LIST OF ABBREVIATIONS
DSC N,N’-disuccinimidyl carbonate
EA Early antigen
EBNA Epstein-Barr nuclear antigen
EBV Epstein-Barr virus
EDC N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
EG Ethylene glycol
ELISA Enzyme-linked immunosorbent assay Fbg Fibrinogen
FBS Foetal bovine serum
FTIR ATR Fourier Transform Infrared attenuated total reflection
FTIR GAATR Fourier Transform Infrared Grazing Angle Attenuated Total Reflection FTIR GASR Fourier Transform Infrared Grazing Angle Specular Reflectance HEMA 2-hydroxyethyl methacrylate
HOEGMA Oligo(ethylene glycol) methacrylate HPLC High performance liquid chromatography HPM 2-hydroxypropyl methacrylate
HPMA N-(2-hydroxypropyl methacrylamide)
HRGP Histidine-rich glycoprotein
HSA Human serum albumin
HTC High capacity trap
I.M. Infectious mononucleosis
IFA indirect fluorescent antibody assay Ig muC Ig mu chain C region
IgG Immunoglobulin G
Kd Disociation constant
Lys Lysozyme
Me4Cyclam 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane Me6TREN Tris[2-(dimethylamino)ethyl]amine
MeOEGMA Oligo(ethylene glycol) methyl ether methacrylate Mn Number average molecular weight
MS Mass spectrometry
Ms-Cl Methanesulfonyl chloride
Mw Weight average molecular weight NAv Neutravidin NC Nancapsules
NC@MeOEGMA Nanocapsules coated with poly(MeOEGMA) brushes
NH2 11-amino undecanethiol
NHS N-hydroxysuccinimide
NMP Nitroxide mediated polymerisation
NMR Nuclear magnetic resonance
NPC 4-nitrophenylchloroformate
NS Nanospheres OEG2 Di(ethylene glycol) undecanethiol OEG4 Tetra(ethylene glycol) undecanethiol OEG6 Hexa(ethylene glycol) undecanethiol OEGn (Ethylene glycol)n undecanethiol ONC Oligonucleotide PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffered saline, pH 7.4 PCL Poly(ε-caprolactone)
PCMA Phosphorylcholine methacrylate
PCR Polymerase chain reaction
PEG Poly(ethylene glycol)
PEG-COOH α-amino ω- carboxy poly(ethylene glycol) PEG-OH α-amino ω- hydroxy poly(ethylene glycol) PEG-OME α-amino ω- metoxy poly(ethylene glycol) PIF Powder infant formula
PP Polypropylene PSN Plasma sputtered nylon
QELS Quasi elastic light scattering
RAFT Reversible addition fragmentation transfer polymerisation
RF Flory's radius
RF Radiofrequency
RMS Root mean square
RNA Ribonucleic acid
Rp Rate of propagation
RαM IgG Rabbit anti-mouse IgG antibody SA Sodium acetate buffer
SAM Self-assembled monolayer
Sav Streptavidin SB Sodium borate buffer
SBMA Sulfobetaine methacrylate
SDS Sodium dodecyl sulphate
SET-LRP Single-electron transfer living radical polymerisation SI-ATRP Surface initiated atom transfer radical polymerisation SPR Surface plasmon resonance
TEM Transmission electron microscopy
THF Tetrahydrofuran
Tos-Cl 4-toluenesulfonyl chloride
TRIS TRIS buffer
TSTU N,N,N’,N’-tetramethyl-o-(N-succinimidyl) uronium tetrafluoroborate VCA Viral capsid antigen
LIST OF ABBREVIATIONS
XPS X-rays photoelectron spectroscopy θadv Advancing water contact angle θrec Receding water contact angle
λres Surface plasmon resonace wavelenght
Appendix I:
Interaction of Blood Plasma with
Antifouling Surfaces
Interaction of Blood Plasma with Antifouling Surfaces
C. Rodriguez Emmenegger,*,†,‡E. Brynda,†T. Riedel,†,§Z. Sedlakova,†M. Houska,† and A. Bologna Alles‡
†Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, v.v.i., 162 06 Prague, Czech Republic,‡Departamento Ingenieria de Materiales, Facultad de Ingenieria, UDELAR, Montevideo
11300, Uruguay, and§Institute of Haematology and Blood Transfusion, 128 20 Prague, Czech Republic Received January 8, 2009. Revised Manuscript Received March 20, 2009
Nonspecific adsorption of proteins is a crucial problem in the detection of analytes in complex biological media by affinity sensors operating with label-free detection. We modified the gold surface of surface plasmon resonance (SPR) sensors with three types of promising antifouling coatings: self-assembled monolayers (SAM)s of alkanethiolates terminated with diethylene glycol and carboxylic groups, poly(ethylene glycol) (PEG) grafted onto the SAMs, and zwitterionic polymer brushes of poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), and poly(phos-phorylcholine methacrylate). Using SPR, we compared the efficacy of the coatings to reduce nonspecific adsorption from human blood plasma and from single-protein solutions of human serum albumin, immunoglobulin G, fibrinogen, and lysozyme. There was no direct relationship between values of water contact angles and plasma deposition on the coated surfaces. A rather high plasma deposition on SAMs was decreased by grafting PEG chains. Fouling on PEG was observed only from plasma fractions containing proteins with molecular mass higher than 350 000 Da. The adsorption kinetics from plasma collected from different healthy donors differed. Poly(carboxybetaine methacrylate) completely prevented the deposition from plasma, but the other more hydrophilic zwitterionic polymers prevented single-protein adsorption but did not prevent plasma deposition. The results suggest that neither wettability nor adsorption of the main plasma proteins was the main indicator of deposition from blood plasma.
Introduction
Surfaces capable of specific binding of selected biological compounds and resistant to nonspecific binding of other com-pounds are required for many biotechnological applications.
In particular, affinity biosensors for label-free detection cannot differentiate between the specific response to the binding of analytes and the nonspecific response to the deposition of other compounds from the tested media. The problem is quite restric-tive when the measurement is to be performed in complex media containing a variety of proteins, such as blood plasma or serum.
Biosensors capable of detection in complex biological fluids can be prepared by the immobilization of biorecognition elements on the transducer surface premodified with a nonfouling coating.
Basic requirements for such a coating are a minimum interfacial free energy with water and zero surface charge to prevent protein adsorption via hydrophobic and ionic interactions, respectively.
The presence of reactive binding sites is necessary for the attach-ment of bioreceptors.
Antifouling surfaces have been mostly tested only for the adsorption of main plasma proteins, particularly serum albumin and fibrinogen, from single-protein solutions. Blood plasma and serum fouling on various antifouling coatings prepared on gold surfaces was studied recently using ellipsometry1and surface plas-mon resonance (SPR)2,3 on various antifouling coatings pre-pared on gold surfaces and using optical waveguide light mode
spectroscopy for serum fouling on silica-titania surfaces coated with poly(L-lysine)-graft-poly(ethylene glycol) or dextran.4
Currently, the most frequently used commercial SPR sensor chips are coated with carboxymethylated dextran covalently attached to a gold surface,5providing an excellent matrix for the covalent attachment of bioreceptors but lacking sufficient resistance to serum fouling.2
Another approach utilizes alkanethiols terminated with func-tional groups that can form self-assembled monolayers (SAM) on a gold surface. Whereas the molecules are chemisorbed on gold via terminal thiol groups, dispersion forces between long hydro-carbon (C11-C15) chains promote their ordering, resulting in the close packing of functional groups on top of the SAM. Mixed SAMs of alkanethiolates terminated with oligo(ethylene glycol) (OEG) and alkanethiolates terminated with reactive carboxylic groups capable of covalent binding bioreceptors are now widely used for the preparation of SPR sensors. The resistance of OEG SAMs to fibrinogen adsorption is controlled by external hydro-philicity due to terminal-OH groups, internal hydrophilicity due to the hydration of oligoether chains,6and the surface packing density, which can be adjusted by the conditions of SAM preparation.7Whereas OEG SAMs appeared to be resistant to the adsorption of fibrinogen from solution, a serious deposition of considerable amounts of proteins from blood plasma and serum has been observed.1,8
Poly(ethylene glycol) (PEG) is a hydrophilic polymer that is frequently used to prevent protein adsorption and cell adhesion.
The two most common theories explaining its resistance are based
*Corresponding author. E-mail: crodrig@fing.edu.uy.
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Published on Web 5/1/2009
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on the steric repulsion and water barrier effects resulting from the structuring of water in the environment surrounding PEG chains.9,10Nanoscale interaction between end-grafted PEG and serum albumin indicated that the repulsive net force is much larger than that predicted by steric theories based on configura-tional entropy.11The attachment of linear PEG (mw = 2000-5000 Da) to gold surfaces prevented albumin adsorption.12SPR measurements of PEG surfaces grafted onto gold and oligo (ethylene glycol) methyl ether methacrylate brushes8 indicated reduced fouling from blood plasma.
Zwitterionic polymers are nowadays the most promising can-didates for the preparation of antifouling surfaces because of their high hydration capacity and electroneutrality. They resist non-specific protein adsorption13,14from model solutions as well as cell adhesion.8,15Ultralow blood plasma and serum fouling on gold SPR sensors coated with various zwitterionic polymers was reported recently.3,16-18
In general, rather little is known about the composition of protein deposits from blood plasma and serum. Deposits from blood plasma onto SAMs terminated with-CH3,-OC(O)CF3, -OSO3-,-COO-, and-OH groups were analyzed using appro-priate antibodies.19The lowest deposits were observed on hydro-xyl surfaces with complement component C3c, FXII, and lipoproteins being the major constituents. Another study showed that SAMs terminated with OEG bound small amounts of fibrinogen from a single protein solution but large amounts of proteins, including C3, from serum and plasma.1
In this work, we investigated the deposition of human blood plasma and model solutions of the main plasma proteins albumin, IgG, and fibrinogen on SPR sensors modified with three typical groups of antifouling coatings containing carboxylic groups for the attachment of bioreceptors: SAMs, PEG grafted onto SAMs, and zwitterionic polymers. The main parameters measured were wettability and the level of deposition. Because there has been no direct comparison of antifouling properties of these coatings in the literature, we compared the effectiveness of these coatings to reduce nonspecific adsorption on SPR sensors and tried to better identify factors affecting plasma fouling and features that might be significant in the preparation of sensors capable of detecting analytes in blood plasma.
Experimental Section
Materials. Reagents. Diethylenglycol alkanethiol HS-(CH2)11-EG2-OH and triethylenglycol alkanethiol carboxy-ter-minated HS-(CH2)11-EG3-OCH2COOH were from ProChimia, Poland. 16-Mercaptohexadecanoic acid (C16, 5 mM solution in EtOH) was purchased from Sigma-Aldrich as Nanothink Acid 16.N,N,N0,N0-Tetramethyl-o-(N-succinimidyl) uronium tetraflu-oroborate (TSTU, 98%) and R-bromoisobutyl bromide (97%) were from Fluka. Glutaraldehyde (25%), 2-(N,N0-dimethyamino)
ethyl methacrylate (DMAEM, 98%), 2-[(methacryloyloxy)ethyl]
dimethyl-3-sulfopropyl ammonium hydroxide (SBMA, 97%), copper(I) bromide (99.999%), 11-mercapto-1-undecanol (97%), and 2,20-bipyridine (BiPY, 99%) were from Sigma-Aldrich.
2-(Methacryloyloxy)ethyl 2-(trimethylammonio) phosphate (PCMA) andβ-propiolactone were from Vertellius Speciallities and Serva electrophoresis GmbH, respectively.R-Aminoω -car-boxy poly(ethylenglycol) (NH2-PEG-COOH, mw 3400 Da) was from Nektar Therapeutics. Initiator ω-mercaptoundecylbromoi-subutyrate was synthesized by reactingR-bromoisobutyl bromide with 11-mercapto-1-undecanol according to the method pub-lished previously,20 and 2-carboxy-N,N-dimethyl-N-(20 -metha-cryloylethyl) ethanaminium inner salt (CBMA) was synthesized according to the reaction described in Zhang et al.21Dried THF was used instead of acetone as the solvent for the reaction.
Biochemicals.Human serum albumin (HSA, 99% by electro-phoresis), fibrinogen (Fbg) and lysozyme (Lys) were from Sigma. Human IgG (IgG) were from Seva Imuno, Prague, Czech Republic. Citrated human plasma from single healthy donors was purchased from the Institute of Haematology and Blood Trans-fusion, Prague, Czech Republic. Plasma was diluted with PBS 1:2 for experiments. Serum was prepared from blood plasma by adding thrombin. A fibrin clot was retracted prior to centrifuga-tion, and serum was stored in a freezer at-18°C.
Solvents.Methanol (99.5%), tetrahydrofuran (THF, 99.5%), and ethanol (96%) were purchased from Lachner, and N,N-dimethylformamide (99.5%) was from Sigma-Aldrich.
Phosphate-buffered saline (PBS, pH 7.4) was used for SPR experiments.
Methods. Fractionation of Blood Plasma.Citrate antic-oagulated plasma was filtrated using a 0.22μm acetate cellulose filter. The filtrate (300μL) was separated by gel filtration on a 301 cm2Superose 12 column (Pharmacia, Uppsala, Sweden) connected to a Shimadzu Prominence HPLC system (Shimadzu, Duisburg, Germany). The column was equilibrated with elution buffer (PBS, pH 7.4). The flow rate was 0.4 mL/min, and eluting peaks were monitored with a UV detector at 280 nm. The fraction size was 0.4 mL, and samples were collected in Eppendorf tubes using Fraction Collector Frac-100 (Pharmacia, Uppsala, Swe-den). The fractions were pooled according to the peaks and checked with SDS-PAGE.
Contact Angle Measurement.The wettability of the samples was examined by a dynamic sessile water drop method using a DataPhysics OCA 20 contact angle system. A 5μL drop was placed on the surface, and advancing and receding contact angles were determined dynamically while the volume of the drop was increased up to 15μL and decreased at a flow rate of 0.5μL/min.
Data were evaluated using a circular fitting algorithm.
Surface Plasmon Resonance (SPR).A custom-built SPR instrument based on the Kretschmann geometry of the attenuated total reflection method and spectral interrogation of SPR condi-tions22was purchased from the Institute of Photonics and Elec-tronics, Academy of Sciences of the Czech Republic, Prague. SPR chips were prepared by vacuum deposition of a gold layer (thickness approximately 50 nm) onto a BK7 glass slide coated with an adhered titanium layer (thickness approximately 2 nm).
The tested solutions flowed via a peristaltic pump through four channels of the flow cell in which SPR responses were simulta-neously measured. The observed shifts in the resonance wave-length, Δλres, reflected changes in the refractive index of the medium at the sensor surface within the penetration depth of the SPR evanescent wave as a result of changes in mass deposited at the sensor surface and the exchanging of solutions of different refractive indexes proportional to different protein and salt con-centrations. The limit of detection (LOD) was estimated to be the (9) McPherson, T.; Kidane, A.; Szleifer, I.; Park, K.Langmuir1998,14, 176–
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baseline noise. The average LOD was 0.03 nm, which roughly corresponds to 6 pg of deposit per mm2. (The value was estimated by comparing the FTIR GASR spectrum of a known amount of HSA deposited on an SPR chip.)
FTIR Grazing Angle Specular Reflectance (FTIR GASR).
FTIR grazing angle specular reflectance spectra of the dried coatings on the gold surface of SPR chips were recorded using a Bruker IFS 55 FTIR spectrometer equipped with a Pike Tech-nologies 80Spec GASR attachment.
Preparation of Surfaces Examined. Gold.The gold-coated SPR chip was rinsed with ethanol and water, dried with nitrogen, and cleaned with a UV-ozone cleaner (Jelight) for 15 min. The procedure was repeated once more, and the chip was immediately used for the SPR experiment or for a subsequent modification.
Self-Assembled Monolayers (SAMs) (I-IV)
(I) 16-mercaptohexadecanoic acid Au-S-(CH2)15-COOH;
(II) diethylene glycol-terminated alkanethiolate, Au-S-(CH2)11-(O-C2H4)2-OH;
(III) mixed Au-S-(CH2)11-(O-C2H4)2-OH/HS-(CH2)15 -COOH, 7:3 (Figure 1);
(IV) mixed Au-S-(CH2)11-(O-C2H4)2-OH/HS-(CH2)11 -(O-C2H4)3-OCH2COOH, 7:3 (Figure 1).
Procedure.The SPR chip was immersed in a 1 mM solution of the respective thiol in ethanol at 40°C for 10 min and then kept in the dark at room temperature for 1 day. The quality of the SAMs did not change if the time was varied from 1 to 7 days.
Carboxy-Terminated Poly(ethylene glycol) (-PEG-COOH) Grafted onto Self-Assembled Monolayers (V-VI).
(V) (III)-PEG-COOH; -PEG-COOH grafted onto SAM III;
(VI) (IV)-PEG-COOH (Figure 1); -PEG-COOH grafted onto SAM IV.
Grafting of NH2-PEG-COOH onto SAMs.An SPR chip that was freshly coated with SAM III or SAM IV was rinsed and sonicated in ethanol and rinsed with DMF. Terminal carboxylic groups of the SAMs were converted intoN-hydroxysuccinimidyl esters by immersing the chips in TSTU solution (2 mg/mL in DMF) for 2 h. The chip was then rinsed with DMF and dichloroethane and incubated overnight with NH2-PEG-COOH solution (1 mg/mL in dichloroethane).
The presence of attached PEG was evidenced by FTIR GASR spectra of the surface (via C-O-C and CdO stretching bands at 1155 and 1730 cm-1, respectively).
Zwitterionic Polymer Coatings (VII-XI).
(VII) poly(carboxybetaine methacrylate) poly(CBMA) (Figure 1);
(VIII) poly(sulfobetaine methacrylate) poly(SBMA) (Fig-ure 1);
(IX) block copolymer poly(SBMA-b-CBMA) (Figure 1);
(X) random copolymer poly(SBMA-co-CBMA);
(XI) poly(phosphorylcholine methacrylate) poly(PCMA) (Figure 1)
The initiator SAMs were formed by soaking the gold-coated SPR chips in a 1.3 mM ethanol solution ofω-mercaptoundecyl bromoisobutyrate for 24 h and then rinsing and sonicating them in pure ethanol. The polymer brushes were grafted from initiator SAMs by surface-initiated atom transfer radical polymerization using BiPy and CuBr or BiPy, CuBr, and CuBr2as catalysts. The monomer was dissolved in the polymerization solvent and degassed by passing a continuous stream of nitrogen through the solution while it was being stirred (20 min). Catalyst was added, and the mixture was further degassed via a nitrogen stream for 15 min.
SPR chips with freshly prepared SAMs of the initiator were place in Schlenk tubes and degassed and refilled with nitrogen.
A polymerization solution was then transferred via syringe into the Schlenk tubes under nitrogen protection. After the
polymer-in Q water. Coatpolymer-ing VII was prepared by polymer-incubatpolymer-ing SPR chips with a solution of CBMA monomer (54.4 mg), CuBr (5.8 mg), and BiPy (12.5 mg) per 1 mL of water/methanol (1:1) for 2 h. Coating VIII was prepared by incubating SPR chips with an aqueous solution of SBMA monomer (83.3 mg), CuBr (5.8 mg), and BiPy (12.5 mg) per 1 mL of water for 1 h. Coating IX was prepared by the two-step block copolymerization of SBMA and CBMA. In the first step, poly(SBMA) was prepared as described for coating VIII, and the chip was rinsed with ethanol. In the second step, the active poly(SBMA) chains attached to the surface were used as macroinitiators for the copolymerization of CBMA from its solution. Polymerization was carried out for 20 min in the same way as described for coating VII. Coating X consisting of a random copolymer of SBMA and CBMA was prepared using the same concentrations of monomer and catalyst as for coat-ing VIII but polymerizcoat-ing from a mixture of 70 mol % SBMA (58.6 mg/mL) and 30 mol % CBMA (19.5 mg/mL). Coating XI was prepared by incubating SPR chips coated with initiator SAM with a solution of PCMA monomer (100 mg/mL), CuBr (9.6 mg/
mL), CuBr2(0.75 mg/mL), and BiPy (20.8 mg/mL) in water for 2 h. Chemical structures of zwitterionic polymers VII, VIII, IX, and XI grown by ATRP fromω-mercaptoundecyl bromoisobu-tyrate initiator attached to gold are shown in Figure 1.
The presence of the polymer coatings was verified by FTIR GASR (SdO and CdO stretching bands at 1210 and 1735 cm-1, respectively).
Results and Discussion
Table 1 shows the values of water-air contact angles measured on the chips coated as described above and deposits remaining on the surfaces after incubation with citrated human blood plasma or model protein solutions and washing with PBS. We prefer to discuss the deposited amounts in terms ofΔλresrather than in terms of the mass of the deposit, although it can be estimated that Δλres= 1 nm is roughly equivalent to 200 pg of deposit per mm2 (Experimental Section).
In the standard SPR experiment, fouling was measured simul-taneously on one chip in four channels. One channel was used for blood plasma, and the other ones were used for solutions of IgG, Fbg, and Lys in PBS (Figure 2). The experiment was started by replacing the PBS buffer in all channels with 5 mg/mL HSA solution in PBS. After reaching a steady-state value ofλres, HSA solution was replaced with PBS. The amount of irreversibly adsorbed HSA was expressed as the difference inλresmeasured in PBS before and after incubation with HSA solution. PBS in one channel was then replaced with HSA solution, and after 5 min, it was replaced with blood plasma. PBS in the other channels was replaced with solutions of IgG, Fbg, and Lys. After 30 min, the protein solutions were replaced with PBS and plasma was replaced with HSA solution and then with PBS. A steady-state value ofλresin blood plasma was usually not reached in 30 min (e.g., it was reached after 12 h on the surface coated with SAM (II)). For experimental feasibility and assuming that shorter measurements would be more convenient for the usual diagnos-tics, differences inλresmeasured on the surfaces in PBS before and after 30 min of incubation with blood plasma and protein solutions were determined as indicators of the surface potential to plasma fouling (Table 1).
The decrease in contact angles indicated that all of the coatings increased the surface wettability of the gold. The increased wettability was accompanied by a decrease in plasma fouling and, particularly, by a significant decrease in adsorption of the tested plasma proteins from PBS, probably because of a con-siderable reduction in hydrophobic interactions. The low Fbg and HSA adsorption from PBS on OEG SAMs II and IV correspond
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to measurements published earlier.3,6,23A higher adsorption of these proteins on 16-mercaptohexadecanoic acid SAM I might have been due to the lack of internal hydrophilicity in SAM I in comparison to that of the hydrated OEG chains in SAMs II and IV.6The high adsorption of IgG on the SAMs, which has not been
reported elsewhere, is unexplained. Coatings V and VI prepared by grafting of NH2-PEG-COOH chains (mw 3400 Da) on SAMs III and IV efficiently prevented the adsorption of all of the tested proteins from PBS and considerably decreased plasma fouling, although they decreased the wettability of the original SAMs measured by contact angles. The higher hydrophilicity of OEG SAM was probably less important for antifouling properties than the steric repulsion of PEG.9
Figure 1. Coatings prepared on gold surface
III-SAM prepared from mixed HS-(CH2)11-EG2-OH/HS-(CH2)15-COOH, 7:3; IV-SAM prepared from mixed HS-(CH2)11-EG2-OH/
HS-(CH2)11-EG3-OCH2COOH, 7:3; VI -carboxy-terminated poly(ethylene glycol) grafted onto a SAM (IV); VII-poly(carboxybetaine methacrylate; VIII - poly(sulfobetaine methacrylate; IX - block copolymer poly(SBMA-b-CBMA); and XI - poly(phosphorylcholine methacrylate).
(23) Zhou, Ch.; Khlestkin, V. K.; Braeken, D.; De Keersmaecker, K.; Laureyn, W.; Engelborghs, Y.; Borghs, G.Langmuir2005,21, 5988–5996.
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There was no or only minor adsorption of the tested proteins from single-protein solutions on surfaces coated with zwitterionic polymers VII-XI; however, only poly(CBMA) was able to achieve ultralow plasma fouling that was below the limit of detection of SPR measurement (0.03 nm). A high degree of fouling was observed on more hydrophilic poly(SBMA) (VIII) and poly(PCMA) (XI), the wettability of which was the highest of all of the surfaces tested. Plasma fouling was decreased on IX prepared by polymerizing nonfouling poly(CBMA) blocks over
poly(SBMA). On the contrary, the coating with a random copolymer poly(SBMA-co-CBMA) (X) was much less efficient.
These results showed that the nonfouling properties were mainly governed by the top layer. The chemical composition might play a central role in the fouling process. It is possible that the presence of carboxylic groups in zwitterions was essential to the antifouling properties of polymer surfaces VII, IX, and X whereas the other ionic groups increased the plasma fouling either directly because of specific interactions with plasma components or indirectly via different morphology of the polymer coatings.
Protein adsorption on unmodified gold, on which high amounts of plasma and proteins from single-protein solutions were deposited (Table 1), was an example of nonspecific adsorp-tion on low-wettability surfaces such as methyl-terminated SAMs.19Plasma deposition on the antifouling coatings suggested more complex mechanisms. The low adsorption of plasma proteins HSA and Fbg from PBS indicated the effective resistance of the hydrated coatings to hydrophobic interactions whereas a low impact of electrostatic attraction between the surfaces and the proteins was indicated by the negligible adsorption of negatively charged HSA and positively charged lysozyme.
We also investigated deposition from fractions of plasma prepared by GPC. Fouling on PEG coating VI was observed only from fractions containing compounds with molecular mass higher than 350 000 Da, and there was no deposition from the other fractions containing only lower-molecular-weight com-pounds. Fractions of plasma collected from two healthy donors behaved in the same way. The composition of deposits from the plasma fractions might have differed from that of deposits from whole plasma; however, it could be derived that the participation of one or more of these high-molecular-mass proteins was necessary for deposition from whole plasma. Such proteins formed whole plasma deposits themselves, or they only promoted the deposition of some other plasma proteins. In other papers, kininogen (mw 120 000 Da), FXII (mw 78 000 Da), prekallikrein (mw 79 500 Da), and C3 complement protein (mw 75 000 Da) were identified as significant components of plasma deposition on carboxy-terminated SAMs,19 and C3 complement protein Protein Solutionsa
contact angle (deg) SPR response to nonspecific adsorptionΔλres(nm)
no. surface advancing receding plasma IgG Fbg HSA Lys
uncoated gold 75.4(0.6 63.2(0.9 20(4.9 20(4.8 21(7.4 7(3.6 10.4(0.3
SAMs
I. HS-(CH2)15-COOH 44(1.1 12(3.0 11(2.7 9(2.5 9(3.2 5.6(0.9 8(1.5
II. HS-(CH2)11-EG2-OH 30(1.1 16(1.0 15(2.4 12(3.7 2(0.9 0.7(0.4 1(0.4
III. HS-(CH2)11-EG2-OH/HS-(CH2)15-COOH 37.1(0.8 11.4(0.7 18(3.1 14(2.3 10(1.5 0 9(1.6 IV. HS-(CH2)11-EG2-OH/HS-(CH2)11-EG3-OCH2COOH 30(2.7 9(1.4 10(5.0 4(3.0 4(1.3 0.4(0.1 0.6(0.2
SAM-PEG
V. (III)-PEG-COOH 42(1.2 28(1.3 6(4.0 0.5(0.25 0.2(0.05 0 0.8(0.01
VI. (IV)-PEG-COOH 40(1.1 27(3.3 7(3.1 0.6(0.48 0 0 0.8(0.3
Zwitterionic Polymers
VII. poly(CBMA) 34(2.4 11(4.1 0 0 0 0 0
VIII. poly(SBMA) 20(2.0 5(1.2 16(3.9 1(0.4 0 0 0
IX. poly(SBMA-block-CBMA) 42(1.1 5(2.6 1.2(0.5 0.9(0.3 1.2(0.03 0 0.9(0.5
X. poly(SBMA-co-CBMA) 35(4.2 8(3.6 9(1.3 * 1.1(0.6 0 0.4(0.2
XI. poly(PCMA) 18(0.4 7(0.4 23(12.5 0.9(0.5 0.5(0.2 0 0
aThe deposits are expressed as differences in SPRλresmeasured in PBS before incubation of the surfaces with plasma or protein solutions for 30 min andλresmeasured after washing with PBS. The standard deviation was calculated from three independent measurements; 0 means that the value was below the detection limit of the instrument, 0.03 nm (approx. 6 pg/mm2); * means that this quantity was not measured. SAM-the self-assembled monolayer; PEG-poly(ethylene glycol); CBMA indicates poly(carboxybetaine methacrylate); SBMA-poly(sulphobetaine methacrylate); and PCMA -poly(phosphorylcholine methacrylate). Solutions: blood plasma 33%, IgG 9 mg/mL, Fbg 1 mg/mL, HSA 5 mg/mL, Lys 1 mg/mL, all solutions in PBS.
Figure 2. Deposition of blood plasma and adsorption of plasma proteins from PBS on a gold sensor surface coated with SAMs of mixed HS-(CH2)11-EG2-OH/HS-(CH2)11-EG3-OCH2COOH, 7:3 (IV).
The deposition was observed by SPR simultaneously in four channels, and each arrow marks an exchange of solutions: (curve 1) HSA 5 mg/mL in PBS, 33% citrated blood plasma; (curve 2) IgG 9 mg/mL in PBS; (curve 3) Lys 1 mg/mL; and (curve 4) Fbg 1 mg/mL in PBS.
DOI:10.1021/la900083s Langmuir 2009, 25(11),6328–6333
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(mw 75 000 Da) was identified in plasma deposits on OEG-terminated SAMs.1We do not have data on individual plasma constituents involved in the fouling on the PEG coating tested in our work; however, we continue our investigation focusing on high-molecular-mass plasma proteins such as coagulant von Willebrand factor (mw 540 000-20 000 000 Da), apolipoprotein B-100 (mw 549 000 Da), thyroglobulin (mw 660 000 Da), and R-2-macroglobulin (mw 718 000 Da), C1q complement protein (mw 459 000 Da), and C4b binding protein (mw 570 000 Da).
Figure 3 (curves 1 and 3) shows the interaction of a diethylene glycol-terminated alkanethiolate SAM II with blood plasma samples collected from two different donors. Although the kinetics were rather different, plasma deposits from the two samples reached similar saturated values after 12 h. The similar shape of the kinetics curves for plasma and serum prepared from the same plasma sample (Figure 3, curves 1 and 2, 3 and 4) suggested that fibrinogen did not take part in plasma deposition.
Different interactions of blood plasma from three donors with a PEG surface (VI) are shown in Figure 4.
Designing affinity biosensors for real-time measurements in blood plasma or serum requires us to consider that (a) the common testing of antifouling properties by the adsorption of plasma proteins, such as HSA, IgG, and Fbg, on sensor surfaces from model solutions is not relevant to plasma fouling. Plasma deposits can be high on surfaces with negligible adsorption of individual proteins. (b) The fouling can be different in plasma and serum collected from different people. Thus, if nonspecific fouling is not completely prevented, then it seems inevitable to correct responses from the detecting sensor surface by the simultaneous measurement of the tested sample on a reference sensor surface that does not capture the detected analytes but provides the same response to nonspecific fouling.
Conclusions
All investigated coatings increased the wettability of the gold surface; however, there was no direct relationship between the contact angles and plasma deposition.
A rather high plasma deposition on OEG-terminated SAMs was decreased by grafting PEG chains onto the SAM surfaces.
Participation of one or more plasma proteins with molecular mass higher than 350 000 Da was necessary for the deposition of whole plasma on the PEG coating. Such proteins formed plasma deposits themselves or only initiated the deposition of other plasma proteins
The kinetics of deposition from plasma collected from dif-ferent donors was difdif-ferent although the final amount of depo-sits was similar. The kinetics of deposition from plasma was similar to that from serum prepared from the same plasma sample, indicating that fibrinogen did not take part in plasma deposition.
Plasma deposition was completely prevented by zwitterionic carboxybetaine poly(CBMA) brushes. More hydrophilic poly-mers with other zwitterionic groups, poly(SBMA), and poly (PCMA) did not prevent plasma deposition, although they prevented the adsorption of main plasma proteins HSA, IgG, Fbg, and lysozyme from single-protein solutions.
The results suggest that neither the wettability nor the adsorp-tion of the main plasma proteins from single-protein soluadsorp-tions was the main indicator of blood plasma deposition on the tested antifouling surfaces.
Acknowledgment.This study was supported by the Academy of Sciences of the Czech Republic (contract KAN20670701 and project AVOZ 40500505) and a grant of the Ministry of Health of the Czech Republic (VZ 2373601).
Figure 3. Interaction of blood plasma and serum samples col-lected from two different donors with a diethylene glycol-terminated alkane self-assembling monolayer (II).
HSA-human serum albumin, 1 mg/mL PBS; curve 1-plasma 33%, donor 1; curve 2-serum 33%, donor 1; curve 3-plasma 33%, donor 2; curve 4 - serum 33%, donor 2 (SPR was measured simultaneously in four channels).
Figure 4. Interaction of blood plasma collected from three dif-ferent donors with a carboxy-terminated poly(ethylene glycol) coating (VI). HSA-human serum albumin, 5 mg/mL PBS; curves 1, 2, and 3-plasma from donors 1, 2, and 3, respectively; curve 4 -HSA 5 mg/mL PBS (SPR was measured simultaneously in four channels).
DOI:10.1021/la900083s
Langmuir 2009, 25(11),6328–6333 6333
Article Emmenegger et al.
Downloaded by CZECH ACADEMY OF SCIENCES on October 13, 2009 | http://pubs.acs.org Publication Date (Web): May 1, 2009 | doi: 10.1021/la900083s