Biological Surface Science

Surface Science 500 (2002) 656–677 www.elsevier.com/locate/susc

Bengt Kasemo *

Department of Applied Physics, Chalmers University of Technology and Go€teborg University, S-41296 G o€teborg, Sweden Received 13 December 2000; accepted for publication 5 July 2001

Abstract Biological surface science (BioSS), as defined here is the broad interdisciplinary area where properties and processes at interfaces between synthetic materials and biological environments are investigated and biofunctional surfaces are fabricated. Six examples are used to introduce and discuss the subject: Medical implants in the human body, biosensors and biochips for diagnostics, tissue engineering, bioelectronics, artificial photosynthesis, and biomimetic materials. They are areas of varying maturity, together constituting a strong driving force for the current rapid development of BioSS. The second driving force is the purely scientific challenges and opportunities to explore the mutual interaction between biological components and surfaces. Model systems range from the unique water structures at solid surfaces and water shells around proteins and biomembranes, via amino and nucleic acids, proteins, DNA, phospholipid membranes, to cells and living tissue at surfaces. At one end of the spectrum the scientific challenge is to map out the structures, bonding, dynamics and kinetics of biomolecules at surfaces in a similar way as has been done for simple molecules during the past three decades in surface science. At the other end of the complexity spectrum one addresses how biofunctional surfaces participate in and can be designed to constructively participate in the total communication system of cells and tissue. Biofunctional surfaces call for advanced design and preparation in order to match the sophisticated (bio) recognition ability of biological systems. Specifically this requires combined topographic, chemical and visco-elastic patterns on surfaces to match proteins at the nm scale and cells at the micrometer scale. Essentially all methods of surface science are useful. High-resolution (e.g. scanning probe) microscopies, spatially resolved and high sensitivity, non-invasive optical spectroscopies, self-organizing monolayers, and nano- and microfabrication are important for BioSS. However, there is also a need to adopt or develop new methods for studies of biointerfaces in the native, liquid state. For the future it is likely that BioSS will have an even broader definition than above and include native interfaces, and that combinations of molecular (cell) biology and BioSS will contribute to the understanding of the ‘‘living state’’. 2001 Elsevier Science B.V. All rights reserved.

Keywords: Adhesion; Biological compounds; Biological molecules – nucleic acids; Biological molecules – proteins; Solid–liquid interfaces

1. Introduction

Imagine the following six situations where the * Tel.: +46-317723370; fax: +46-317723134. properties of solid surfaces are or may become E-mail address: [email protected] (B. Kasemo). important in practice:

(i) A patient with severely degraded dental sta- (iii) A small number of living cells of a partic- tus is treated by a surgical procedure, where one or ular kind, maybe a tissue culture or so-called stem several dental implants, made from metal or ce- cells for a certain type of human tissue, are placed ramic, are implanted into the jawbone so that they in a scaffold made from some synthetic mate- after some healing in period can function as arti- rial, with the intention to make the cells grow in ficial teeth (Fig. 1). Other types of implants are number and differentiate ex vivo into a new func- shown in Fig. 2. tional tissue, which later is placed in a patient, in (ii) A blood or urine sample is distributed over a order to repair a lost or degraded body function suitably prepared biosensor or biochip surface in (Fig. 4). order to diagnose a patient’s health status or al- (iv) Neural cells (neurons) are placed on a ternatively for a forensic identification (Fig. 3). micropatterned surface, where they self-organize into a functioning neural network, which can be addressed chemically and/or electronically by in– out (I/O) connections so that a cell-based bioelectronic circuit is achieved. (v) A particular kind of photosensitive, charge transfer proteins are attached to and organized on a specially designed material surface in order to harvest the energy of sun light with a high efficiency, thereby converting the light into chemical or electrical energy in a process mimicking the photosynthetic process of green plants or certain bacteria. (vi) A surface is microfabricated with an array of specially architectured, soft protrusions in the micrometer size range, mimicking a shark or dolphin skin and thereby providing a dramatic re- duction in hydro- or aerodynamic friction. The medical implant example [1,2] is a clinical reality since many years and hundreds of thou- sands of patients have been treated with a major increase in life quality. There are many other examples of medical implants (Fig. 3), each one un- iquely different from the others in the biological/ clinical details e.g. artificial hip and knee joints, artificial blood vessels and heart valves, and synthetic intraocular lenses. Together they represent a large number of industries with total turnover approaching or even exceeding a hundred billion dollars per year. The biosensor example [3–5] is representing an area, where commercial products already exist both for single sensors and array type sensors [6] (e.g. so-called DNA chips) which are used clini- Fig. 1. Schematic illustration of the interface between a dental cally or at the R&D level in biomedical industries implant and the jawbone into which it is implanted, at different magnifications. After the surgical procedure the surface is first and academia. Figs. 4 and 5 illustrate some of the exposed to water, then to proteins, and eventually to cells (see current sensing principles and surface immobili- Fig. 7). See Fig. 2 for other types of implants. zation strategies. This area is in extremely rapid 658 B. Kasemo / Surface Science 500 (2002) 656–677

Fig. 2. A selected set of diﬀerent medical implants.

Fig. 3. Some common detection principles for biosensors and biochips. B. Kasemo / Surface Science 500 (2002) 656–677 659

Fig. 3 (continued)

Fig. 4. Tissue engineering scaﬀold. A template of a synthetic material forms the ‘‘host’’ for a cell or tissue culture. The scaﬀold should promote the development of the culture into a functioning tissue. 660 B. Kasemo / Surface Science 500 (2002) 656–677

Fig. 5. Immobilization strategies for biomolecules on surfaces to achieve biosensor and biomedical diagnostic functions. development with an estimated market growth for e.g. for real-time control of drug administra- labchips/bioarrays of 40% per year. Most of its tion. One such example is the search for a reliable impact, technically and commercially, is yet to glucose sensor for real-time control of insulin come in the wake of the so-called molecular bio- administration for diabetes patients, via an im- logy or biotechnology revolution. The product will planted, sensor controlled insulin pump. have important input from modern microelec- Tissue engineering [7,8] is an emerging area tronics, materials and surface science, advanced where most of the impact is also still to come, very micro- and nanofabrication, and bioinformatics. much fueled by the molecular and cell biology The perspective for the future is unprecedented revolution. The basic strategy for repair of lost precision and speed in multiple (broad band), and body functions by way of tissue engineering is dif- individualized clinical and eventually ‘‘near pa- ferent compared to medical implants: In the latter tient’’ (home) diagnostics. Other important appli- case a synthetic component is fabricated that re- cations are expected in e.g. environmental control places the function of the lost or degraded part of and food production. the body, such as the metal ball head on the hip Although the focus up till now has been pri- implant, and a matching counterpart made from marily on DNA-based diagnostics by large sensor high molecular weight polyethylene (HMWPE), arrays, the future sensing arrays will also address constituting an artificial hip joint. In the tissue biologically more ‘‘down stream’’ components in engineering strategy the idea (Fig. 4) is instead to the cell expression and cell–cell communication produce a real functioning tissue from a cell cul- processes, typically peptides and proteins (the lat- ture ‘‘seed’’, by making it grow ex vivo in a suit- ter area is sometimes referred to as ‘‘proteomics’’). able environment. The latter requires a synthetic Even further down the line lies (potentially) cell- scaffold or template for the growing cells/tissue, based diagnostics (cellomics). whose macroscopic geometry, microscopic topo- Another type of sensors is the kind that can be graphy and surface chemical properties cooperate implanted and used in feed back systems in vivo with other stimuli such as extracellular signal B. Kasemo / Surface Science 500 (2002) 656–677 661 substances and the composition of the physiolog- have new unique characteristics originating from ical solution etc., to make the cell culture develop the biomolecules’ highly specialized functions, in- into a functioning tissue, ready for implantation. cluding their replication and recognition ability, Practical tissue engineering is today realized only and self-organization. The cell level, reverse engi- for a few ‘‘simple’’ tissues like skin and, to some neering approach will result in entirely new circuit, extent, cartilage, while large research efforts are component, and functional concepts. The idea is focussed on e.g. liver, pancreas, blood vessel, to ‘‘automatically’’ achieve signal- and informa- bladder, and heart valve tissues. tion-storage pathways from the internal self- Bioelectronics [9–11] is still only at the very organization, architecture and multiple functions early, explorative research stage and it is too early of living cells, and from the biosystem’s inter- and to judge if it will or will not become a practical intracellular communication pathways. The latter reality in the future. The basic idea is to take ad- have evolved over billions of years, and thus one vantage of some unique aspects of nature’s own does not need to invent these functions and ar- way of information storage and processing. This chitectures again (the latter is the prime reason for can alternatively be done at the cellular level calling this approach reverse engineering). (sometimes referred to as ‘‘reverse engineering’’; As in the bioelectronics case, biomimetic or see below), where the primary building stones are artificial photosynthesis [12] is still an area at its networks of real cells, or by ‘‘forward engineering’’ infancy. The basic mechanism is known by prin- at the biomolecular level, where the building ciple (Fig. 6); it is based on electron excitation by stones are peptides, proteins, DNA strings etc., incident, photons in suitable proteins or protein and combinations of them. Both in the cellular and arrays, followed by charge separation (preventing molecular approach the biological components are charge recombination and concomitant energy likely to be steered into a suitable pattern in or on loss), and finally redox reactions creating useful, a microengineered template or substrate surface, energy rich (fuel) molecules. However, biomimetic providing chemical and/or electrical I/O commu- structures that have the quantum efficiency, sus- nication. Bioelectronic components and circuits tainability, and a self-repair ability approaching made by the molecular level, forward engineering those of natural systems, and that can produce approach are likely to resemble today’s micro- useful fuels (like hydrogen) are still just a vision electronics circuits in some respects, but will also and goal for future research. A strong driving

Artificial Photosynthesis

LUMO - e Re Cat - e- red A e- Electron energy A (Qualitative) (Acceptor)

D+ PIGMENT - D e Catox - R (Donor) e h e- HOMO

Re = Electron Relay Rh =Hole Relay

Fig. 6. The principle of photon to electron energy conversion in photosynthesis. 662 B. Kasemo / Surface Science 500 (2002) 656–677 force is the rapidly growing need for sustainable living cells) are briefly discussed. The same para- energy systems. One strategy to realize such struc- graph also discusses different types of surfaces and tures is to immobilize the photon-harvesting and surface modifications, and analytical and prepa- redox protein arrays of real plants, or their syn- ration methods for biofunctional surfaces. How- thetic counterparts, on surfaces with suitable ever, these paragraphs are intentionally very short, chemical and electronic properties. These surfaces since the focus is on general aspects and concepts need to be functionilized both with respect to rather than specific surfaces or methods. Finally nano- and microtopography and with respect to some comments are attempted regarding the likely surface chemistry. Another approach is to build future development of BioSS. entirely synthetic nanostructures mimicking the The intention with the article is not to present a photosynthetic systems. comprehensive review of the field, but rather to The low friction mimic of a sharkskin surface identify some important trends, directions, and constitutes just one example of biomimetic mate- concepts in BioSS and particularly to outline the rials science [13]. In biomimetic materials science opportunities for graduate students and surface the idea is to mimic the functional properties of scientists to contribute to this exciting field. Ref- biological materials/components or the processes erences are therefore chosen primarily to provide by which they are manufactured in nature, in order starting points for further reading, and there is no to achieve new, superior materials properties or ambition to cover all previous work in BioSS (i.e. manufacturing advantages. A major driving force the reference lists in the quoted references are just to attempt mimicking the sharkskin is the poten- as important as the textual content). tially much lower energy consumption of airplanes and sea vehicles that would be obtained. A more exotic example, already realized in real products, is 2. The role of surfaces swimsuits for faster swimming and low friction hull coatings on sailboats. Other biomimetic ma- 2.1. Biorecognition terials examples are antifreeze materials, utilizing the special features of some water holding proteins In most of the examples above, especially for that can lower the freezing point of water in cer- (i)–(iv), biorecognition is a central component. Any tain arctic fishes below 0 C, and self-cleaning attempt to make a sophisticated, functional sur- surfaces like some green leaves (the lotus leaf), and face for biointeractions must take into account the self-repairing surfaces. highly developed ability of biological systems to recognize specially designed features on the 1.1. Biological surface science molecular scale. Famous examples are antibody– antigen, enzyme–substrate, and receptor–trans- In all six examples above, each representing mitter recognition (e.g. in cell membranes). The large and growing fields of applications and asso- recognition is programmed into the molecules ciated basic R&D, surfaces play a very important through the combination of their 3D topographic role. As a generic name for this branch of surface architecture, the superimposed chemical architec- science I use biological surface science (BioSS) [14] ture, and the dynamic properties. Consequently an (it includes the sub-area of medical applications; optimally designed surface for specific biological biomedical surface science). function must take these aspects into account. The importance and function of synthetic solid Although the fundamental interactions occur surfaces in biological and medical contexts, is the on the molecular scale there is an interesting and subject of the next paragraph (Section 2), with unique synergistic connection between the na- special focus on the six examples or sub-areas nometer and the micrometer length scales [1,15,16] mentioned above. In Section 3 the large variety of when cells are present, as e.g. in the cases of model systems available for BioSS studies, of dif- medical implants, tissue engineering and cell-based ferent complexity (from water and amino acids to bioelectronics. This is schematically illustrated in B. Kasemo / Surface Science 500 (2002) 656–677 663

Fig. 7. Schematic illustration of the successive events following after implantation of a medical implant. The first molecules to reach the surface are water molecules (ns time scale). The water shell that is formed affect the protein interaction starting on the micro- to millisecond time scale, and continuing for much longer times. The water shell on the surface affects the protein interaction. Eventually cells reach the surface. Their surface interaction takes place via the protein coating whose properties is determined by the surface and water adlayer properties.

Figs. 7 and 8. The first picture (Fig. 7) shows the may determine if proteins denature or not, their sequence of events after a surface has suddenly orientation, and coverage etc. been placed in a biological milieu containing cells. When cells arrive at the surface they ‘‘see’’ a The first molecules to reach the surface (time scale protein-covered surface whose protein layer has of order ns) are water molecules. Water is known properties that were initially determined by the to interact and bind very differently at surfaces preformed water shells. Thus, when we talk about depending on the surface properties (see Section cell–surface interactions, it is ultimately an inter- 3). The properties of the surface water ‘‘shell’’ are action between cells and surface bound proteins an important factor influencing proteins and other (or other biomolecules). The latter is illustrated in molecules that arrive a little later. These water- Fig. 8, where the clean surface in the illustration is soluble biomolecules also have hydration (water) deliberately covered with a native or artificial shells and the interaction between the surface biomembrane (supported bilayer), containing em- water shell with the biomolecular water shell in- bedded receptors that can specifically interact fluences the fundamental kinetic processes and the (bind to and/or provide I/O signals etc.) with cells thermodynamics at the interface. For example, it approaching the surface. 664 B. Kasemo / Surface Science 500 (2002) 656–677

only marginally applying surface analytical techniques to study medical implant surfaces, to a current situation where such use is the rule and mandatory for high quality research, and also for legal control, standardization and quality control of commercial products. Let us briefly inspect how surfaces come into play. For a dental implant (Fig. 1), which is just one of many examples of so-called bone anchored implants, the clinical goal is to obtain a long term (i.e. life long) secure anchoring of the implant. The latter includes e.g. the implant’s ability to carry and sustain the dynamic and static loads that it is subject to. It is obviously (for the patient and for cost reasons) important to achieve this function with the shortest possible healing time, with a very small failure rate and with minimal uncomfort for the patient. The implant is put in place after a surgical procedure typically involving drilling a hole in the cheekbone, threading of the hole, and then placing the screw-shaped end of the implant into the threaded hole. The surface design is one of many components contributing to the clinical goal fulfillment. An inappropriate material choice or surface coating might lead to a too strong inflammatory response that adds on top of the already initiated inflammatory process by the surgical procedure. Then Fig. 8. A conceptual approach to convert a synthetic surface to connective tissue rather than bone is formed a biomimetic surface by coating it with a supported biomem- around the implant, with concomitant loss of brane with built-in functional membrane-bound proteins. mechanical stability and eventual implant failure. A less perturbing surface may lead to a bone healing process, which is similar to the one in the Figs. 7 and 8 emphasize the above mentioned absence of an implant i.e. as if the implant were a large size range that the functional units in biology fairly passive spectator (sometimes this is referred cover, from water molecules and small proteins at to as inert materials or bone accepting materials, sub-nanometer sizes, via cell membranes and su- although no material is really inert in an absolute pra-molecular complexes in the 10 nm range, to sense). Some surface coatings appear to have the cells in the micrometer range and finally fully or- ability to induce bone formation, for example the ganized tissue and organs at the macroscopic size calcium phosphate materials usually referred to as level. hydroxyapatites. More recently there has also been attempts to 2.2. Medical implants speed up the bone healing beyond the normal healing rate by adding so-called bone growth fac- In the case of medical implants (Figs. 1 and 3) tors. The latter can e.g. be implemented by using a the importance of surface science is quite obvious porous surface with a slow release of the desired [1,2,14–16]. In 15–20 years time biomaterials re- biomolecules. Engineering of such surfaces research and development has transformed from quires a whole set of surface science tools. Exam- B. Kasemo / Surface Science 500 (2002) 656–677 665 ples of used surface science methods are coating that ‘‘detector molecules’’ are attached/immobi- technologies to deposit the desired surface material lized on a solid surface in such a way that a specific on a bulk carrier; surface spectroscopies like XPS, signal is obtained from the sensor when the de- AES, and SIMS to control e.g. the Ca/P ratio; tector molecules selectively react with (bind to) the SEM or scanning probe techniques (SPMs) to biomolecules they are designed to detect. In other control surface structure and chemistry, both of words, the detection relies on the biorecognition which influence the biological response; micro- between pre-designed native or synthetic reagent and nanofabrication or other techniques to make molecules and unknown sample molecules. The porous surfaces; surface impregnation by mole- biorecognition event typically occurs when the pre- cules that are released at a pre-programmed rate to coated sensor is exposed to blood or urine samples promote healing/bone formation, FTIR to mea- or extracts thereof. sure adsorbed biological molecules etc. The detector read-out signals may be electrical, Another challenging task for surface engineer- optical, or some other signal that is convertible ing of biomaterials is blood-compatible materials, into a measurable electrical signal. The detection such as the inner walls of artificial blood vessels, may occur in real time, i.e. just when the recog- and the blood contacting surfaces of heart valves, nition event occurs, or the sensor can be read-off dialyses equipment, and heart–lung machines. at a later time. Two common examples are (i) Normal blood is, naively speaking, a metastable antigens are pre-adsorbed on the sensor surface to state stabilized between two opposing driving detect the corresponding antibody(ies) in a clinical forces––coagulation and anticoagulation. Almost sample for diagnostics; (ii) a single strand DNA any external perturbation in the form of an arti- segment (i.e. a nucleic acid chain without cross- ficial surface tends to trigger the coagulation sys- linking), representing a specific, known part of the tem, i.e. blood clotting may occur. The extent to genetic code, is attached to the surface so that all which this happens, and is detrimental for the the dangling bonds of the non-hybridized, single patient, depends on a number of factors such as strand are exposed and available for binding to a where the surface is placed in the blood stream, the matching DNA strand. When the sensor is ex- local hydrodynamics, the flow speed, contact time posed to a DNA sample to be analyzed, one wants between the surface and blood and, of course, on to obtain a specific response when there is per- the surface chemistry. fect matching between the detector molecule and There is today clear evidence that the latter has some sample molecules, while a different response a major influence on blood compatibility and that should be obtained when the matching is imper- surfaces can be engineered to suppress blood co- fect. On a DNA chip many different such DNA agulation, at least for considerable time. However, segments are placed at different locations and thus there is still a long way before surfaces that mimic many different DNA segments can be analyzed in the blood compatibility of the endothelial cells of one ‘‘shot’’ (bioarray, biochip). When the sensor normal blood vessel walls are achieved. For the array is combined with a sophisticated microflui- surface scientist this task involves designing du- dics system often utilizing functional hydropho- rable surface coatings that have the right chemical bic–hydrophilic surfaces––we call the device a and micromechanical (visco-elastic) properties, lab-on-a-chip or just labchip. which in turn may require a better understanding Other sensing principles are to e.g. employ en- than today of how the native system works, so that zymes that are immobilized on a surface with a true biomimetic approach can be implemented. preserved function, and which can recognize specific biochemical substances, or the use of MAL- 2.3. Biosensors and biochips DI-MS to identify proteins and antibody–antigen complexes. A variety of surface based detection principles Surface science tools and expertise come into are employed for biosensors/biochips (Figs. 2 and play in many ways in the preparation and use of 3). A common principle of a biosensor/biochip is biosensors and biochips. Immobilization of the 666 B. Kasemo / Surface Science 500 (2002) 656–677 detector molecules in such a way that they preserve material (Fig. 4), on which the original cell cul- their original recognition specificity, requires sur- ture(s) are deposited and eventually develop into a face pre-coatings that simultaneously allow suffi- tissue, must be specifically designed to promote, ciently strong binding of the detector molecules, and not compromise, the tissue evolution. This and sufficiently weak and gentle perturbation that calls first of all for a non-toxic surface for the cell the molecules retain their functionality (Fig. 5). culture, and for suitable protein adsorption prop- For example, the strong polarization forces at erties, since the protein adsorption layer that metal surfaces and the strong ionic or covalent always forms on the surface influences the cell– interactions on many inorganic metal oxides and surface interactions. Since differentiated tissue is semiconductor materials may cause denaturation an ultimate goal, different parts of the scaffold may and loss of specificity of biomolecules. At the same require different surface functionalities, i.e. differ- time such materials may be desirable ‘‘platforms’’ ent surface chemistries and topographies. for sensor arrays. A solution is then deposition of Cells in a tissue culture communicate with each special spacer and/or linker molecules (typically other by sending out and receiving signaling mol- some type of polymer or a native or synthetic ecules to/from the extracellular matrix. The total biomembrane) on the original sensor substrate. spectrum of such signals is influenced by the sur- The preparation and characterization of such faces onto which cells attach. Thus, the surfaces of layers for useful deposition of detector molecules, tissue engineering scaffolds are an integral part of obviously require sophisticated surface science in- the communicating and self-organizing tissue. put. The same holds for the preparation of the part A common––but not the only one––concept in of the sensor that provides a measurable signal. tissue engineering is a scaffold that eventually is The most common detection approaches so far are dissolved as the tissue ‘‘matures’’. This calls for a based on optical detection in the visible or infra- sophisticated ‘‘programming’’ of the material so red. Optimization of the optical detection requires that it dissolves into harmless products at the right deposition of optically suitable layers e.g. regard- pace, and maybe also exposes different functional ing reflectivity, absorption properties, plasmon surfaces at different times. (The latter may also be properties, field enhancements etc., which are all a desired function of medical implants.) Many typical components in thin film design of optical different surface technologies are potentially suit- coatings. Consequently advanced surface spec- able for such surface functionalization, especially troscopy and microscopy come into play both for coating technologies that can be applied to 3D understanding of how the matrix and detector curved surfaces and porous surfaces, but also the molecules bind to the sensor surfaces and for standard surface characterization methods come practical diagnostics. Regarding biochips, arrays into play. The 3D surface topography, which is of detector ‘‘spots’’ are required and here surface very important for cell–surface interactions, may science plays a similar role as it does for the mul- require micro- and nanofabrication methods. tiple microfabrication steps in semiconductor/mi- croelectronics technology. 2.5. Bioelectronics

2.4. Tissue engineering Bioelectronics based on living cells (neurons etc.) call for patterned surfaces where the cells can To reach the goal of tissue engineering––to be confined to certain geographic locations (in grow a functional tissue ex vivo that can later be order to make a reproducible circuit), and where implanted into a human body––requires control they can be kept alive and functional, which in over a large number of external parameters. Ex- turn requires suitable surface chemistries and to- amples are the nutrients, extracellular signal sub- pographies. Regarding the basic control of cells stances, antibiotics, and the dynamic mechanical there is actually large similarities between cell- force fields etc. in the bioreactor where the tissue is based electronics and tissue engineering. The sur- grown. The surface of the template or scaffold face patterns for bioelectronics require in addition B. Kasemo / Surface Science 500 (2002) 656–677 667 adequate I/O connections for electrical or chemi- amples involving surfaces as key elements are cal communication. Similar requirements are at mimics of the lubrication and wear resistance of hand for bioelectronic devices based on biomole- artificial joints, the low drag friction of shark and cules rather than whole cells, albeit at a generally dolphin skin, the self-cleaning action of some smaller length scale (see also the text under artifi- green leaves, and threads made to mimic the cial photosynthesis below). Consequently bioelec- strength and flexibility of spider web. tronic circuits require a combination of molecular cell biology with surface preparation/characterization and micro- or nanofabrication techniques. 3. Model systems, research topics, and ongoing research 2.6. Artificial photosynthesis 3.1. Biological model systems From a surface design point of view there are many similarities between biomolecule-based bio- In this section model systems are discussed and electronics and artificial photosynthesis based on brief references are made to recent or ongoing re- biomolecules. One common denominator is charge search, illustrating the diversity of research areas transfer within or between biomolecules, and be- and the challenging research problems in BioSS. tween biomolecules and surfaces, either induced by The biologically relevant model systems available external electrical fields (voltage bias) or by the for surface science approaches cover such a broad electromagnetic field of light in the visible regime range that any taste should find its topic of choice. (e.g. photoexcitations of electron hole pairs). The Some of these areas––such as water and simple relationship between the two areas becomes espe- amino acids––are immediately approachable by cially obvious when the bioelectronics concepts are existing UHV- and surface science preparative and extended towards opto(bio)electronic devices. analytical techniques. Others require entirely new For the surface scientist a major contribution or modified experimental approaches, such as can be to produce chemical–topographic patterns those requiring wet preparation and in-liquid an- on surfaces that adsorb and retain the biomolec- alyses or e.g. freeze-drying techniques, to maintain ular building blocks of the circuit elements at the biological properties and function of the prescribed locations and in the right orientations studied systems. The model systems contained in (self-organization), without loss of functionality. the following list should be read with the accom- In artificial photosynthesis with e.g. proteins (Fig. panying label ‘‘... at surfaces’’: 6) there is also a major challenge to find and prepare surfaces that match the electronic states of the • water charge transfer proteins, e.g., as electron/hole ac- • amino acids, nucleic acids, lipids ceptors or donors. Further requirements of the • peptides, DNA segments surface may be to make field enhancement struc- • proteins tures that increase the quantum efficiency of • DNA ‘‘photon harvesting’’; perhaps similar to the ones • lipids, biomembranes that produce the recently discovered giant surface • cells enhanced Raman effect. Other ideas that come • tissue, in vitro into play are to use optically active quantum dot • tissue, in vivo arrays to catch and convert photons to energetic • microorganisms and ‘‘useful’’ electrons. The list covers most but not all biointerfaces 2.7. Biomimetic materials where there is an interface between a synthetic (i.e. non-native) material surface and some component(s) This heading covers a large number of widely of native biological systems. I call them hybrid different scientific and technical applications. Ex- biointerfaces, in contrast to native biointerfaces, 668 B. Kasemo / Surface Science 500 (2002) 656–677 because they consist of one native biological rent progress in the modeling of water hydration component and one synthetic, non-native com- shells on protein resistant self-assembled mono- ponent. I will not treat native biointerfaces, layers by Gruntze and co-workers [19], which is an such as cell–cell or microorganism–cell interfaces, important component both for understanding of although they are closely related to the hybrid what the requirements protein resistant surface is, interfaces; it is actually very likely that the and for understanding protein adsorption. It is knowledge and methods for these two types of also a central component for blood-compatible interfaces will mutually interact and cross-fertilize surfaces. in the future. For example, studies of properties and processes in real cell membranes and in so- 3.1.2. Amino acids and peptides called supported biomembranes (see below) are Amino acids are the building stones of one of clearly benefiting from each other. the most elementary functional units in biology, namely proteins. Mapping their interactions with 3.1.1. Water surfaces is therefore, apart from the inherent basic Water at interfaces is an area where surface research interest, a precursor to the understanding science studies will contribute significantly in the of how peptides and proteins interact with sur- future, e.g. to the understanding of what is some- faces. In the recent years a number of papers have times called ‘‘biological water’’. The latter refers to been published [20–24] addressing how sub- the special water structures that are formed near monolayer to multilayer deposits of simple amino surfaces or at narrow interfaces between biological acids (glycine, alanine, ...) interact with single components, such as the hydration shells of pro- crystal surfaces or polycrystalline surfaces in teins, DNA and biomembranes. Biological water vacuo. These studies include standard spectro- at surfaces, or in small confined volumes, is in scopic techniques, such as SIMS, HREELS, FTIR, principle closely related to the special water and synchrotron light based spectroscopies applied structures that form at solid synthetic surfaces. to amino acid adsorbate layers. Some of these They obtain their unique interfacial properties systems have also been characterized by LEED through a combination of the local physical– and TDS. There are also recent attempts to de- chemical interactions (the thermodynamics) at the scribe the amino acid–substrate interactions (in- interface, including the spatial constraints caused cluding adsorbate–adsorbate interaction) by first by the surface, and the kinetic constraints. They principle calculations. are also related to the hydration shells of solvated The amino acids––at least the simpler ones––are ions and to the Helmholtz layers at electrode sur- compatible with UHV evaporation techniques. faces in electrochemical cells, an area where sur- For example glycine and several other amino acids face science has contributed with significant new can be evaporated from a simple Knudsen source insight in the past two decades. at low temperature. More complex (fragile) amino Since the presence of water has such a profound acids may require deposition from the liquid influence on both the thermodynamics and kinetics phase, and then similar techniques are needed at biointerfaces it is a safe prediction that it will be as have been developed for UHV studies of elec- a major topic in BioSS for a decade or more ahead. trode double layers in electrochemistry. Important Recent conceptual and theoretical considerations questions concern (i) how the amino acids bind to can be found in the series of papers published by the surface, if the bonding is non-dissociative or Vogler and collaborators (see Ref. [17] and refer- dissociative, where and how the bonds are estab- ences therein). Experimental work includes both lished (at the amine group, and/or at the car- direct studies of the force interactions at hybrid boxylic group, and/or at the R-side group etc.), (ii) interfaces, as described by e.g. Israelachvili and strength and character of adsorbate–adsorbate collaborators [18], and kinetic and spectroscopic interactions, (iii) molecular orientations, (iv) 2D studies of interfacial water, using both biological crystal structure (when ordered layers are formed), and non-biological substances. There is also cur- and (v) the influence of water molecules on these B. Kasemo / Surface Science 500 (2002) 656–677 669 properties. Another class of questions regards ki- is gradually covered by a single protein type or by netic processes, e.g. to what extent can surfaces proteins that compete for the surface sites. Com- catalyze polymerization (e.g. forming peptide mon techniques are surface plasmon resonance, bonds) and other reactions between amino acids ellipsometry, fluorescence detection, and other and co-adsorbates. optical methods, and gravimetric (QCM, QCM-D, The same questions as above can be formulated SAW-devices) and radiolabelling methods. Pro- for peptides, which are polymer chains of amino tein adsorption is for most surfaces irreversible acids. They are important functional units in through additive contributions from e.g. van der biology per se, and also constitute the amino-acid- Waal’s interaction, polar and charged groups on chain sub-units of proteins. Studies of peptides are the proteins, hydrogen bonding, and hydrophobic therefore one step up in complexity from amino interactions. acids, but also a step closer towards ‘‘real bio- The relative strengths of these contributions logy’’. (By analogy the step from single nucleic depend on protein type, on surface properties and acids at surfaces to DNA segments at surfaces is a on pH and salt concentration. Binding energies are step both in complexity and towards biological difficult to measure for irreversible adsorption. A relevance.) A natural extension of studies of amino few surfaces bind proteins sufficiently weakly that acid monolayers is thus di-peptide, tri-peptide reversible adsorption occurs, whereby heats of etc. adsorption studies. Up to date no systematic adsorption can be obtained from the kinetic mea- peptide studies, like the ones for amino acids, have surements. to my knowledge been done in UHV, with state of Very few surfaces are so-called protein resis- the art surface science techniques. In contrast there tant surfaces (see also under biomembranes be- is large ongoing activity focussing on liquid phase low). Protein resistance means that the adsorbed deposition of various peptide depositions and amount, even at relatively high protein concen- formulations on biomaterial surfaces in order to tration in the bulk liquid, is below or near the make them more functional for e.g. medical im- detection limit. Theoretically it is still a major plant and tissue engineering applications. These challenge to understand the mechanisms that con- depositions still lack the rigorous control that can tribute to protein resistance of surfaces, and even be achieved in UHV, but surface spectroscopies more so to understand the detailed contributions like XPS and FTIR can and are applied, after to strong(er) protein–surface bonds. Recent ex- deposition, to control the deposited layer compo- perimental work by Whitesides and co-workers sition. [40–42] and by Gruntze and co-workers [19,39] constitute important steps towards understanding of protein adsorption, and protein resistance and 3.1.3. Proteins ultimately of protein binding more generally. Proteins, which are (polymerized) peptide Important challenges, not at all clarified at the chains wrapped folded into special 3D structures molecular level, are to quantify the relative and (conformations), have since long been, and is absolute contributions of the various protein– currently the subject of extensive surface studies surface interaction terms, and to ultimately [25–42] due both to their central role in all bio- understand how the relatively fragile protein logical processes and due to their importance in structures (usually they denature well below 100 the context of bioengineering, e.g. for biosensors, C) are affected by the perturbation exerted by a biofouling and tissue engineering. The vast ma- solid surface. There is no doubt that the water shell jority of past studies have focussed on the (mac- of the surface and that of the (water soluble) roscopic) kinetics, and to some extent on the proteins, and their mutual interaction, play an energetics of adsorption on various surfaces, and important role, which can be expressed in terms of under different conditions of liquid properties hydrophobicity–hydrophilicity at the macroscopic (protein concentration, pH, salinity, ...). A typical scale, and in terms of water–water versus water– experiment records the kinetics by which a surface surface bonding strengths at the molecular level. 670 B. Kasemo / Surface Science 500 (2002) 656–677

For example, a material surface of low polariz- techniques are already addressing some of these ability in the bulk with a strongly bound water questions. There are major challenges to adopt mono- or bilayer on the surface, is likely to only these techniques to the special ‘‘wet’’ requirements weakly perturb a protein with predominantly hy- for proteins. The theoretical work ahead is equally drophilic surface domains, while a hydrophobic demanding and important as the experimental surface is likely to strongly perturb a protein with work and will be absolutely necessary for progress mixed hydrophilic and hydrophobic domains, via in this area. hydrophobic interactions with the latter. These ideas have recently been quantified theoretically in 3.1.4. DNA some simple cases. Furthermore there are impor- Studies of the DNA molecule at surfaces [44,45] tant entropy effects in the energetics of protein is driven both by the inherent interest in under- bonding at surfaces due to the highly organized standing different aspects of this molecule, and by and multiple state structures of proteins, and also its importance for medical diagnostics using DNA due to the many different water structures that are biochip arrays. More recently this interest has been possible. stirred by potential applications of DNA in (bio- Finally kinetic effects are of primary impor- mimetic) materials science and in molecular elec- tance; the final structure of an adsorbed single tronics. Generally, much less has been done protein, or protein adlayer, is almost always a regarding basic adsorption studies of nucleic acids long-lived metastable state, rather than the lowest and DNA compared to amino acid, peptide and energy state. In other words there is usually a protein adsorption, except in the sub-area related sufficiently large activation barrier towards for- to DNA biochips. As a rule direct adsorption of mation of the lowest energy states, so that further DNA on such surfaces is of less interest than ad- conformational changes are prevented. A common sorption on pre-adsorbed spacer layers. The rea- experimental manifestation of this is that the final son is that the surface perturbation from, say a structure of a protein adlayer often depends on the metal, oxide or semiconductor surface, turns out rate by which it is formed; at low bulk concen- or is suspected to be too strong to preserve the full tration of proteins the adsorption is slow and there function of the DNA molecule. For example, it is is then longer time available for conformational very likely that the dangling bonds of a non- changes to occur, in comparison with high bulk hybridized DNA segment will bind to the surface concentration and high adsorption rates, when and then be useless for detection of matching neighboring molecules may sterically prevent slow segments. The maintenance of the full recognition conformational changes. The issue of protein function of e.g. a single strand DNA segment is conformation and denaturation at surfaces con- crucial for diagnostic applications as on biochips. nects protein adsorption to the broad field of Therefore various spacer layer and linker molecule protein folding–unfolding in the bulk phase (see strategies are currently developed to achieve both e.g. Refs. [38,43] and references therein). localization of the diagnostic DNA segments at A quantitative first principle description of different spots on a surface array, and preserved protein bonding at surfaces lies far into the future. functionality. It will be preceded by corresponding descriptions The molecular electronics potential of DNA is of adsorbed amino acids and di- and tri-peptides currently a hot topic. Specifically there is intense (and the influence of water on such bonding). It research addressing the question whether the DNA will also require a quantitative first principles total molecule is a 1D conductor or not, and in that energy description of proteins in the bulk phase, case, what the conductivity mechanisms are. Re- including the description of dynamics and multiple cent literature reports both high conductivity and states. Spectroscopic techniques such as non-linear insulating properties. Although some reports may optical techniques and other laser based techniques be plagued by experimental difficulties there is (SFG, SHG,...), synchrotron based spectroscop- another aspect; the question of conductivity or not ies, neutron scattering, NMR, and scanning probe must be more specific, and discriminate between B. Kasemo / Surface Science 500 (2002) 656–677 671 different types of molecules, and take into account tures that minimize the water interaction of the if the measurement is made with or without pres- hydrophobic tails with surrounding water, and ence of the water shell of DNA etc. Microscopic maximize the exposure of the hydrophilic end (e.g. by SPMs) and spectroscopic characterization groups towards the water. This can result in many of the actual DNA configurations, preferably in different self-assembled structures of which so- the liquid phase, is thus vital for progress in this called unilamellar or bilayer membranes are the field. most interesting in the present context. They typically consist of a double layer of phospholipid 3.1.5. Biomembranes molecules, with opposite orientations of the two Biomembranes are and will be a future ‘‘hot’’ layers i.e. with two consecutive layers of parallel topic for BioSS and many other sub-disciplines, hydrophobic tails in the interior, terminated on due to their enormous importance as functional both sides by the hydrophilic end groups. units in living cells, and because of their potential A specific structure occurs when the bilayer role in many applications [46–53]. Biomembranes forms a closed spherical shell, with water both on (Fig. 8 lower picture, Figs. 9 and 10) are self- the inside and the outside. This structure is named organized structures of amphiphilic lipid mole- a vesicle or liposome, an entity that is a first cules, the latter consisting of a hydrophobic crude approximation of a closed cell membrane, (weakly interacting with water) lipid tail (linear however, lacking the functional units of real cell hydrocarbon chain) and a hydrophilic (strongly membranes such as membrane-bound proteins, interacting with water) terminal group such as ion channels etc. One could refer to them as a a phosphate group (phospholipid molecules). In primitive, ‘‘empty cell’’ that can be filled by both water solutions the thermodynamics drive these membrane-bound functional units (membrane amphiphilic molecules to (self)assemble into struc- proteins) and intracellular components, in order to

Fig. 9. Formation of SPB from vesicles (schematic; not proven by experiments). 672 B. Kasemo / Surface Science 500 (2002) 656–677

Fig. 10. A sequence of preparation steps that have been experimentally implemented [50] to build up a biofunctional surface for e.g. cell culturing or biosensing. The first step consists of formation of a biotin doped, phospholipid bilayer from vesicles, on top of which (2nd step) a 2D crystalline protein layer (streptavidin) is adsorbed. The third step is adsorption of DNA or PNA molecules, which are biotin coupled to the remaining free attachment sites on the streptavidin molecules. This platform can then be used e.g. for dis- criminative recognition of perfectly and non-perfectly matching DNA segments. successively build up more cell-like structures. brane proteins or protein or DNA layers on top of Another common structure is the planar bilayer the membrane, can also be formed [48–53]. Pro- membrane, which is called a supported (bilayer) teins are e.g. incorporated into the membrane by membrane when deposited on a suitable surface. placing them inside the vesicles/liposome mem- Such supported membranes are of extensive in- brane prior to surface deposition. The proteins terest both as key elements of biosensor devices, incorporated in the membrane can then in turn act and as mimics of ‘‘empty’’ or real cell membranes, as a binding site for other proteins on top of the the latter after they have been ‘‘doped’’ by incor- membrane, e.g. through antibody–antigen binding. porating membrane proteins etc. When the bi- A beautiful example reported by Reviakine and layer is made up of phospholipids, it is called a Brisson is the 2D protein crystals on top of a biotin supported phospholipid bilayer or biomembrane doped bilayer imaged by AFM [49]. By suitable (SPB). Such bilayer membranes are also of interest linker chemistry one can continue to add additional as functional coatings on medical implants, cell layers, to achieve a specific desired function. One culture surfaces, and tissue engineering scaffolds. such sequence of preparation steps executed by Supported bialyer membranes can be formed on Ho€ook€ et al. [50] is shown in Fig. 11, beginning with a surface starting from vesicles/liposomes (see Refs. a supported membrane containing biotin, followed [46–50], and references therein). Fig. 10 shows in by binding a 2D (crystal) of streptavidin on top, schematic form how such supported bilayers may and completed by binding another biotin layer form. Supported bilayers with incorporated mem- linked to a single strand DNA or PNA segment in B. Kasemo / Surface Science 500 (2002) 656–677 673 the outermost layer. This structure can then be 3.1.7. Tissue used to detect recognition events between the single Tissues at surfaces, in vitro and in vivo, have strand DNA segment and its matching counter- been demonstrated to be sensitive to the adjacent part, forming a double helix. For sensor applica- surfaces, but the mechanisms are understandably tions it is important to find methods to pattern cells even less known than in the case of single cells at surfaces. Such patterning has recently been and cell monocultures. Current research on tissue- demonstrated by several groups, see e.g. Ref. [53] material interfaces in vivo follows several parallel and references therein. lines. One major direction is chemical modification of surfaces to enhance their biocompatibility 3.1.6. Cells and/or to provide controlled release of substances Cells at surfaces is a fundamental ingredient that promote the healing and sustained function of both with regard to the cultivation of cell cultures implants. Another one focuses on topographic in vitro, tissue engineering, and medical implants. modification of surfaces to promote desired cell– In the future it may also be employed in cell-based surface responses, and to some extent also pro- biosensors and bioelectronics. The role of the tein–surface interaction. In some studies these surface chemistry and topography for the evolu- surface modifications, i.e. chemical and topo- tion and function of single cells and cell assemblies graphic, are combined to achieve optimal perfor- has been demonstrated in many experiments [54– mance of the surface. Additional functions of the 63] but is still poorly understood. The recent rapid surface may be to e.g. act as a reservoir that can development in the area of so-called stem cells is release certain desired substances, such as growth particularly interesting in this context. Stem cells hormones, in a time-programmed manner. are cells that have not reached a high degree of In the rapidly expanding tissue engineering field specialization, but can ‘‘choose’’ to differentiate in the questions and challenges are to some extent different specialized directions depending on the similar as for the in vivo situation with medical local environment. Depending on the latter stimuli implants. There are however also entirely new the cell can thus differentiate into different func- challenges connected with the fact that tissue en- tions. It has also been shown that some stimuli can gineering demands that a given cell or tissue culture make a cell ‘‘go backwards’’ from a specialized to shall evolve into a desired tissue without the sup- less specialized state. port from the living organism that is the host for The chemistry and topography of surfaces on in vivo implants. Thus tissue engineering demands which cells grow/divide are part of the total envi- scaffolds that cooperatively with other factors ronment for the cells and can potentially influence (nutrients, extracellular chemicals, force fields, their differentiation, i.e. they are part of the total temperature, ...) steer the tissue culture in the re- cell–cell signaling/communication system. One key quired direction. In some sense one can say that research area for the future is therefore how tai- the bioreactor must do for the tissue engineering lored surfaces should be designed to act as stimuli scaffold þ cell culture, what the living organism to guide cell differentiation, e.g. in tissue engi- does for the medical implant during the healing-in neering, bioelectronics and cell-based sensors. For period. However, the total demand is much higher, the surface scientist the chemical and topographic namely to make the orininal cell/tissue culture de- patterning of surfaces, and the associated charac- velop into a functioning organ. This may require terization is a major opportunity and challenge for special 3D architectures ranging from macroscopic future research. Another is to use SPMs to probe dimensions to micrometer and nanometer dimen- or stimulate specific cell functions. Yet another sions, with superimposed chemical patterns and one is non-invasive, spatially resolved spectro- (bio)chemical signals, and probably with a time- scopies and microscopies to study special fractions programmed degradation rate of the templates as of living cells or cell membranes at high spatial the tissue matures. The research opportunities for resolution (bioimaging) and sensitivity (single surface scientists in this area are similar to those for molecule detection). cell–surface interactions (see above). 674 B. Kasemo / Surface Science 500 (2002) 656–677

3.1.8. Microorganisms terns derives from the very advanced recognition Microorganisms at surfaces are important e.g. power of biological systems discussed earlier. in marine biofouling and bacterial infections on Native material surfaces are very unsophisti- implant surfaces or in tissue cultures. They are also cated compared to the biomolecular chemical þ potentially of interest for growing simple organism topographic architectures they are intended to in- cultures as model systems for a variety of biolog- teract with, and will in most cases at best cause a ical experiments e.g. related to food production, mild negative response in the biological system. In development and evaluation of pharmaceuticals, order to obtain a positive and selective response and biomimetic energy production. Colonization the molecular architecture should in general match of surfaces by e.g. bacteria (biofouling) is surface some recognition sites of biomolecules on the bi- specific and can be influenced by surface chemis- ological side of the hybrid interface, so that it try, surface topography, and surface visco-elastic- generates desired signal patterns to adjacent bio- ity. The settling down of microorganisms on a molecules or cells or sensing devices. This type surface is––as in the case of cell adhesion––almost of topographic þ chemical microarchitectures are always preceded by protein adsorption. There is necessary both for applications and understand- thus a causal relationship between protein ad- ing-oriented basic research of biointerfacial pro- sorption and biofouling by microorganisms, in a cesses. similar way as sketched earlier for cell adhesion on Consequently the future development of bio- a surface. Also the basic research opportunities logically functional surfaces demands a very so- and tools are similar as for cell culture growth and phisticated machinery for surface preparation and tissue engineering, while the more applied research characterization, including dry and wet chemical may require much more specific and specially adlayer depositions, and nano- and microfabrica- adapted tools, e.g. to study ‘‘real’’ marine bio- tion. fouling or bacterial colonization on medical implants. 5. Methods

4. Surfaces Since this article is more concept and idea oriented than method oriented this paragraph is de- The material surfaces include almost all ma- liberately very brief. A general statement about terial types; metals, ceramics, carbon materials, surface science methods and biointerfaces would polymers and composite materials. Although be too trivial, since it would conclude that essen- many native materials are currently used in prac- tially all methods of surface science come into play tice, such as titanium for dental implants, stainless in some contexts. steel for orthopedic implants, PTFE for blood Well-established examples on the analytical side vessel replacements, silicones for internal drainage, are the use of established surface spectroscopies PMMA for intraocular lenses, and so on, future (XPS, AES, SIMS,...) to characterize medical more advanced materials and applications will implants after manufacturing and sterilization, but often require the build up of sandwich type over- before clinical insertion, and also after varying layers, with specific topographies and patterns, on periods in vivo (retrieved implants). Synchrotron the native surfaces, to obtain the desired function. based spectroscopies will no doubt be important This is actually already the case e.g. for intraocular tools both for spectroscopic characterization, lenses made from PMMA and almost always the structure determination, and for imaging of bio- case for advanced biosensors. In addition we know logical components on surfaces. With regard to in that surface patterns, topographically and chemi- situ characterization of surface bound biomole- cally, on different length scales generate different cules and larger entities, and especially for real- responses of the biological system. This require- time recording of kinetic processes, the optical ment of sophisticated surface structures and pat- techniques are of prime importance. This includes B. Kasemo / Surface Science 500 (2002) 656–677 675 both linear and non-linear (laser) techniques, and probe techniques and time resolved laser spec- all spectral regions from UV and down in wave- troscopies will be central tools in the development length to the far IR. There is no doubt that scan- of BioSS. ning probe techniques will continue to increase in importance for studies of biomolecules at surfaces. This includes both STM and AFM [64,65] and 6. A look into the future spatially resolved optical techniques like SNOM. Complementary techniques that provide unique When surface science started to take off as a information in special cases, when sufficient sur- research field on its own, over thirty years ago, face sensitivity is achieved are e.g. NMR, neutron little was known even about the simplest model scattering and ESR. systems and the number and sophistication of the Preparation by thin film deposition methods experimental and theoretical tools were very lim- (PVD, CVD) and by glow discharge plasma ited compared to today. The electronic structure of methods are common in R&D on hybrid surfaces was largely unknown, we did not know biointerfaces. Wet chemical methods such as e.g. how low energy electrons were scattered by self-assembled monolayers, colloidal chemistry, surface atoms, hampering the use of LEED as a microemulsions, and micelles and vesicles come quantitative tool, the positions and orientation of into play because they have the ability to produce CO on any surface were totally unknown, and we interesting chemical and topographic patterns on were very far from any quantitative potential en- surfaces. In addition they are usually fast and ergy surfaces for dissociation or reaction dynam- often inexpensive. Synthetic polymer, protein and ics (even the 1D cuts were unknown). Total oligonucleotide chemistry are increasingly impor- energies of chemical accuracy were impossible to tant because they can produce ligands that can calculate. selectively anchor desired polymer chains, pep- For simple systems the situation is today re- tides, oligonucleitides or whole proteins to sur- versed; the total energies can be calculated, the faces. electronic and atomic structures, and the lattice The whole range of nano- and microfabrication dynamics of surfaces are known in great detail. methods constitute important tools for the func- Potential energy surfaces can be calculated for tional patterning of surfaces. They include electron dissociation and reaction of simple molecules, and beam and photolithography and the soft lithog- the understanding is conceptually transparent even raphies, like stamping and imprinting. for much more complex systems. Important fac- Interesting to note are the special demands on tors have been the development of new experi- methods for BioSS, set by the fact that one deals mental probes, advanced preparation of well with very fragile matter, often requiring a liquid defined model systems, development of theoretical environment for meaningful studies. This calls for methods and simulation schemes to describe both new developments and/or to adaptations of ex- the studied model systems and how the experi- perimental techniques to work non-invasively with mental probes (electrons, photons, ions, SPM tips e.g. cells under liquid environments, or perform etc.) interact with surfaces, and a vast increase in sophisticated freeze drying to retain the features to computational power. This development has al- be studied by methods that require a vacuum lowed a broad and systematic, theoretical and environment. For smaller entities like adsorbed experimental, exploration of model systems of proteins and biomembranes, both the microscopic varying complexity, which successively paved the inspection by SPMs and the spectroscopic char- way for a genuine, quantitative understanding to- acterization under water are key issues. In order to day of the simpler model systems, and a semi- address the important and challenging task of quantitative and/or conceptual understanding of dynamics of e.g. proteins and supported mem- quite complex systems. branes, femtosecond techniques are required, in This is the platform from which BioSS launches the aqueous environment. Consequently scanning just now. We can foresee an exciting development 676 B. Kasemo / Surface Science 500 (2002) 656–677 of new knowledge about biomolecules at surfaces, References initially mainly conceptual and qualitative rather than quantitative, except for the simplest model [1] B. Kasemo, J. Gold, Advances in Dental Research 13 systems like water, and amino and nucleic acids. (1999) 8, and references therein. [2] B.D. Ratner, Molecular Recognition 9 (1996) 617. Successively we will map out and understand the [3] W. Goopel,€ Biosensors and Bioelectronics 13 (1998) 723. structure, dynamics and biological functions of [4] A.F. Collings, F. Caruso, Reports on Progress in Physics supported biomebranes with and without mem- 60 (1997) 165. brane-bound proteins, charge transfer in electron [5] H.A. Fishman, D.R. Greenwald, R.N. Zare, Annual transfer proteins, DNA and ion channels attached Review of Biophysics and Biomolecular Structure 27 (1998) 165. to surfaces etc. Eventually (probably a decade or [6] D.J. Graves, Tibtech 17 (1999) 127. two) we will see the beginning of a detailed un- [7] R. Langer, Accounts of Chemical Research 33 (2000) 94. derstanding of cell–surface interactions. [8] J.A. Hubbell, Current Opinion in Solid State and Material These basic research endeavors will be driven Science 3 (1998) 246. both by curiosity and the large number of appli- [9] R. lyengar, Science 283 (1999) 381. [10] P. Fromhertz, A. Offenhaausser,€ T. Vetter, J. Weis, Science cations of this knowledge; tissue engineering, 252 (1991) 1290. biosensors, DNA and proteomic chips for drug [11] R.F. Service, Science 284 (1999) 578. development and unprecedented precision and in- [12] A.J. Bard, M.A. Fox, Accounts of Chemical Research 28 dividualization in medical diagnostics, and bio- (1995) 141. elctronics based on cell–cell self-organization and [13] M. Sarikaya, Proceedings of the National Academy of Sciences 96 (1999) 14183. communication. [14] B. Kasemo, Current Opinion in Solid State and Materials On the way there will be a dramatic im- Science 3 (1998) 451. provement of existing experimental methods, and [15] B. Kasemo, J. Lausmaa, CRC Critical Review on Bio- development of new ones, to prepare and charac- compatibility 2 (1986) 335. terize surfaces on the atomic and mesoscopic scale, [16] B. Kasemo, J. Lausmaa, Osseointegration in Oral Reha- bilitation, 1993. including measurements of the dynamics and ki- [17] E.A. Vogler, Advances in Colloid and Interface Science 74 netics. These methods must, to a large extent, be (1998) 69. adopted for real-time measurements at solid– [18] J. Israelachvili, H. Wennerstroom,€ Nature 379 (1996) 219. liquid interfaces. The influence of the theoreti- [19] A.J. Pertsin, M. Grunze, Langmuir 16 (2000) 8829. cal methods and simulations is likely to be even [20] S.M. Barlow, K.J. Kitching, S. Haq, N.V. Richardson, Surface Science 401 (1998) 322. stronger than in the development of surface sci- [21] M. Nyberg, J. Hasselstroom,€ O. Karis, N. Wassdahl, ence referred to above. M. Weinelt, A. Nilsson, L.G.M. Pettersson, Journal of The development will take place through Chemical Physics 112 (2000) 5420. strong interaction with adjacent fields to surface [22] N.A. Booth, D.P. Woodruff, O. Schaff, T. Gießel, R. physics and chemistry, for example molecular bi- Lindsay, P. Baumgartel, A.M. Bradshaw, Surface Science 397 (1998) 258. ology, nanoscience and polymer science. Ulti- [23] X. Zhao, Z. Gai, R.G. Zhao, W.S. Yang, T. Sakurai, mately, it is likely that these studies will not only Surface Science 424 (1999) L348. produce a deep understanding of material-biosys- [24] P. Loofgren,€ A. Krozer, J. Lausmaa, B. Kasemo, Surface tem interfaces, but also about the biosystems Science 370 (1997) 277. themselves. [25] I. Lundstroom,€ Progress in Colloid and Polymer Science 70 (1985) 76. [26] J.L. Brash, P.W. Wojciechowski, Interfacial Phenomena and Bioproducts (Edited by McGregor WC), Marcel Acknowledgements Dekker, New York, 1996. [27] T.A. Horbett, T.L. Brash, ACS Symposium Series 602, I am very grateful to all members of the ACS, Washington, DC, 1995. Chemical Physics Group at the Department of [28] V. Hlady, J. Buiks, Current Opinion in Biotechnology 7 (1996) 72. Applied Physics, Chalmers, who have so kindly [29] W. Norde, Macromolecular Symposium 103 (1996) 5. helped me with invaluable reference searches and [30] T. Zoungrana, G.H. Findenegg, W. Norde, Journal of constructive comments on the manuscript. Colloid and Interface Science 190 (1997) 437. B. Kasemo / Surface Science 500 (2002) 656–677 677

[31] H. Arwin, Thin Solid Films 313 (1998) 764. [49] I. Reviakine, A. Brisson, Langmuir 16 (2000) 1806. [32] F. Ho€ook,€ M. Rodahl, B. Kasemo, P. Brzezinski, Proceed- [50] F. Ho€ook€ et al., Langmuir, submitted for publication. ings of the National Academy of Sciences 95 (1998) 12271. [51] W. Knoll, M. Zizlsperger, T. Liebermann, S. Arnold, A. [33] F. Ho€ook,€ M. Rodahl, P. Brzezinski, B. Kasemo, Langmuir Badia, M. Liley, D. Piscevic, F.J. Schmitt, Physiochemical 14 (1998) 729. and Engineering Aspects 161 (2000) 115. [34] F. Caruso, D.N. Furlong, P. Kingshott, Journal of Colloid [52] T. Stora, Z. Dienes, H. Vogel, C. Duschl, Langmuir 16 and Interface Science 186 (1997) 129. (2000) 547. [35] P. Tengvall, I. Lundstroom,€ B. Liedberg, Biomaterials 19 [53] J.S. Hovis, S.G. Boxer, Langmuir 16 (2000) 894. (1998) 407. [54] G.M. Whitesides, J.P. Mathias, C.T. Seto, Science 254 [36] H. Elwing, Biomaterials 19 (1998) 397. (1991) 1312. [37] V. Hlady, J.N. Lin, J.D. Andrade, Biosensors Bioelectron- [55] R.S. Kane, S. Takayama, E. Ostuni, D.E. Ingber, G.M. ics 5 (1990) 291. Whitesides, Biomaterials 20 (1999) 2363. [38] V.P. Zhdanov, B. Kasemo, Surface Review Letters 5 (1998) [56] C.S. Chen et al., Science 276 (1997) 1425. 615. [57] R.G. Flemming, C.J. Murphy, G.A. Abrams, S.L. Good- [39] P. Harder, M. Gruntze, J.H. Waite, Journal of Adhesion man, P.F. Nealey, Biomaterials 20 (1999) 573. 73 (2000) 161. [58] Y. Xia, J.A. Rogers, K.E. Paul, G.M. Whitesides, Chem- [40] R.G. Chapman, E. Ostuni, S. Takayama, R.E. Holmlin, L. ical Reviews 99 (1999) 1823. Yan, G.M. Whitesides, Journal of the American Chemical [59] A.S. Curtis, C.D. Wilkinson, Journal of Biomaterials Society 122 (2000) 8303. Science Polymer Edition 9 (1998) 1313. [41] R.G. Chapman, E. Ostuni, L. Yan, G.M. Whitesides, [60] D. Brunette, B. Cheroudi, Journal of Biomechanical Langmuir 16 (2000) 6927. Engineering 121 (1999) 49. [42] M. Mrksich, G.M. Whitesides, Annual Review of Bio- [61] M. Mrksich, Cellular and Molecular Life Sciences 54 physics and Biomolecular Structure 25 (1996) 55. (1998) 653. [43] V.P. Zhdanov, B. Kasemo, Proteins 29 (1997) 508. [62] R.S. Kane, S. Takayama, E. Ostuni, D.E. Ingber, G.M. [44] H.W. Fink, C. Schoonenberger,€ Nature 398 (1999) 407. Whitesides, Biomaterials 20 (1999) 2363. [45] E. Braun, Y. Eichen, U. Sivan, G. Ben-Yoseph, Nature 391 [63] D. Qin, Y.N. Xia, B. Xu, H. Yang, C. Zhu, G.M. (1998) 775. Whitesides, Advanced Materials 11 (1999) 1433. [46] E. Sackman, Science 271 (1996) 43. [64] A. Engel, H.E. Gaub, D.J. Muller, Current Biology 9 [47] J. Salafsky et al., Biochemistry 35 (1996) 14773. (1999) R133. [48] C.A. Keller, B. Kasemo, Biophysical Journal 75 (1998) [65] H.G. Hansma, K.J. Kirn, D.E. Laney, et al., Journal of 1397. Structural Biology 199 (1997) 99.