F
ACULTY OFM
ATHEMATICS ANDP
HYSICSD OCTORAL T HESIS
S ENSITIVE L AYERS FOR O PTICAL B IOSENSORS AND P ROTEIN C HIPS
C ÉSAR R ODRÍGUEZ E MMENEGGER
Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic
Supervisor: RNDr. Eduard Brynda, CSc.
Associate supervisor: Prof. Dr. Aldo Bologna Alles Associate supervisor: Ing. Zdeňka Sedláková, CSc.
Study programme: F-4. Biophysics, chemical and macromolecular physics
Prague 2012
First, and foremost, I am deeply indebted to Dr. Eduard Brynda for providing me with the freedom to pursue challenging goals in a self-directed manner and for being an inexhaustible source of advice and help every time I needed. His impeccable and rigorous way of understanding science will always be a model for me.
I am equally grateful to my co-advisor Prof. Aldo Bologna Alles, who was the reason why I have got engaged in science back in 2003. He has kept his trust on me even in the hardest times being a constant source of support. Not only have I learnt from him about science but I had the opportunity to receive from him very important moral advice which certainly helped me to grow.
I would also like to thank Dr. Zdeňka Sedláková for helping me in my first steps in the world of polymer synthesis and her kindness.
I must certainly acknowledge the members of our group who have always had time to help me with so many things and questions while I tried to integrate to Czech lifestyle. I certainly appreciate the friendly atmosphere of our group; Dr. Milan Houska, Dr. Tomáš Riedel, Ognen Pop- Georgievski, Andrés de los Santos, Zdeňek Plichta and Oxana Avramenko. Milan introduced me to FTIR spectroscopy of polymers and has made a significant contribution to the chemical characterisation of the thin films prepared. Ognen and Tomáš have been a lot more than outstanding coworkers but excellents friends to whom I am very grateful. I also want to express my gratefulness to Dr. Sergii Pochekailov for being such an excellent friend and sharing with me his always fresh and interesting point of view.
My sincere gratitude goes to Prof. Wilhelm T.S. Huck and his group at Melville Laboratory; Dr. Erol Hasan and Dr. Julien Gautrot. My stays full of exciting research at the University of Cambridge have helped me to make quantitative advancements in my research. To Erol, I thank for his sharing his ideas and endless discussions about science.
My research stay at Prof. Christopher Barner-Kowollik group (Karlsruhe Institute of Technology) was not less enjoyable and productive. The friendly atmosphere and great number of challenging projects run at his group made from it an ideal place to work. I must acknowledge Bernhard V.K.J. Schmidt for fruitful collaboration and all the members for welcoming me to their group.
To Dr. Ondřej Kylian, I thank for performing the depositions of amino rich RF-plasma sputtered nylon films, Ognen Pop-Georgievski for preparing the poly(dopamine) films and Dr.
Tomáš Riedel for collaboration in the projects of identification of proteins and the preparation of sensors for clinical samples. Prof. Ivan Krakovský, Prof. Jiři Homola, Prof. František Rypáček and Dr. Andrej Choukourov are acknowledged for adapting their courses to English so that I could participate.
A special note of gratitude goes for Andrés de los Santos has made extensive proof-reading of this manuscript. In his short time at our group, he has naturally become a friend and an outstanding coworker.
I would also like to thank my parents; Nilza Emmenegger and Julio Rodríguez as well as my dear grandmother; Nidia dos Santos who have done so much for me. Their unconditional support laid the foundations of who I am.
Finally, I thank to my dearest Nina Kostina who has been a tremendous source of strength and support over the past few years. Your kindness and understanding have been an immensurable help throughout my sleepless nights engaged with work. Only you can love me enough to endure my particular personality and my constant lack of free time.
I declare that I carried out this doctoral thesis independently, and only with the cited sources, literature and other professional sources.
I understand that my work relates to the rights and obligations under the Act No. 121/2000 Coll., the Copyright Act, as amended, in particular the fact that the Charles University in Prague has the right to conclude a license agreement on the use of this work as a school work pursuant to Section 60 paragraph 1 of the Copyright Act.
Prague, 29 March 2012
César Rodríguez Emmenegger
Autor: César Rodriguez Emmenegger
Ústav: Ústav makromolekulární chemie, Akademie věd České Republiky v.v.i.
Vedoucí doktorské práce: RNDr. Eduard Brynda, CSc. Ústav makromolekulární chemie, Akademie věd České Republiky v.v.i.
Abstrakt:
Cílem této disertační práce byl vývoj citlivých povrchů pro optické afinitní biosenzory vhodné k detekci analytů v komplexních biologických médiích. Praktické aplikaci těchto technik brání tvorba nespecifických proteinových depozitů na površích senzorů (fouling), zvláště při styku s krevní plazmou, kde se vyskytuje velká většina relevantních analytů.
Práce byla zaměřena na tři hlavní oblasti:
vývoj a příprava povrchů, které významně snižují nebo zamezují vzniku depozitů (antifouling, resp. non-fouling povrchy);
hodnocení a konceptualizace odolnosti povrchů proti vzniku depozitů z krevní plazmy a séra a dalších biologických tekutin;
příprava citlivých vrstev pro detekci v komplexních biologických médiích.
K přípravě povrchů odolných proti vzniku proteinových depozitů byly použity tři přístupy:
i) samoorganizované (self-assembled) monovrstvy s koncovou funkční skupinou, ii) na konci ukotvené polymerní řetězce (end-tethered polymers) a iii) polymerní kartáče připravené povrchově iniciovanou kontrolovanou radikálovou polymerací. Zkoumání proteinů v depozitech krevní plazmy vznikajících na površích na bázi PEG ukázalo, že vzniku depozitů na tomto typu povrchů nelze zcela zabránit.
Byl připraven nový typ non-fouling polymerních kartáčů na bázi poly[N(2-hydroxypropyl) methakrylamidu], které se v některých ohledech vymykají dosud přijímaným představám o površích odolných proti tvorbě proteinových depozitů.
Poprvé byl optický afinitní biosenzor nevyžadující použití značky úspěšně použit k diagnostice v reálných klinických vzorcích.
Klíčová slova: afinitní biosensory, nespecifická adsorpce krevní plasmy, polymerní kartáče, radikálová polymerace s přenosem atomu
Title: Sensitive layers for optical biosensors and protein chips Author: César Rodriguez Emmenegger
Institute: Institute of Macromolecular Chemistry. Academy of Sciences of the Czech Republic v.v.i.
Supervisor of the doctoral thesis: RNDr. Eduard Brynda, CSc. Institute of Macromolecular Chemistry. Academy of Sciences of the Czech Republic v.v.i.
Abstract:
The goal of this thesis was the development of sensitive surfaces for optical affinity biosensors detecting in complex biological media. The practical application of these surface-based technologies has been hampered by protein fouling from biological media, in particular blood plasma, where the vast majority of relevant analytes are present. The work of the thesis was centred on three main foci:
Design and preparation of antifouling and non-fouling surfaces
Evaluation and conceptualisation of their resistance to fouling from blood plasma and serum as well as other biological fluids
Preparation of sensitive layers for detection in complex biological media
Three approaches were used to prepare protein resistance surfaces, i) ω-functional self- assembled monolayers (SAM), ii) end-tethered polymers and iii) polymer brushes prepared by surface initiated controlled radical polymerisation. Investigation of proteins in the blood plasma deposits on PEG- based surfaces revealed that some fouling is unavoidable in PEG-based surface modifications. A novel type of non-fouling polymer brushes based on poly[N-(2-hydroxypropyl) methacrylamide] challenged the accepted ideas for the design of protein resistant surfaces.
For the first time a label-free optical affinity biosensor was successfully applied to diagnosis in real clinical samples.
Keywords: affinity biosensors, blood plasma fouling, polymer brushes, atom transfer radical polymerization.
1 Introduction ... 1-2 1.1 Label-free optical affinity biosensors ... 1-4 1.2 State of the art in the field of affinity biosensors at the commencement of the Thesis ………1-14
1.3 References……… ……… 1-14
2 Goals of the Thesis ... 2-1 2.1 Experimental design ... 2-1 3 Synthetic strategies and Characterisation ... 3-1
3.1 Building blocks for surface preparation ... 3-1 3.2 Monomer synthesis... 3-4 3.3 Synthesis of polymer brushes ... 3-4 3.4 Characterisation of the antifouling surfaces ... 3-12 3.5 References ... 3-13 4 Self-assembled monolayers ... 4-1 4.1 Surface preparation and characterisation ... 4-1 4.2 Fouling studies ... 4-4 4.3 Summary ... 4-7 4.4 References ... 4-8 5 End-grafted polymers ... 5-1 5.1 Surfaces prepared ... 5-3 5.2 Summary ... 5-6 5.3 References ... 5-7 6 Non-ionic polymer brushes ... 6-1 6.1 Non-ionic polymer brushes prepared ... 6-1 6.2 Fouling studies ... 6-6 6.3 Summary ... 6-8
6.4 References ... 6-9 7 Protein identification in deposits from blood plasma on PEG ... 7-1 7.1 Fouling studies ... 7-3 7.2 Identification of proteins in blood plasma deposits ... 7-4 7.3 Summary ... 7-6 7.4 References ... 7-7 8 Toward non-fouling brushes: Polymeric betaines ... 8-1 8.1 Selection of monomers ... 8-2 8.2 Synthesis of poly(betaine) brushes ... 8-3 8.3 Characterisation of the brushes ... 8-5 8.4 Fouling studies ... 8-7 8.5 Summary ... 8-9 8.6 References ... 8-9 9 Poly(HPMA): Rethinking the accepted ideas ... 9-1 9.1 Poly(HPMA) brushes ... 9-1 9.2 Fouling studies ... 9-3 9.3 Stability and reusability of of poly(HPMA) brushes. ... 9-4 9.4 Summary ... 9-5 9.5 References ... 9-6 10 Fouling from biological fluids ... 10-1 10.1 Antifouling surfaces ... 10-1 10.2 Fouling studies ... 10-2 10.3 Summary ... 10-5 10.4 References ... 10-5 11 Antifouling coatings via surface-independent approaches ... 11-1 11.1 Strategies ... 11-2 11.2 Amino-rich plasma sputtered nylon ad-layers ... 11-2 11.3 Poly(dopamine) ad-layers ... 11-8 11.4 Summary ... 11-10 11.5 References ... 11-11 12 Nanocarriers stable in biological fluids ... 12-1 12.1 Synthetic strategy ... 12-1
12.4 References ... 12-5 13 Sensitive antifouling surfaces ... 13-1 13.1 Functionalisation of SAMs ... 13-2 13.2 Functionalisation of hydroxy-functional polymer brushes ... 13-4 13.3 Functionalisation of carboxybetaine polymer brushes ... 13-7 13.4 Sensing platforms in real biological media ... 13-10 13.5 Diagnosis of EBV in clinical samples ... 13-16 13.6 Summary ... 13-18 13.7 References ... 13-18 14 Concluding remarks and outlook ... 14-1 14.1 Conclusion ... 14-1 14.2 Outlook ... 14-2 List of abbreviations
Appendices
List of publications
1-1
1 Introduction
Advancements in life sciences, pharmaceutical discovery, medical diagnostic methods and food safety rely heavily on modern analytical techniques. These technologies provide scientists with the capability not only to detect analytes or pathogens, but also to evaluate biochemical and biophysical interactions among biomolecules and cells.
Modern health care, for example, increasingly involves diagnostic methods based on monitoring biomarkers in body fluids. This development can allow the early identification of pathologies, even before any symptoms can be detected. Monitoring the concentration of some biomarkers is also a tool to determine the predisposition to a disease or its progression. Furthermore, the precise detection of biomarkers is expected to lead to a paradigm shift in the treatment of disease. Therapies will move from the traditional practises based on the symptomatology of the patient to a more rational molecular basis.
Direct detection of biological analytes through their intrinsic physicochemical properties, such as mass, size, electrical impedance, or dielectric permittivity, has proven very difficult. Therefore, biological research has historically relied upon attachment of a
‘label’ to one or more of the molecules, viruses or cells being studied. The label is designed to be easily detected and acts as a surrogate to indirectly indicate the presence of the analyte to which it has been attached. Typical labels include molecular fluorescent probes, nanoparticles, quantum dots, enzymes, and radioisotopes. Currently, most routine methods for the determination of biomarkers in body fluids include enzyme-linked immunosorbent assay (ELISA), chemiluminescent, immunofluorescent, radioimmunoassays, immunoenzymatic, immunoprecipitation and immunoagglutination.[1]
However, these methods are very laborious, expensive, require highly trained staff and offer limited possibilities for automation and integration.[2-4] In practise, label-based assays also require a high degree of development to assure that the label does not block an important active site on the tagged molecule or negatively modify its conformation resulting in false negative results. Additionally, the use of tagged reagents makes it impossible to study multi-step sequential processes with these techniques. This is a serious drawback for research in molecular biology, where most reactions occur as a cascade of events and require real-time monitoring.
Current trends suggest that modern medicine is facing a paradigm shift in which early detection and treatment will be based on the detection of biomarkers. This will be enabled by high-throughput point-of-care techniques integrated with computerised systems, allowing easy evaluation of the results by the physician. In addition, emerging key disciplines such as proteomics have the urgent need for novel technologies able to
CHAPTER 1.INTRODUCTION
1-2
perform rapid and multiplexed analysis of biological samples in a highly specific and sensitive manner.[2, 5-7] As previously stated, label detection techniques are not able to encompass these changes and methods reducing costs and complexity while providing more quantitative information with high throughput will be required. Label-free biosensor technology has emerged as the most promising candidate to meet the challenges of modern diagnostics and medicine. There are now various methods allowing direct detection of biological analytes without labels. Biosensors are devices incorporating a biologically active component, the bioreceptor, able to interact with the selected analyte; and a physical transducer. The sensor functions as a transducer that can convert a change in physical property after a biorecognition event on the sensor’s active surface into a quantifiable signal (Figure C1-1). In this way, label-free detection avoids the experimental uncertainties induced by the effect of the label on molecular conformation, blocking of active binding epitopes, steric hindrance, or the inaccessibility of the labelling site. Furthermore, label- free detection methods greatly simplify the time and effort required for assay development while removing experimental artefacts from quenching, shelf life, and background fluorescence, generally found on label-based assays.
Various physical principles have been used to design biosensors, being the most sensitive those sensors based on optical,[3, 8-10] electrochemical,[11, 12] piezoelectric effect,[13-
15] acoustic waves,[16] film bulk acoustic resonators,[17, 18] cavity ring-down resonators[2]
and nano-electro-mechanic detection.[2, 19] The vast majority of biosensors are optical because the measurement does not require any physical or electrical contact between the sensor and the detection instrument, i.e spectrophotometer, simplifying the preparation of multisensing surfaces. A single multisensing surface has the potential to assess a huge number of analytes by illuminating at different zones of the sensor decorated with an array of biorecognition elements in discrete positions. This allows the inexpensive preparation of high throughput biosensors.[20-22]
Figure C1-1. Scheme of a label-free biosensor. The binding event occurs at the biorecognition element (A). This reaction is converted in an output by the transducer (B) which is amplified (C), processed (D) leading to a read-out (E). Modified after Chaplin, 2004 (http://www.lsbu.ac.uk/biology/enztech/biosensors.html)
Reference
(A) (B)
(C)
(D) (E)
1-3
The optical affinity biosensors are frequently divided in two conceptual classes, functional and analytical biosensors.[20] Functional biosensors examine the biological activity, such as binding activity and avidity and reactivity, of a set of immobilised biomolecules; while in analytical biosensors, biorecognition elements act as probes to detect and quantify an analyte.[5, 7]
1.1 Label-free optical affinity biosensors
1.1.1 The concepts
An optical sensor is a sensing device which, by optical means, converts the quantity being measured to an output which is typically encoded into one of the characteristics of the light.[3, 4]
The key behind optical biosensors’ ability to detect biological analytes is that all biological molecules, including proteins, cells, and DNA, have dielectric permittivity greater than that of air and water. Therefore, all of these materials reduce the propagation velocity of electromagnetic fields that pass through them.[2-4, 7] Optical biosensors are designed to translate changes in the propagation speed of light through a medium that contains biological material into a quantifiable signal proportional to the amount of biological material present on the sensor surface. When a solution containing analyte molecules is brought into contact with the biosensor, the analytes in solution bind to the biorecognition elements. Consequently, a small number of water molecules are displaced and replaced with molecules that are more easily polarised by electromagnetic fields associated with light. Therefore, the design goal for all optical biosensors is to provide a transducer with some externally measurable characteristic that is modified by changes in dielectric permittivity on its surface. In this way, optical biosensors do not measure the mass of adsorbed material, although often the mass of deposited material is related to the change in dielectric permittivity.
For most optical biosensors, the transducer confines an electromagnetic wave in such a way that the wave interacts with the test sample. For the light to be guided by the sensor structure while interacting with the external medium, the structure must be designed so that the light wave can extend from the sensor surface into the test sample. The electromagnetic fields bound to an optical device that can couple some energy to an external medium are called evanescent fields. The field intensity decays exponentially with the distance from the transducer surface, with the penetration depth being directly proportional to λ.[2, 4]
Consequently, for wavelengths of typical biosensors (λ = 600 to 900 nm) the evanescent field extends only ca 100 to 150 nm into the test sample. Therefore, these transducers are only sensitive to interactions in the vicinity of their surface.
Various optical affinity biosensors have been prepared, including those based on surface plasmon resonance (SPR), Young interferometers, grating couplers, micro-ring
CHAPTER 1.INTRODUCTION
1-4
resonators and Mach Zehnder interferometers. SPR biosensors present a mainstay technology for research of biomacromolecules and their interactions. In addition, SPR holds potential for many other applications of paramount importance, such as food safety, environmental monitoring, detection of toxins, etc.[4, 23, 24] However, existing commercial SPR sensors are not designed to face detection in real complex biological media, suffering a series of drawbacks.
1.1.2 Biosensing in complex biological fluids
Detection of biomarkers for various diseases; pathogens and monitoring biomolecular interactions in complex matrices is of key importance in the analytical field.
Remarkably, label-free affinity biosensors have the potential to reduce the time of analysis while having and excellent sensitivity and selectivity.[23-26] However, contrary to other multi-step analytical methods or biosensors based on labelled reagents, the in real-time direct detection cannot differentiate between the specific response due to the capture of analytes by the immobilised bioreceptors and the non-specific adsorption of other components, hereafter termed as fouling.[27, 28] In order to utilise the surface of a transducer as a biosensor, it must have the ability to selectively capture the desired analyte from the test sample while preventing fouling of other biomolecules or biological entities (cells, bacteria, etc). Typically, the analytes of biomedical interest are present in body fluids or complex biological liquids. In particular, the vast majority of them are present in blood, therefore biosensors able to operate in blood plasma and serum are of the outmost importance.
Most cells of a multicellular organism cannot move around to obtain oxygen and nutrients or eliminate carbon dioxide and other wastes. Instead, these needs are met by two fluids: blood and interstitial fluid. Blood is a liquid connective tissue that consists of cells surrounded by a liquid extracellular matrix. The extracellular matrix is called blood plasma, and it suspends various cells and cell fragments. Interstitial fluid is the fluid that bathes body cells and is constantly renewed by the blood. Blood transports oxygen from the lungs and nutrients from the gastrointestinal tract, which diffuse from the blood into the interstitial fluid and then into body cells. Carbon dioxide and other wastes move in the reverse direction, from body cells to interstitial fluid to blood. Blood then transports the wastes to various organs—the lungs, kidneys, and skin—for elimination from the body. It also plays a vital role in homeostasis of the organisms.[29]
Whole blood has two components: blood plasma, a watery liquid extracellular matrix that contains dissolved substances, and formed elements, which are cells and cell fragments. If a sample of blood is centrifuged, the cells sediment to the bottom of the tube while the plasma forms a layer on top.[30]
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1-7
Some authors have proposed the use of reference channels to separate the specific from the non-specific signal by subtracting the reference to the sensing channel. Two strategies have been applied, (i) to inject in the same sample in a channel modified with dummy bioreceptors (not active toward the analyte) and (ii) to inject blood plasma from a different donor not containing the analyte of interest. The first approach in general fails due to the impossibility to prepare an absolutely identical ‘dummy reference’, the second and most commonly used in scientific works leads to bigger errors since plasma fouling from different donors can be very different as we recently showed (Refer to Figure C1- 3).[31]
1.1.3 Rationale behind biosensor design
The success of affinity biosensors is strongly linked to the ability of researchers to finely engineer the interface between the physical transducer and the medium where it should operate. A bioactive surface, interfacing the physical transducer and the test medium must be designed to achieve maximum specificity and selectivity toward the desired analyte while preventing the fouling. The former is achieved utilising highly specific biorecognition molecules while the latter requires a coating with a layer resistant to fouling. Remarkably, to maximise the sensitivity of the sensor, the interface occupies a region of space of no more than few tens of nanometers. Consequently, its preparation often requires a combination of tools from nanotechnology, litography, polymer and supramolecular chemistry, and biochemistry.
The preparation of affinity biosensors can be tackled with a modular approach, where the building blocks, bioreceptors and ultrathin coatings, are conjugated to create bioactive surfaces able to perform the desired function (Figure C1-4). A general strategy for achieving an effective bioactive surface comprises two main tasks:
a) Modification of the transducer’s surface with a protein-resistant ultra-thin layer to rule out any undesired interactions. Not only should this surface be antifouling but also provide an adequate microenvironment to ensure the retention of the activity of the attached bioreceptors, thus hydrophilic surfaces are preferred.
b) Functionalisation of the surface with the desired bioactive molecules. This requires: (i) functional-group-specific chemical reactions to activate or generate functional groups to immobilise biomolecules, (ii) to attract the bioactive molecule to the surface which repels it and (iii) optimisation of the amount and activity of bioreceptors avoiding overcrowding.
Combining the ‘building blocks’ -surface modifications and bioreceptors- with immobilization techniques a plethora of surfaces can be obtained for each application.
However, only an appropriate rationalisation of the selection of the surface modification and immobilisation technique will ensure bioactive surfaces able to meet the needs of medicine and bioapplications.
CHAPTER 1.INTRODUCTION
1-8
Figure C1-4. Scheme of an optical affinity biosensor; light interacts with the physical transducer (A) generating an evanescent field (depicted as the pink curve) which penetrates the vicinity of the tested sample (E). By means of specific bioreceptors (C) the specific binding is observed (D) while other components of the matrix are effectively repelled (F) from the antifouling surface (B).
1.1.3.1 Biorecognition elements
The choice of the appropriate biorecognition element and its immobilisation is critical with direct impact in the performance of any bioactive surface.[22, 24, 32] Various bioreceptors have been used including antibodies (monoclonal, polyclonal and recombinant),[32] Fab antibody fragments,[33-35] single-chain antibody fragments,[36]
recombinant proteins, peptides,[37] DNA, ARN or peptidic aptamers,[38-40] lectins, and more recently antibody mimetics such affibodies.[41, 42] Typically antibodies offering high affinity and specificity are the most widely used bioreceptors.[21]
1.1.3.2 Fouling and antifouling surfaces
Non-specific protein adsorption or fouling from complex biological fluids, in particular blood plasma and serum, have posed an insurmountable challenge affecting affinity biosensors and prevents real medical applications of this technology.
The contact between biological media and an artificial object commonly starts with protein adsorption.[43-47] Various amino acid residues on the peptide backbone can mediate adsorption of proteins via hydrogen bonds, ionic, and other polar or hydrophobic interactions.[46, 48] The protein adsorption from biological media is a complex and dynamic process in which proteins can change their conformation thus, firstly adsorbed proteins can be subsequently replaced with proteins of higher affinity to the surface (Vroman effect).[47,
Physical transducer Antifouling layer (A) (B)
(C)
(D)
(E)
(F)
1-9
49] Protein fouling in complex biological fluids, and in particular in whole blood, plasma, and serum, is an adverse event that impairs the properties and functions not only of biosensors[31, 45] but of various biotechnological and biomedical devices.[31, 45, 50] Some examples include stopping flow through separation columns and porous membranes,[51]
reduced circulation time of nanocarriers in blood stream due to colloidal instability[52] or opsonisation,[53, 54] attachment of microorganisms on contact lenses[55] and synthetic grafts,[56] and disabling cardiovascular devices by thrombus formation.[56] Importantly, protein fouling at the surface of implants may impair its functions or even lead to thrombus formation.[57] The adsorbed layer of protein at these interfaces provides a suitable surface for bacteria attachment being a mayor concern for its risk of infection.[58] The ubiquity and relevance of the problem of fouling from blood plasma have resulted in an intense research for over 35 years pioneered by Andrade[46, 59, 60], Brash[61-63], Brynda[64-66] and later by Whitesides[67-69], Chilkoti[50, 70] and Jiang.[71, 72] Various types of surfaces have been shown to reduce the fouling from proteins to a certain extend. In this work, surfaces with a reduced protein adsorption from blood plasma will be referred as ‘antifouling’ while the term ‘non-fouling’ will be solely used for those surfaces with a fouling from undiluted human blood plasma lower than 3 pg·cm-2 (Figure C1-5).
Figure C1-5. Classification of bioactive surfaces according to their level of fouling.
Research in antifouling and non-fouling surfaces has been focussed in the following types of surfaces:
Poly(ethylene glycol) coatings (PEG)
Bioartificial surfaces
Self-assembled monolayers (SAM)
Polymer brushes
Bioactive surface
Complex biological fluid
Fouling surface Antifouling surface Non-fouling surface
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eoretical tre fting densit s of Brash a surfaces ha
D),[93, 94] ch mercial SPR
transducer rs,[93, 94] its gher than the
preparation o
ces and prob ). SAMs are
in the use erial.[48, 60] I e protein fou s physical a Typical exa
nd reaction/
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led to cros
86]
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which was eatment pre ty and cha and others.[8 ave been itosan [95] an R biosenso r. Although use in blo e specific re
of a SAM
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uling,[74, 75]
and chemic mples are interaction fting-to’). D h, the vast m nct strategy
by plasma polymerizat hermal deco
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reduction in very few stu s in turn, a edicted imp ain length.[4
81, 89-92]
proposed nd protein m ors are coa
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most widely dered and or
HAPTER 1.
toxic, nonim used for the
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]
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INTRODUC
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1-11
exactly one molecule thick formed at a surface by spontaneous adsorption of molecules from solution.[99] The organization of these monomolecular assemblies at solid surfaces provides a rational approach for the preparation interfaces with well-defined composition, structure, and thickness allowing to control the chemical and physical properties of the interface.[100] From all the types of SAMs two systems are the most widely used in the analytical field; adsorption of alkanethiols on gold and silver and reaction of alkyltrichlorosilanes on silicon or glass.[101, 102] These molecules upon physisorption bind to the surface via thiolate or trichlorosilane. A long hydrophobic alkane chain, typically longer than 11 methylene units, is imperative for close packing driven by van der Waals forces.[99, 100]
Whitesides has pioneered the research of antifouling properties on SAMs. Thorough studies trying to unravel the mechanisms behind resistance to fouling from single protein solutions were carried out. Chapman and Ostuni surveyed the fouling from fibrinogen and lysozyme solutions on more than 50 SAMs bearing various terminal groups prepared by the anhydride method[67] and proposed a set of conditions considered essential to prevent the fouling from single protein solution:[68, 103]
i) hydrophilicity, ii) electroneutrality,
iii) presence of hydrogen bond acceptors iv) lack of hydrogen bond donors.
Other hypotheses, such as the one based on kosmotropes, have also tried to explain the resistance to fouling from single protein solutions with limited success.[104, 105]
It must be highlighted that the research by Whitesides’ group was carried out utilising model solutions of a single protein dissolved in phosphate buffered saline.
Although the results are consistent with the fouling from simple model single protein solutions, they fail to explain the fouling from blood plasma in surfaces resistant to the fouling from fibrinogen suggesting that the mechanisms governing the fouling from blood plasma and serum are far more complex. In spite of this, the requisites suggested by Whitesides et al. have been widely accepted as sine qua non conditions for the design of surfaces resistance to blood plasma fouling.[45, 106, 107]
With the advent of new surface controlled polymerisation techniques a new class of ultra-thin coating emerged, the polymer brushes.[108, 109] These ultra-thin polymeric films consist of stretched polymer chains tethered by one end to a surface (Figure C1-7).[108-110]
Since the grafting density is very high, the polymer chains adopt an unusual conformation wherein the individual polymer coils overlap.[109, 111] Under these conditions the polymer molecules are strongly stretched away from the surface and achieve a molecular conformation very different from the random coil observed in solution.[109, 110] From a structural point of view polymer brushes can be regarded as the most defined type of polymer coating that can be currently realised. Especially the combination of surface
CHAPTER 1.INTRODUCTION
1-12
polymerisation with modern, controlled radical polymerisation methods thereby allows tailoring the structure of the polymer brush almost at the molecular level. Specifically, precise control over the composition, thickness, architectures, gradients and grafting density can be achieved.[108, 109]
Figure C1-7. Scheme of polymer brushes on a planar surface
Fouling resistance was predicted in hydrophilic brushes.[108, 109] For an unstretched chain, a slight molecular deformation leads to a moderate increase of the energy stored in the system (entropy elasticity) [109, 112]. However, when the molecules are strongly stretched, as in brushes, the energy penalty is large.[109, 112] It was expected that if a protein adsorbs on a hydrophilic polymer brush, water molecules associated with the polymer chains will be released into the bulk, and the chains will be compressed leading to an increase in enthalpy due to dehydration and a decrease in entropy due to chain compression.[108]
1.1.3.3 Functionalisation
The design of biosensors requires that one of the interacting molecules (biorecognition element or target molecule) be immobilised at the surface of the sensor.[32,
74] One of the paramount challenges for the preparation of bioactive surfaces is the correct choice of surface chemistry and immobilisation, compatible with a diverse set of bioreceptors while preventing their loss of activity. The immobilisation of bioreceptors on inert antifouling layers requires driving forces to attract them to the surface, which is in general preactivated for the binding. Immobilisation must be engineered in order to achieve maximum activity with a minimum loss of the antifouling properties.[24, 32, 38, 74]
Various immobilisation techniques have been developed based on three strategies: i) physical, ii) covalent and iii) bioaffinity immobilisation. The microenvironment, orientation and crowding of bioreceptor molecules at the interface are also important factors on the final performance of the sensor.
Br Br Br
Br Br Br
1-13
1.2 State of the art in the field of affinity biosensors at the commencement of the Thesis
Before the beginning of the Thesis work (October 2007) no surface fully resistant to the fouling from blood plasma had been presented. Commercial biosensors from Biacore (CMD-based) could be utilised as excellent surfaces for immobilisation of biorecognition elements and subsequent studies in model solutions but never in complex biological matrices or fluids. Some examples of biosensing in blood plasma and serum utilising SAMs for detection of cancer biomarkers and other analytes were put forward later.[10, 24,
113, 114] In all of these examples, the presence of spiked analytes in blood plasma or serum was detected, however detection and quantification in medical samples of blood plasma samples is far more cumbersome. The high fouling observed on SAMs[28, 115, 116] requires subtraction of the signal of the sensing channel to that of a reference channel to separate the specific from a very high non-specific signal. Even at concentration of analytes much higher than those medically relevant, the non-specific signal was higher than the signal of the sensing channel [117] which indicates that the use of SAMs for quantification of biomarkers typically present at very low concentration is impossible. In addition, variation in the plasma used as reference or differences in the reference surface result that this subtraction is not accurate enough for biomarkers at relevant concentrations. The situation was not better for the detection in other body fluids or food stuff. For example, a fouling from milk equivalent to about 20% of a protein monolayer was reported on a SAM-based biosensor for detection of staphylococcal enterotoxin B.[118] Improved results were obtained by Brynda et al. preparing a biosensor based on multilayers of antibodies against horseradish peroxidase or methotrexate on a grating coupler or Young interferometer.[119]
The resulting sensor showed a remarkably lower fouling than SAMs, however, it still needed to be substracted a reference channel and the robustness was poorer than those based on polymers or SAMs.
In view of this, it was clear for my advisor, Dr. Brynda, that if affinity biosensors for medical diagnostic were to exist, the fouling from blood plasma and other biological fluids must be prevented. Consequently, the topic of my PhD work was set.
1.3 References
[1] K. Murphy, P. Travers, M. Walport, "Janeway's Immunobiology", 7 edition, Taylor &
Francis, New York, 2008.
[2] F. S. Ligler, C. R. Taitt, "Optical Biosensors, Second Edition: Today and Tomorrow ", Elsevier Science, 2008.
[3] M. A. Cooper, "Label-Free Biosensors. Techniques and applications", Cambridge University Press, Cambridge, 2009.
[4] J. Homola, "Surface Plasmon Resonance Based Sensors", in Springer Series on Chemical Sensors and Biosensors, O.S. Wolfbeis, Ed., Springer-Verlag, Berlin, 2006.
CHAPTER 1.INTRODUCTION
1-14
[5] M. Zourob, S. Elwary, A. P. F. Turner, "Principles of Bacterial Detection: Biosensors, Recognition Receptors and Microsystems", Springer, 2008.
[6] A. Sadana, "Binding and Dissociation Kinetics for Different Biosensor Applications Using Fractals", Elsevier, 2006.
[7] K. R. Rogers, A. Mulchandani, "Affinity Biosensors. Techniques and Protocols", Humana Press Inc, Totowa, New Jersey, 1998.
[8] K. Hegnerová, M. Bocková, H. Vaisocherová, Z. Kristofiková, J. Rícný, D. Rípová, J.
Homola, Sensors Actuators B: Chem. 2009, 139, 69.
[9] F. Fernández, K. Hegnerová, M. Piliarik, F. Sanchez-Baeza, J. Homola, M. P. Marco, Biosens. Bioelectron. 2010, 26, 1231.
[10] T. Špringer, M. Piliarik, J. Homola, Anal. Bioanal. Chem. 2010, 1.
[11] M. Ortiz, E. M. Wajs, A. Fragoso, C. K. O'Sullivan, Chem. Commun. 2012.
[12] Y. Hasebe, K. Yokobori, K. Fukasawa, T. Kogure, S. Uchiyama, Anal. Chim. Acta 1997, 357, 51.
[13] C. Ayela, F. Roquet, L. Valera, C. Granier, L. Nicu, M. Pugnière, Biosens.
Bioelectron. 2007, 22, 3113.
[14] M. Salmain, M. Ghasemi, S. Boujday, J. Spadavecchia, C. Técher, F. Val, V. Le Moigne, M. Gautier, R. Briandet, C. M. Pradier, Biosens. Bioelectron. 2011, 29, 140.
[15] W. Pang, H. Zhao, E. S. Kim, H. Zhang, H. Yu, X. Hu, Lab on a Chip - Miniaturisation for Chemistry and Biology 2012, 12, 29.
[16] F. J. Gruhl, K. Länge, Anal. Biochem. 2012, 420, 188.
[17] J. Weber, W. M. Albers, J. Tuppurainen, M. Link, R. Gabl, W. Wersing, M. Schreiter, Sensors and Actuators A: Physical 2006, 128, 84.
[18] G. Wingqvist, Surf. Coat. Technol. 2010, 205, 1279.
[19] M. Alvarez, L. M. Lechuga, Analyst 2010, 135, 827.
[20] G. MacBeath, S. L. Schreiber, Science 2000, 289, 1760.
[21] J. S. Albala, I. Humphery-Smith, "Protein Arrays, Biochips and Proteomics. The next Phase of Genomic Discovery", Marcel Dekker, Inc., New York, 2003.
[22] P. Jonkheijm, D. Weinrich, H. Schröder, C. M. Niemeyer, H. Waldmann, Angew.
Chem. Int. Ed. 2008, 47, 9618.
[23] J. Homola, Anal. Bioanal. Chem. 2003, 377, 528.
[24] J. Homola, Chem. Rev. 2008, 108, 462.
[25] K. De Vos, J. Girones, S. Popelka, E. Schacht, R. Baets, P. Bienstman, Biosens.
Bioelectron. 2009, 24, 2528.
[26] L. M. Lechuga, K. Zinoviev, L. Fernandez, J. Elizalde, O. E. Hidalgo, C. Dominguez,
"Biosensing microsystem platform based on the integration of Si Mach-Zehnder interferometer, microfluidics and grating couplers", 2009, p. 7220/.
[27] C. Rodriguez-Emmenegger, O. A. Avramenko, E. Brynda, J. Skvor, A. Bologna Alles, Biosens. Bioelectron. 2011, 26, 4545.
[28] H. Vaisocherova, W. Yang, Z. Zhang, Z. Cao, G. Cheng, M. Piliarik, J. Homola, S.
Jiang, Anal. Chem. 2008, 80, 7894.
1-15
[29] D. W. Fawcett, R. P. Jensh, "Bloom and Fawcett: Concise Histology", 2 edition edition, Hodder Arnold Publishers, London, 2002.
[30] J. Schaller, S. Gerber, U. Kämpfer, S. Lejon, C. Trachsel, "Human blood plasma proteins. Structure and functions", John Wiley & Sons, 2008.
[31] C. Rodriguez-Emmenegger, E. Brynda, T. Riedel, Z. Sedlakova, M. Houska, A. B.
Alles, Langmuir 2009, 25, 6328.
[32] F. Rusmini, Z. Zhong, J. Feijen, Biomacromolecules 2007, 8, 1775.
[33] Y. Iwasaki, Y. Omichi, R. Iwata, Langmuir 2008, 24, 8427.
[34] I. Vikholm-Lundin, Langmuir 2005, 21, 6473.
[35] A. Kausaite-Minkstimiene, A. Ramanaviciene, J. Kirlyte, A. Ramanavicius, Anal.
Chem. 2010, 82, 6401.
[36] L. Dunne, S. Daly, A. Baxter, S. Haughey, R. O'Kennedy, Spectrosc. Lett. 2005, 38, 229.
[37] H. Vaisocherová, K. Mrkvová, M. Piliarik, P. Jinoch, M. Šteinbachová, J. Homola, Biosens. Bioelectron. 2007, 22, 1020.
[38] S. Balamurugan, A. Obubuafo, S. Soper, D. Spivak, Anal. Bioanal. Chem. 2008, 390, 1009.
[39] Z. Wang, T. Wilkop, D. Xu, Y. Dong, G. Ma, Q. Cheng, Anal. Bioanal. Chem. 2007, 389, 819.
[40] C. Rodriguez-Emmenegger, O. Kylian, M. Houska, E. Brynda, A. Artemenko, J.
Kousal, A. Bologna Alles, H. Biederman, Biomacromolecules 2011, 12, 1058.
[41] A. Orlova, M. Magnusson, T. L. J. Eriksson, M. Nilsson, B. Larsson, I. Höidén- Guthenberg, C. Widström, J. Carlsson, V. Tolmachev, S. Ståhl, F. Y. Nilsson, Cancer Res.
2006, 66, 4339.
[42] B. Renberg, J. Nordin, A. Merca, M. Uhlén, J. Feldwisch, P.-Å. Nygren, A. Eriksson Karlström, Journal of Proteome Research 2006, 6, 171.
[43] I. Banerjee, R. C. Pangule, R. S. Kane, Adv. Mater. 2011, 23, 690.
[44] D. F. Williams, Biomaterials 2009, 30, 5897.
[45] S. Jiang, Z. Cao, Adv. Mater. 2010, 22, 920.
[46] J. D. Andrade, "Surface and Interfacial Aspects of Biomedial Polymers", Plenun Press, New York, 1985.
[47] L. Vroman, Colloids Surf. B. Biointerfaces 2008, 62, 1.
[48] J. D. Andrade, V. L. Hlady, R. A. Vanwagenen, Pure Appl. Chem. 1984, 56, 1345.
[49] A. H. Schmaier, L. Silver, A. L. Adams, G. C. Fischer, P. C. Munoz, L. Vroman, R.
W. Colman, Thromb. Res. 1984, 33, 51.
[50] A. Hucknall, S. Rangarajan, A. Chilkoti, Adv. Mater. 2009, 21, 2441.
[51] D. Rana, T. Matsuura, Chem. Rev. 2010, 110, 2448.
[52] C. Rodriguez-Emmenegger, A. Jäger, E. Jäger, P. Stepanek, A. B. Alles, S. S.
Guterres, A. R. Pohlmann, E. Brynda, Colloids Surf. B. Biointerfaces 2011, 83, 376.
[53] A. Verma, F. Stellacci, Small 2010, 6, 12.
[54] I. Lynch, K. A. Dawson, Nano Today 2008, 3, 40.
CHAPTER 1.INTRODUCTION
1-16
[55] H. Thissen, T. Gengenbach, R. du Toit, D. F. Sweeney, P. Kingshott, H. J. Griesser, L. Meagher, Biomaterials 2010, 31, 5510.
[56] B. D. Ratner, Biomaterials 2007, 28, 5144.
[57] M. Amiji, K. Park, J. Biomater. Sci. Polym. Ed. 1993, 4, 217.
[58] L. Hall-Stoodley, J. W. Costerton, P. Stoodley, Nat Rev Microbiol 2004, 2, 95.
[59] S. I. Jeon, J. D. Andrade, J. Colloid Interface Sci. 1991, 142, 159.
[60] S. I. Jeon, J. H. Lee, J. D. Andrade, P. G. De Gennes, J. Colloid Interface Sci. 1991, 142, 149.
[61] C. A. LeDuc, L. Vroman, E. F. Leonard, Industrial & Engineering Chemistry Research 1995, 34, 3488.
[62] Y. J. Du, J. L. Brash, J. Appl. Polym. Sci. 2003, 90, 594.
[63] L. D. Unsworth, H. Sheardown, J. L. Brash, Biomaterials 2005, 26, 5927.
[64] E. Brynda, J. Drobnik, J. Vacik, J. Kalal, J. Biomed. Mater. Res. 1978, 12, 55.
[65] E. Brynda, M. Houska, J. Kalal, N. A. Cepalova, Ann. Biomed. Eng. 1980, 8, 245.
[66] E. Brynda, N. A. Cepalova, M. Stol, J. Biomed. Mater. Res. 1984, 18, 685.
[67] R. G. Chapman, E. Ostuni, L. Yan, G. M. Whitesides, Langmuir 2000, 16, 6927.
[68] E. Ostuni, R. G. Chapman, R. E. Holmlin, S. Takayama, G. M. Whitesides, Langmuir 2001, 17, 5605.
[69] X. Qian, S. J. Metallo, I. S. Choi, H. Wu, M. N. Liang, G. M. Whitesides, Anal. Chem.
2002, 74, 1805.
[70] H. Ma, J. Hyun, P. Stiller, A. Chilkoti, Adv. Mater. 2004, 16, 338.
[71] L. Li, S. Chen, J. Zheng, B. D. Ratner, S. Jiang, The Journal of Physical Chemistry B 2005, 109, 2934.
[72] Z. Zhang, S. Chen, Y. Chang, S. Jiang, The Journal of Physical Chemistry B 2006, 110, 10799.
[73] K. Knop, R. Hoogenboom, D. Fischer, U. S. Schubert, Angew. Chem. Int. Ed. 2010, 49, 6288.
[74] Q. Yu, Y. Zhang, H. Wang, J. Brash, H. Chen, Acta Biomater. 2011, 7, 1550.
[75] J. H. Lee, H. B. Lee, J. D. Andrade, Prog. Polym. Sci. 1995, 20, 1043.
[76] R. P. Quirk, R. T. Mathers, T. Cregger, M. D. Foster, Macromolecules 2002, 35, 9964.
[77] K. C. Popat, T. A. Desai, Biosens. Bioelectron. 2004, 19, 1037.
[78] S. Pasche, S. M. De Paul, J. Voros, N. D. Spencer, M. Textor, Langmuir 2003, 19, 9216.
[79] T. McPherson, A. Kidane, I. Szleifer, K. Park, Langmuir 1998, 14, 176.
[80] S. Sharma, R. W. Johnson, T. A. Desai, Langmuir 2004, 20, 348.
[81] J. L. Dalsin, L. Lin, S. Tosatti, J. Vörös, M. Textor, P. B. Messersmith, Langmuir 2005, 21, 640.
[82] R. C. Gunawan, J. A. King, B. P. Lee, P. B. Messersmith, W. M. Miller, Langmuir 2007, 23, 10635.
[83] S. Zurcher, D. Wackerlin, Y. Bethuel, B. Malisova, M. Textor, S. Tosatti, K.
Gademann, J. Am. Chem. Soc. 2006, 128, 1064.
1-17
[84] M. Shen, L. Martinson, M. S. Wagner, D. G. Castner, B. D. Ratner, T. A. Horbett, J.
Biomater. Sci. Polym. Ed. 2002, 13, 367.
[85] H. Muguruma, Plasma Processes and Polymers 2010, 7, 151.
[86] A. Choukourov, I. Gordeev, O. Polonskyi, A. Artemenko, L. Hanyková, I. Krakovský, O. Kylián, D. Slavínská, H. Biederman, Plasma Processes and Polymers 2010, 7, 445.
[87] I. Szleifer, Physica A: Statistical Mechanics and its Applications 1997, 244, 370.
[88] I. Szleifer, Curr. Opin. Solid State Mater. Sci. 1997, 2, 337.
[89] L. D. Unsworth, H. Sheardown, J. L. Brash, Langmuir 2005, 21, 1036.
[90] L. D. Unsworth, H. Sheardown, J. L. Brash, Langmuir 2005, 21, 1036.
[91] L. D. Unsworth, Z. Tun, H. Sheardown, J. L. Brash, J. Colloid Interface Sci. 2005, 281, 112.
[92] D. Leckband, S. Sheth, A. Halperin, Journal of Biomaterials Science-Polymer Edition 1999, 10, 1125.
[93] B. Johnsson, S. Löfås, G. Lindquist, A. Edström, R. M. Müller Hillgren, A. Hansson, Journal of molecular recognition : JMR 1995, 8, 125.
[94] F. Yu, B. Persson, S. Löfås, W. Knoll, J. Am. Chem. Soc. 2004, 126, 8902.
[95] J. Benesch, P. Tengvall, Biomaterials 2002, 23, 2561.
[96] E. Brynda, M. Houska, J. Colloid Interface Sci. 1996, 183, 18.
[97] E. Brynda, M. Houska, Macromol. Rapid Commun. 1998, 19, 173.
[98] J. F. Masson, T. M. Battaglia, M. J. Davidson, Y. C. Kim, A. M. C. Prakash, S.
Beaudoin, K. S. Booksh, Talanta 2005, 67, 918.
[99] A. Ulman, Chem. Rev. 1996, 96, 1533.
[100] M. D. Porter, T. B. Bright, D. L. Allara, C. E. D. Chidsey, J. Am. Chem. Soc. 1987, 109, 3559.
[101] C. D. Bain, J. Evall, G. M. Whitesides, J. Am. Chem. Soc. 1989, 111, 7155.
[102] J. Sagiv, J. Am. Chem. Soc. 1980, 102, 92.
[103] R. G. Chapman, E. Ostuni, S. Takayama, R. E. Holmlin, L. Yan, G. M. Whitesides, J. Am. Chem. Soc. 2000, 122, 8303.
[104] R. S. Kane, P. Deschatelets, G. M. Whitesides, Langmuir 2003, 19, 2388.
[105] M. V. Athawale, J. S. Dordick, S. Garde, Biophys. J. 2005, 89, 858.
[106] A. R. Statz, R. J. Meagher, A. E. Barron, P. B. Messersmith, J. Am. Chem. Soc.
2005, 127, 7972.
[107] S. Chen, L. Li, C. Zhao, J. Zheng, Polymer 2010, 51, 5283.
[108] R. l. Barbey, L. Lavanant, D. Paripovic, N. Schuwer, C. Sugnaux, S. Tugulu, H.-A.
Klok, Chem. Rev. 2009, 109, 5437.
[109] R. C. Advincula, W. J. Brittain, K. C. Caster, R. Jürgen, "Polymer Brushes:
Synthesis, Characterization, Applications", Wiley Interscience, 2004.
[110] W. J. Brittain, S. Minko, J. Polym. Sci., Part A: Polym. Chem. 2007, 45, 3505.
[111] A. Halperin, Langmuir 1999, 15, 2525.
[112] M. Rubinstein, R. H. Colby, "Polymer Physics", OUP Oxford, 2003.
[113] J. Ladd, H. Lu, A. D. Taylor, V. Goodell, M. L. Disis, S. Jiang, Colloids Surf. B.
Biointerfaces 2009, 70, 1.
CHAPTER 1.INTRODUCTION
1-18
[114] N.-P. Huang, J. Vörös, S. M. De Paul, M. Textor, N. D. Spencer, Langmuir 2002, 18, 220.
[115] O. R. Bolduc, J. N. Pelletier, J.-F. o. Masson, Anal. Chem. 2010, 82, 3699.
[116] O. R. Bolduc, C. M. Clouthier, J. N. Pelletier, J. F. Masson, Anal. Chem. 2009, 81, 6779.
[117] J. Zhang, R. Lao, S. Song, Z. Yan, C. Fan, Anal. Chem. 2008, 80, 9029.
[118] J. Homola, J. Dostalek, S. F. Chen, A. Rasooly, S. Y. Jiang, S. S. Yee, Int. J. Food Microbiol. 2002, 75, 61.
[119] E. Brynda, M. Houska, A. Brandenburg, A. Wikerstål, Biosens. Bioelectron. 2002, 17, 665.
2-1
2 Goals of the Thesis
The main objective of my Thesis is to prepare sensitive layers for optical affinity biosensors. Striving to provide a solution, having surfaces fully resistant to blood plasma fouling able to be functionalised with bioactive molecules was critical. As explained in the previous chapter, at the commencement of the work no non-fouling surfaces existed, so finding a way to prepare them was the key for advancing in the work.
The work has 3 main foci:
Design and preparation of antifouling and non-fouling surfaces
Evaluation and conceptualisation of their resistance to fouling from blood plasma and serum as well as other biological fluids
Preparation of sensitive layers for detection in complex biological media
The first focus was tackled by preparing surfaces utilising both classical techniques, such as SAMs, and the most modern techniques for surface initiated controlled polymerisation of brushes. The techniques were expanded to various substrates utilising two surface-independent approaches and to polymeric nanocapsules.
The fouling from blood plasma and other biological fluids on the prepared surfaces was evaluated as a function of physicochemical properties, i.e. chemical composition, architecture, thickness and wettability. The results were contrasted with the accepted ideas for the design of antifouling surfaces.
The prepared surfaces were functionalised and prototypes of real biosensors for detection in complex biological media were prepared.
2.1 Experimental design
The logic of the experimental design is minimalistic. Surfaces of increasing synthetic complexity were prepared only after simpler ones were not able to meet the challenges posed by undiluted blood plasma. Correlation and conceptualisation of fouling with the physicochemical properties of the antifouling surfaces was the basis for selecting the following more complex surface to be tested. Investigation of the composition of fouling was of great aid for following designs. Once suitable surfaces were obtained, different functionalisation techniques were used to yield non-fouling bioactive surfaces. Since the problem of fouling is also detrimental to many other fields we extended the work to the
2-2 surface addition polymer While th ones sho
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2-3
The author envisions that in the near future the construction of hybrids of biological entities (virus, cells, etc) and synthetic macromolecules will play an important role in medicine and biology. In particular, conjugation of biological entities with antifouling polymers could render new biomaterials invisible to the immune system by avoiding protein adsorption. This has motivated efforts to develop new polymerisation techniques of non-fouling polymers in mild conditions, compatible with biological molecules and entities. A controlled polymerisation in biological media was presented for the first time (Appendix VII). In addition reversible addition fragmentation transfer polymerisation, a class of process that does not require any catalyst, organic solvent or elevated temperature was used to synthesise novel polymers.
For ease of navigation, Figure C2-1 provides an overview of the experimental design and how the steps interrelate.
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3 Synthetic strategies and characterisation
This chapter describes the general methods utilised in this thesis for the preparation of the coatings studied exposing the reasons behind the selection of the applied methodology. Important stress is placed on the chemical strategies used to achieve surfaces with the desired physicochemical properties and even brushes with tuned architecture.
The majority of the coatings prepared on this work were on gold coated substrates.
The main reason for choosing gold is its compatibility with surface plasmon resonance spectroscopy. This technique is central in the present work being used both for in-real-time assessment of the fouling as well as for the preparation of biosensors. This apparent lack of generality was ruled out by extending these results to various substrates of interest only utilising an adlayer which could link the antifouling layer to the substrate as described in Appendix IV.
Due to the comprehensive description of synthetic and characterisation methodologies in the publications attached, the following chapter only aims at introducing the reader to the main synthetic concepts behind the surface design and preparation.
Precise details of the synthetic and characterisation methods can be found in the Appendices attached.
3.1 Building blocks for surface preparation
Three types of surfaces were prepared, i.e. i) self-assembled monolayers (SAMs), ii)
‘grafted to’ α-amino, ω-carboxy poly(ethylene glycol) (PEG) and α-mercapto poly[N-(2- hydroxypropyl methacrylamide)] and various polymer brushes.
3.1.1 SAMs
Figure C3-1 depicts the alkanethiols used for the preparation of the SAMs in this work, i.e. 16-mercaptohexadecanoic acid (C16), carboxy-capped (ethylene glycol)n
undecanethiol (COEGn, where n denotes the number of ethylene glycol units), (ethylene glycol)n undecanethiol (OEGn, where n denotes the number of ethylene glycol units), and ω-mercaptoundecyl bromoisobutyrate.
SAM were prepared by immersing gold coated substrates in a 1 mM solution of thiols in ethanol according to the procedures described in Appendices I, II, III and V.
CHAPTER 3.SYNTHETIC STRATEGIES AND CHARACTERISATION
3-2
Figure C3-1. Alkanethiols utilised for the preparation of SAMs
3.1.2 Grafted-to Polymers
α-amino, ω-carboxy poly(ethylene glycol) (PEG, Mn 3400 g.mol-1) and α-mercapto poly[N-(2-hydroxypropyl methacrylamide)] (α-mercapto poly(HPMA), Mn 100 000 g.mol-
1) with narrow polydispersity were grafted on SAMs or directly on gold. α-mercapto poly[N-(2-hydroxypropyl methacrylamide)] was synthesised by reversible addition fragmentation transfer polymerisation as described in Appendix VIII. The dithiobenzoate end-group was cleaved by aminolysis to yield a thiol useful for immobilisation on gold surface.
Figure C3-2. Polymers used for grafting-to approach
3.1.3 Monomers for surface-initiated polymerisations
Three groups of monomers have been used for the preparation of antifouling and non-fouling surfaces, i) highly wettable zwitterionic betaines, ii) monomers with oligo(ethylene glycol) (OEG) side chains and iii) hydroxy-functional monomers (Figure C3-3).
Zwitterions are dipolar species, in which the cation and the anion are separately bound to the same monomer unit and can be completely dissociated thus maintaining the
SH
O
OH SH O
O OH n
O
SH O
OH n
SH O
O
Br
C16 COEGn
OEGn ω-mercaptoundecyl bromoisobutyrate (ATRP initiator)
HO
O
NH2
O
n
PEG
HO
SH O
CN
HN O
HO n
poly(HPMA)
