface modifications are ∼10–15 nm thick,9 while the poly OEGMA surfaces have a thickness of 20–25 nm, as measured by ellipsometry. XPS results corroborate this thickness difference. The Au percentage, seen from the underlying gold film on the sensor chip, from analyzing a survey scan of the zwitterionic polymers was ∼1.5%, while the Au
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percentage for the poly OEGMA surface was
