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Surface Propertiesof BiomaterialsMS 413100_Biomedical materials
Date: 2016.01.08
Tzu-Wei Wang, PhD.
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• Biomaterial surface is extremely important in determining thebiological response and thus implant success.
• The surface of a material can be considered a type of planar defect.Because the atoms at the surface are not bonded on all sides toother atoms, there is extra energy associated with this region due tounfilled valence shells. Because this state is thermodynamically
unstable, there is a driving force to minimize the surface tension bythe adsorption of atoms or molecules, which satisfy the unfilledbonds at the material surface.
• Adsorption is the adhesion of molecules to a solid surface.
• Absorption, which is the penetration of molecules into the bulk ofanother material, such as water is absorbed by a sponge.
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MCD:sintered apatite grit
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Surface Properties Governing
Protein Adsorption• Two surface properties have the largest effect on the
favorability of adsorption:
– 1. Surface hydrophobicity – 2. Surface charge
• To quantify hydrophobicity, a biomaterial surface is
subjected to contact angle analysis. Synthetic polymersparticularly those containing pendant methyl [e.g.,poly(methyl methacrylate)] or styrene [e.g., poly(styrene)]groups are examples of hydrophobic biomaterials.
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• A significant surface charge can have the additional
effect of attracting or repelling charged areas of proteins.Surface charge occurs via dissociation of ionizablesurface groups or through specific adsorption of ionsfrom the solution. Again, how this property affectsprotein adsorption depends on both the charge ofthe surface and that of the protein.
• It has been found that protein adsorption generallyincreases with increasing hydrophobicity of the surfaceand the protein.
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• The physical characteristics of the biomaterial surfaceare also important. These include – steric concerns – surface roughness
• For example, adding large, flexible hydrophilic polymerchains such as poly(ethylene glycol) (PEG) to abiomaterial surface will result in a decrease in proteinadsorption. – This is due to the fact that a large volume at the surface is taken
up by these bulky chains that are in constant motion. Since theymove too quickly to allow the proteins to adsorb to them and aretoo large for the proteins to move through, they form a type ofwall, using steric repulsion to prevent adsorption to the surface.
Fig. Schematic of steric hindrancedue to PEG chains. a) On a materialwithout steric hindrance proteins havefree access to adhere to the materialsurface. b) PEG chains inhibit theattachment of proteins by physicallyblocking access to the surface.
H(OCH2CH2)nOH
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• A surface with a high degree of roughness may
promote protein adsorption in certain areas byphysically "trapping" the proteins in the valleyson the surface.
• The hydrophobicity, charge, steric hindranceand/or roughness of a biomaterial surface canbe altered during formation or processing to
change the protein adsorption profile of the finalmaterial.
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• Q:Consider the following hypothetical protein and twomaterials (A) and (B). Assume that in this case the watercontact angle correlates directly with the hydrophobicity
of the material surface. To which material would greateradsorption of the given protein be expected? Why?
• A:Material B has a greater contact angle than A. Material B
is more hydrophobic than Material A. Consequently, onewould expect greater adsorption of the highlyhydrophobic protein X to the surface of Material B,through hydrophobic interaction, assuming that the
surface charges of Materials A and B are comparable.
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Physicochemical Surface Modification Techniques
• Surface modification possesses the advantagethat bulk characteristics such as mechanicalproperties are not altered. However, the
possibility of delamination in vivo is a seriousconcern.
• An ideal technique would produce a surfacetreatment with the following characteristics: – 1. Thin (to minimize effects on bulk properties) – 2. Resistant to delamination
– 3. Simple and robust (to promote commercialization) – 4. Prevent continuous surface rearrangement
Fig. Low-voltage scanning electron
micrograph of stainless-steel
endoluminal vascular stent with
conventional silicone coating
showing peeling and delamination ofcoating. Photo courtesy of the
University of Florida (Gainesville).
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• Medical coatings and surface modifications are as
diverse as the products they seek to enhance.Some of the more common techniques includeplasma deposition, physical vapor deposition,chemical vapor deposition, ion bombardment,
ion-beam sputter deposition, ion-beam-assisteddeposition, sputtering, thermal spraying, anddipping. Qualities that can be altered include
hardness, wear resistance, lubricity, wettability,bond strength, and resistance to bacterialattachment.
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Covalent Surface Coatings• Plasma Treatment
• Chemical Vapor Deposition• Physical Vapor Deposition
• Radiation Grafting• Photografting
• Self-Assembled Monolayers
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Plasma Treatment• Plasma refers to an assembly of species in an
atomically/molecularly dissociated gaseous environment.
• The ionized gas can include positive and negative ions,free radicals, electrons, atoms, molecules, andphotons.
• Plasma discharge can occur at a range of temperatures(usually 25°C and higher) and is most often createdunder vacuum.
• Exposure to a plasma environment can also be used asa pretreatment to other surface modification techniques.
持續的從外部施加能量,例如施加高溫 高速電子或離子以及放射線等具有能
量的粒子之撞擊,會導致中性物質離子化,解離為電子 離子 中性粒子 受
激粒子 而這種使氣體物質解離為陰陽電荷粒子的狀態即稱為電漿 電漿為激
發狀態粒子之集合體
.
氣體的離子化狀態
.
離子氣體
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Fig. Schematic of plasma discharge treatment. A plasma environment is obtained byapplying an electric potential across a gas.The cathode is the surface to be treatedand has a negative potential relative to theanode. Electrons must traverse the gas in
the chamber to travel from the cathode tothe anode. During this stage, they collidewith molecules in the gaseousenvironment to form gaseous ions andradicals. These species can then interactwith the sample and cause a variety ofsurface reactions. The plasma is sustainedbecause electrons flow from the samplewhile positive ions flow toward the sample.
http://en.wikibooks.org/wiki/Microtechnology/Additive_Processes
電漿的產生是藉由電子在電場中加速,使帶
有極高動能的電子
(e-)撞擊氣體原子或分子
(N2)而產生離子化反應,被撞擊的氣體原子或分子被解離為帶正電的離子
(2N+)與二個
電子
(2e-),此兩個電子會再被加速而撞擊其
它氣體分子或原子,使其解離後可產生更多
的自由電子,由此連續反應將反應區域的氣
體電漿化(離子化)
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• 電漿蝕刻(Plasma etching) (Ar, O2, N2)電漿蝕刻主要應用於清除表面污染物,具有清潔之功效;並且造成基材表面氧化、分解或進行其它化學反應,進而在高分子表面植入新的官能基,造成基材表面之交聯。
• 電漿濺鍍 (Plasma sputtering)利用惰性氣體原子受高速運動的電子相互碰撞,藉由電場與磁場作用,正離子撞到陰極或靶材表面,靶材原子被撞擊並沉積在基材進而產生大量的電漿以撞擊靶材,使其沉積在基材上。
• 電漿鍍膜 (Plasma coating)將可聚合性之單體(Ex: Acrylic Acid, N-Vinyl 2-Pyrrolidone, HEMA, NIPAAm, etc )通入電漿中進行電漿處理反應,導致聚合反應形成,而在基材表面形成一層高分子薄膜(具高交聯密度)。
• 電漿接枝 (Plasma grafting)經電漿處理過之高分子表面會產生自由基,若適時在系統中通入其它反應性氣體,氣體會與自由基發生反應,造成高分子表面接枝現象(Ex: -OH)。惰性氣體原子的碰撞機會,產生更大量正離子以撞擊靶材,以增加被撞擊出的靶材原子的數量,藉此提高鍍
層的成長速率。
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• Plasma discharge is often used for cleaning or addition of hydroxyl(OH) or amine (NH2) groups to biomaterials as a precursor to further
modification.• At the sample surface, there is competition between deposition and
ablation/etching. – If this process is very rapid, no deposition will be observed.
– Deposition can occur via at least two possible methods. For example,free radicals may polymerize other molecules from the gas phase ontothe surface, or small molecules may combine into larger particulatesthat settle on the surface
• The energetic plasma may also be employed to directlypolymerize molecules on the sample. For example, a copolymercan be placed on the surface of another polymer and exposedto plasma to crosslink the copolymer to the surface.
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Plasma torch (Thermal plasma)• To melt substances with high melting points, such as
ceramics, and accelerate the molten particulates to ahigh velocity.
• Using this method, the torch can produce plasma spraycoatings. This technique is commonly used inbiomaterials to add ceramic coatings to metallicorthopedic or dental implants to improve their integrationwith the surrounding bone.
Dr. Maria Virgínia Alves, Associated Plasma Laboratory
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Corning® Cell Culture Surfaces:
Standard Tissue Culture Treated Polystyrene Surface
• Standard Corning® polystyrene cell culture vessels are surfacemodified using either corona discharge (flasks, dishes and
microplates) or gas-plasma (roller bottles and culture tubes). Theseprocesses generate highly energetic oxygen ions which graft ontothe surface polystyrene chains so that the surface becomeshydrophilic and negatively charged when medium is added (Hudis,
1974; Amstein and Hartman, 1975; Ramsey et. al., 1984). The moreoxygen that is incorporated on to the surface the more hydrophilic itbecomes and the better it is for cell attachment and spreading.
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Chemical Vapor Deposition (CVD)• Chemical vapor deposition (CVD) is a surface treatment
in which a mixture of gases is exposed to a sample at a
high temperature.
• This environment causes a variety of reactions resultingin the decomposition of one or more components of the
gas mixture and subsequent deposition on the substrate.
• CVD techniques are most commonly used inbiomaterials applications to deposit pyrolytic carbon
coatings on substrates. In this case, the gases arehydrocarbons and they undergo thermal decomposition,or pyrolysis, within the reaction chamber, allowingcarbon deposition on the surface of the material.
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Figure 2. A) Low-magnification SEMimage of 3D pyrolyzed carbon structure.B.) Medium-magnification SEM image of3D pyrolyzed carbon structure.
Small 2009, 5, No. 24, 2792–2796
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Prof. Ko-Shao Chen, Dept. of Materials Engineering, Tatung University
Plasma CVD system
GAS SOURSE
DIFFUSION
PUMPROTARY
PUMP
REACTOR VACUUMGAUGE
MATCH
BOX
R.F
GENERATOR
MONOMER
SOURCE
sample
(nitrogen, argon, oxygen, water vapor)
(RF, microwave, acoustic)
• In order to reduce the reaction temperature,plasma environments are often used to increasethe reactivity of the gaseous species. This istermed plasma-assisted chemical vapordeposition.
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Physical Vapor Deposition (PVD)• PVD is surface coating via deposition of atoms
generated through physical processes onto the sample.
• This class of techniques includes sputtering and thermalevaporation.
• Metal alloy coatings have been deposited using thismethod to increase wear resistance of metallic hipimplants.
• Sputtering techniques are often employed to coatnonconductive samples with a thin layer of metal beforeimaging with an electron microscope.
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• Sputter deposition is a two-step process.
– First, energetic ions or atoms bombard a targetmaterial and transfer their momentum to atoms withinthe target. This causes the ejection of a certainnumber of target surface atoms.
– In the second step, the released target atoms strikethe sample surface and condense to form a thin film.Both covalent and non-covalent coatings are possiblevia this method.
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• Plasma-assisted PVD techniques
Fig. Schematic of a type of physical vapor deposition.
The formation of plasma is used to create high-energy specieswhich collide with the target in plasma-assisted physical vapordeposition. The target is held at a large negative potential comparedto the sample to be coated. An environment of sufficient vacuum willinitiate formation of plasma near the target. Species from the plasma
then strike the target to release atoms that can be deposited on thesubstrate surface.
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Radiation Grafting• The substrate is exposed to a radiation source of high
energy, which forms reactive species at the surface to
create covalent bonding of the coating to the underlyingmaterial.
• These methods are often employed to bind hydrogels tohydrophobic substrates.
• This technique also provides a means to easily tailor the
properties of the coating since a mixture of monomers orother precursors can be used.
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Prof. Masao Tamada, Environmental and Industrial Materials Research Division, Japan Atomic Energy Agency
For the removal of toxic heavy metals from industry waste water
Radiation-induced graft polymerization
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胺腈化
鈾
anionic carbonate complexes
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Photografting• Photografting is similar to radiation grafting, but, the
radiation is UV or visible light.
• A number of photoresponsive chemical moieties havebeen developed to facilitate this type of surfacemodification (viz., phenyl azide 疊氮苯 or benzophenone二苯酮 ).
• If these functional groups are present in the coating
precursor, they will be excited by exposure to the lightand form free radicals or other reactive species. Theactivated molecules can then participate in reactions atthe substrate surface resulting in covalent linkage of the
coating to the underlying biomaterial.
BP
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Self-Assembled Monolayers (SAM)• In this technique, the molecules composing the coating
are designed so that it is thermodynamically favorable for
them to align on and form covalent bonds with the surfaceof the biomaterial.
• Therefore, in contrast to the previous treatment methods,no specialized equipment is required, and the modificationcan be carried out at room temperature and under normalatmospheric pressure.
• Self-assembling molecules are amphiphilic, meaning theyhave both hydrophilic (polar) and hydrophobic (nonpolar)areas.
Th l l th k i
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• These molecules possess three key regions: – 1. Attachment group – 2. Assembly group (long hydrocarbon (alkyl 烷基)
chain): nonpolar – 3. Functional head group: polar
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• Of these, both the attachment group and the nonpolarhydrocarbon chains play a large role in self-assembly.
• The functional group can be used to alter thehydrophobicity of the substrate material, or it can be apoint of further chemical reaction to attach biologicallyactive molecules.
• Silanes (矽烷) are commonly used attachment groups,because they react readily with amine (NH
2) or
hydroxyl (OH) groups. Therefore, materials containinglarge amounts of hydroxyl groups, such as glass andmetal oxides, are preferred substrates for these types ofSAMs.
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• Fig. Self-assembly of alkyl silanes on substrates containinghydroxyl groups. A strong, exothermic reaction between thehydroxyl groups on the substrate and the silane attachmentgroup is the driving force for self-assembly.
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• Alternatively, other biomaterials may be pretreated (viaplasma discharge or other methods) to increase the
number of appropriate reactive groups present on thesurface.
• The advantages of SAMs as a surface modificationtechnique include – 1. the ease of their formation – 2. the chemical stability of the coating – 3. the variety of chemical moieties that can be included either in
the attachment or the functional groups – 4. form molecularly smooth surfaces
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Non-Covalent Surface Coatings
• Solution Coatings• Langmuir-Blodgett (LB) Films
• Surface-Modifying Additives
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Solution Coatings• In this technique, the substrate is dipped in
a solution containing the dissolved coatingmaterial (usually a polymer or proteinsdissolved in an organic solvent or aqueous
solution). The substrate is then left to dryand, as the solvent evaporates, thecoating is deposited on the surface.
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Langmuir-Blodgett (LB) Films• Like SAMs, the coating molecules are
amphiphilic with two regions-a hydrophilichead and a hydrophobic tail (example:fatty acids, phospholipid).
• Using a piece of equipment called a
Langmuir trough, these molecules can betransferred to a biomaterial surface.
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Kibron Inc., Finland
(a) gaseous phase(b) liquid-expanded phase(c) solid (condensed) phase
Langmuir trough
• As illustrated in the figure, the substrate to be coated is
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As illustrated in the figure, the substrate to be coated isplaced into aqueous media and the amphiphilicmolecules are added so that the polar head groupsinteract with the water and the remainder of the moleculerests in the air.
• By changing the position of the moveable barrier, the
coating is slowly compressed until all of the moleculesare orientated so they are standing on end.
• At this point the area per molecule reaches a minimum
and is nearly constant, even as the barrier is furthercompressed (the surface pressure increases). This valueis called the critical area and is a function of the size andtype of hydrophobic tail on the molecule.
• By maintaining a surface pressure corresponding to thecritical area as the material to be coated is slowlyremoved from the trough, a homogenous, well-orientatedcoating can be deposited.
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• In the gaseous phase, there is minimal pressureincrease for a decrease in area. This continues
until the first transition occurs and there is aproportional increase in pressure withdecreasing area. Moving into the solid region isaccompanied by another sharp transition to amore severe area dependent pressure. Thistrend continues up to a point where themolecules are relatively close packed and havevery little room to move. Applying an increasingpressure at this point causes the monolayer tobecome unstable and destroy the monolayer.
( Brazilian Journal of Physics, vol. 22, no. 2, 1992)
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• The advantage, like SAMs, is to provide manypossibilities for altering the chemistry of thecoating molecules.
• A major disadvantage is the relative instabilityof the coating, due in large part to the fact thatit is not chemically bonded to the surface. Theaddition of moieties on the head groups of the
LB coatings to allow for crosslinking to othercoating molecules or to the biomaterial surfaceis one way to overcome this limitation.
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Surface-Modifying Additives (SMAs)
Fig. A depiction of the use of surface-modifying additives in a polymeric
material. In this example the additive is ablock copolymer in which block A iscompatible with the bulk polymer, whileblock B is incompatible and possessesan affinity for the surface (has a lowersurface energy than the bulk polymer/Ablock). Block A then serves as an"anchor “ to hold the molecule in thebulk, while the surface characteristicswill be provided by block B, as seen inthe postfabrication diagram.
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• Surface-modifying additives (SMAs) are atoms ormolecules that, when added in the material bulk, will
spontaneously rise to the surface, thus producing acoating with characteristics dictated by the properties ofthe SMA.
• The driving force for this rearrangement is the reductionof surface free energy.
• It should be noted that, unlike the surface modification
techniques previously discussed in this chapter, SMAtreatment is not a post-fabrication procedure, butrather a part of the formation/synthesis of thebiomaterial.
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• Examples of SMAs for metals include copper in
gold alloys, chromium in stainless steel(chromium will move preferentially to the surfaceof steels to impart corrosion resistance).
• SMA systems for ceramics are less likelybecause there is less atomic mobility within the
bulk due to the nature of the ionic bonds that arefound within these materials.
Surface Modification Methods
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Surface Modification Methods
with No Overcoat – To alter the surface properties of biomaterials
without the formation of a separate coating
• These techniques are designed tomodify existing atoms at the surface,but not attach a distinct coating layer .
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• Ion beam implantation – a process whereby accelerated ions with high energies are
directed at the surface of a biomaterial – This method is usually employed for metals and ceramics
• As the ion interacts with the material surface, there are
many possible results. – Formation of a cascade of vacancies and interstitials is triggered. – Atoms may be sputtered from the substrate due to the high energy
of the bombarding ions. Such changes can affect the overallsurface roughness of the sample.
– Localized heating at the surface also occurs, which can altercrystal structure or kinetics of defect formation in this area.
Ion beam implantation
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• Fig. Schematic representation of ion beam implantation.Ions with high energy states are projected at the surface of thematerial at high speed, where they break the surface. Once withinthe material, some atoms from the material are ejected, while otherscollide with each other and create vacancies and changes within thecrystal structure. Using different ions at various concentrations
can alter the degree of modification.
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• Examples of ion beam implantation for biomaterialapplications
– the addition of nitrogen to titanium to increase wear resistance – the implantation of boron and carbon into stainless steel to
improve fatigue life
• The advantages of ion beam implantation – almost all elements can be subjected to ionization, there exists avariety of possibilities to affect properties such as hardness,wear, corrosion, and biocompatibility.
• The disadvantages of ion beam implantation – vacancy and interstitial defects often remain after treatment, so
subsequent heating to remove them may be necessary.
S f b t t tt i
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Surface or substrate patterning
• Surface patterning can be used with a variety ofsurface-active molecules, and common substrates
include both metals and polymers. Two of the mostwidely used patterning techniques aremicrocontact printing and microfluidics.
Fig. Surface substratepatterning can be used toalter the surface propertiesof a material in controlled,
well-defined areas, as seenin the circular pattern in thephoto. The inset presents alower magnification view ofthe same surface.
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Adv. Funct. Mater. 2005;15(5):739
Adv. Mater. 2006;18:165–169.
Angew. Chem. 2003;42:1262–1265 .
1 Selective adhesion
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1. Selective adhesion
[Biomaterials, 29, 2008, 94-102]
2. Guidance
[Sensors and Actuators B, 106, 2005, 843-850]
Ridge valley
Microcontact printing
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Microcontact printingFig. In microcontact printing, a mold ofthe desired pattern is first created, oftenvia photolithographic techniques on asilicon wafer (a-e). A silicone rubbermaterial (PDMS) is then polymerized in
the mold to make a positive "stamp“ (f-h). The stamp is "inked“ with thesurface-modifying substance bydipping it into a solution containing themolecule of interest (i) and the inkedstamp is then pressed onto the
substrate (j). After carefully removing thestamp, the process can be repeated tomodify the portion of the substrate thatwas not stamped the first time, thuscreating multifunctional surfaces. In thiscase, a biomaterial could be modified
to expose one or more proteins with awell-controlled spatial orientation, thuspotentially modulating cell attachment andimproving interaction of the implant withthe native tissue.
PDMS: polydimethylsiloxane
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• 微接觸式印刷術的製作過程(A) 矽基模板經由微影與蝕刻技術製作。先將光阻旋轉塗
佈在矽基上,接著UV隔著一個光罩對矽基模板進行曝光顯影。之後再進行蝕刻,形成圖案結構。將此矽基板當作模仁,塗佈彈性物質PDMS在70℃下處理,印章會凝固
成型,再進行脫模。(B)印章的表面是非常疏水的,但是經由簡單的電漿處裡,就可以轉變成親水的表面,使得印章可以沾上蛋白質溶液。經過去除多餘的液體後,印章就可以和將要用作培養的材質表面接觸。
羅韻晴1 劉宏文2 楊忠諺1 林奇宏1,2 1.國家奈米元 實驗室; 2.陽明大學 微生物及免疫學研究所
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Scheme 1. Schematic diagram of the HA-PLL layering approach to pattern co-cultures.
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Fig. 4. Patterned co-cultures of ES cells with fibroblasts.(A) represents light (left) and fluorescent (right) imagesafter 1 day. (B) and (C) illustrate that co-cultures remainedstable for 3 and 5 days. (D) represents the reversal in theorder of cell seeding.
ES cells
NIH-3T3 cells
NIH-3T3 cells
ES cells
AML12 cells(hepatocytes)
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Fig. 5. Patterned co-cultures ofhepatocytes with fibroblasts.
(A) represents the light andfluorescent images after 1 day.The cultures remained stable forat least 5 days (B). (C)demonstrates that the same cell
type could be patterned in co-cultures.
NIH-3T3 cells
(hepatocytes)
Microfluidics
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Microfluidics
• Fig. For the micro fluidics technique, (a) the mold is fabricated as described inmicrocontact printing, but the mold is a positive, rather than a negative, image of thedesired design. (b) PDMS is then polymerized in the mold. (c) The formed PDMS isthen removed from the mold and pressed against a glass slide. This setup isthen plasma-treated to increase the hydrophilicity of inner areas of thechannels only, while the regions between channels remain hydrophobic to maintainthe integrity of each channel. Like for microcontact printing, (d) the PDMS form isthen pressed against the substrate and a small amount of a solution containingthe molecule of interest is injected or placed near the opening of a channel,where capillary action pulls the liquid throughout the channels. The areas of thesubstrate under the channels are appropriately modified. (e) After the molecule hasreacted with the surface, the PDMS form is removed and the surface is rinsed.
A distinct advantage of this process is that it takes very little fluid volume, so it can beeasily used with expensive or difficult-to-produce biologic reagents.
DNA/Protein microarray, Lab-on-a-Chip technology"L b Chi " i di t ll th li f i l lti l l b d t hi f t
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http://en.wikipedia.org/wiki/Microfluidics
"Lab-on-a-Chip" indicates generally the scaling of single or multiple lab processes down to chip-format
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• Detection principles may not always scale down in a
positive way, leading to low signal-to-noise ratios.• Physical and chemical effects that become more
dominant on small-scale sometimes make processes inLOCs behave more complex than in conventional labequipment (like capillary forces, surface roughness,chemical interactions of construction materials onreaction processes)
• The behavior of fluids at the microscale can differ from'macrofluidic' behavior in that factors such as surfacetension, energy dissipation, and fluidic resistance start todominate the system.
Biological Surface Modification
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Techniques• Biological surface modification techniques involve
attachment of biologically active molecules to a
substrate through a variety of means, including many ofthe physicochemical methods described in the previoussections. The attached molecules are then free tointeract with specific target areas on cells or other tissuecomponents.
• A primary concern with these techniques is that themolecule of interest remain attached whilemaintaining its biological activity. Since manybiologically based molecules are sensitive to changes inconformation, particular attention must be paid to theorientation and rotational ability of individual moleculesafter coating.
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蛋白質的構形是所有生命機能的根本
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• Biomolecules for surface treatment are often eitherproteins or carbohydrates derived from various tissuesand are attached to facilitate interactions with certain celltypes.
• Other major categories include nucleic acid derivatives(DNA or RNA) or drugs, which are added to the surfacein order to alter specific cellular functions in a controlledmanner.
• Although biological modification of all types ofbiomaterials is possible, most of the work in this areahas centered on polymeric substrates. Biomoleculeattachment has been successfully achieved on solublepolymers, solid polymers, solid polymers containingpores to form three-dimensional scaffolds, and hydrogels.
Covalent Biological Coatings
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Covalent Biological Coatings
• Like physicochemical techniques, covalentlylinked coatings may impart additional stability
and thus are preferred in many applications.
• However, they require the presence of a reactivesubstrate surface, often containing hydroxyl(OH), carboxyl (COOH), or amine (NH2) groups.
• If these are not found on the chosen biomaterial
surface, it may be modified (via plasmadischarge, for example) to add appropriatefunctionality before proceeding with the reaction.
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Fig. Methods for the covalent
attachment of biomolecules toa biomaterial surface.(a-c)attachment via postfabrication methods, (d-e)attachment during synthesis.The biomolecule may beattached with or without aspacer arm in any of thesemethods.
Crosslinker
Direct coupling
A type of binding agent is oftenused to facilitate interaction of themolecule with the surface The
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The biomolecule may be bound toa precursor (monomer) that is thenpolymerized in three dimensions or as a surface coating.
molecule with the surface. The
binding agent can then remain as"glue" or catalyze the reaction andbe released.
An activated precursor (monomer)
containing groups with affinity forthe biomolecule is polymerizedand the formed biomaterial issubsequently exposed to themolecule of interest
Crosslinker
• In any of these, the molecule can be bound to thesubstrate directly or by a spacer arm, which is an inertmolecule that provides physical space between the
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molecule that provides physical space between the
biomolecule and the substrate.
• The spacer arm may allow for greater rotational freedomand thus improve the activity of the biological molecule.
• In addition, it is possible to design biodegradablespacer arms to release the biomolecule in a localizedregion after biomaterial implantation.
poly-ethylenimine (PEI)
PEG
poly(divinylbenzene) (polyDVB)
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Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 48, 99–108 (2010)
poly tert-butyl acrylate (PtBA)
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• Q: A researcher has created a biodegradable polymericimplant material that degrades through a surfacedegradation mechanism. The material is intended toserve as a tissue engineering scaffold, in which long-
term cell adhesion to the material is important. Initialstudies have found that cells will not adhere to thesurface of the material. The researcher is consideringcovalent attachment [with a poly(ethylene glycol)spacer of 3400 Da] of a peptide sequence known toimprove cell attachment to other materials to the surfaceof this material. The researcher asks if you support theidea. Will you support the idea? Why or why not? Wouldbulk modification with the peptide sequence be a more
or less appropriate method of modification for theintended result?
H(OCH2CH2)nOH
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• A:The idea is not feasible to yield the intended effect for the givenapplication and should not be supported. Recalling that degradation
by surface erosion involves the continual loss of the material at thesurface of the construct (much like a bar of soap disappears withtime of exposure to water as the surface continually erodes away), itfollows that any modification to the surface of this material wouldonly be viable for a short period of time until the modified surfacedegrades away. Following the initial surface degradation, the cellswill be exposed to the bulk unmodified material, to which cellattachment has been shown to be nil. Thus, although the cells mayattach initially, long-term cell attachment could not feasibly beattained with this modification technique. Bulk modification, however,would be a more appropriate technique because the cell adhesion
peptide would be present throughout the bulk of the material, unlikesurface modification. As a result, cells could potentially attach to thematerial throughout the stages of degradation.
Non-Covalent Biological Coatings
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g g
• This usually involves adsorption of the biomoleculeto the biomaterial, and then possible crosslinking toimprove the coating stability.
• It has been found that whether or not a certainmolecule will gather at a given surface is oftendictated by hydrophobic and electrostaticinteractions. These forces can therefore beexploited to couple biomolecules to particularsubstrates.
• For example, one method to coat heparin, a very
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hydrophilic carbohydrate based biomolecule important inanticoagulation, on a hydrophobic surface is the additionof a hydrophobic region to the heparin. Thus, interactionof the hydrophobic regions of the biomolecule and thesubstrate in an aqueous environment will result in
extension of the heparin portion away from the surface,effectively coating the biomaterial.
• In contrast, adsorption of heparin to positively charged
surfaces requires no modification of the biomolecule.Because heparin possesses significant negative charge,electrostatic attraction drives the formation of a heparinlayer at the surface of positively charged biomaterials.
Heparin
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• Fig. Two methods for coating a surface with heparin, a hydrophilicbiomolecule. (a) A hydrophobic region is added to the heparin. Thus,interaction of the hydrophobic regions of the biomolecule and the substratein an aqueous environment will result in extension of the heparin portionaway from the surface, effectively coating the biomaterial. (b) The
adsorption of heparin to positively charged surfaces requires nomodification of the biomolecule. Because heparin possesses significantnegative charge, electrostatic attraction drives the formation of a heparinlayer at the surface of positively charged biomaterials.
Immobilized Enzymes
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– enzymes are a subclass of proteins that act to promote specificchemical reactions involving other biomolecules.
• Many of the methods discussed above can be used toattach enzymes to solid substrates (carriers).
• This process, called enzyme immobilization, has avariety of applications in such areas as biosensors,controlled release devices, and protein analysis. Muchresearch has been devoted to immobilization techniques,
which range from simple adsorption to more complexmechanisms involving covalent linkages with spacerarms.
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Scheme shows the design of an enzyme-modified
nanocrystalline diamond electrode. The bilayer of twoenzymes, glucose oxidase and horseradish peroxidase,is covalently immobilized to the diamond surface.The cascade of electrochemical reactions initiated by thepresence of glucose is converted to an electrical current
measured at the diamond electrode.
Nanotechweb.org
controlled release
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• Both hydrophilic hydrogel carriers, such aspoly(acrylamide) or poly(ethylene glycol), and
hydrophobic carriers, such as nylon or poly(styrene),have been explored, with optimal results dependent onthe properties of the enzyme of interest.
• An additional concern for these devices is that thebiomolecule that the enzyme targets (its substrate)must be able to diffuse into the area to physicallyinteract with the immobilized enzyme. Thus, the
geometry of the carrier is crucial to allow a sufficientlylarge surface area for enzyme contact.
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Techniques for Surface Characterization
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• Surface characterization of biomaterials isimportant, both to determine the quality of the
surface treatments and also to provideinformation about the extent of proteinadsorption to a material.
• Because surfaces are chemically active, manysurface techniques require special preparation
equipment or conditions such as high vacuum toprevent contamination.
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• Fig. Surface penetrationof various surfaceanalysis methods.Some surface analysistechniques actually
penetrate relativelydeeply into the bulk ofthe material.
AFM: Atomic Force Microscope 原子力顯微鏡
SEM: Scanning Electron Microscope 掃描電子顯微鏡
STM: Scanning Tunneling Microscope 掃描穿隧電子顯微鏡
SIMS: Secondary Ion Mass Spectrometry 二 次 離 子 質 譜 儀
ESCA: Electron Spectroscopy for Chemical Analysis 化學分析電子能譜儀
ATR-IR: Attenuated Total Reflection Infrared Spectroscopy 衰減全反射紅外光譜儀
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AFM: Atomic Force Microscope
STM: Scanning Tunneling MicroscopeSEM: Scanning Electron Microscope
SIMS: Secondary Ion Mass Spectrometry
XPS: X-Ray Photoelectron Spectroscopy
ESCA: Electron Spectroscopy for Chemical Analysis
ATR-IR: Attenuated Total Reflection Infrared Spectroscopy
Contact Angle Analysis
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• Contact angle analysis is often used to provide overallinformation about the hydrophobicity of a surface.
• The surface free energy or surface tension (γ) of amaterial can be defined thermodynamically as the workof making a unit area of new surface.
• In most cases, the liquid chosen for testing of biomedicalmaterials is water. The energetics at each of theinterfaces causes the water droplet to assume aparticular shape. Therefore, by accurately measuring the
angle between the drop and the solid surface (thecontact angle, θ ), the surface tension can then becalculated.
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• Fig. (a) Schematic ofcontact angle testing. Around the waterdroplet, there are
three importantinterfaces: liquid-vapor ( γ LV ), solid-liquid ( γ sL ), andsolid-vapor ( γ sv ).(b) Change in
wettability via surfacemodification. Thewater droplet spreadsmore on the modifiedsurface because themodification
decreases thesurface tension ofthe liquid/solidinterface, thusreducing the contactangle.
• This equation contains two unknowns, γ sv and γ sL . γ sv isoften approximated using the critical surface tension (γ c ),as developed by Zisman. γ c is determined by testing the
t i l i i li id ith f γ l
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material using various liquids with a range of γ LV values. A plot of contact angle vs. γ LV is generated andextrapolated to θ = 0 (complete spreading on thesurface). The value of γ LV at this point is called γ c .
Fig. Diagram showinghow the critical surface
tension (γ c ) isdetermined. The contactangle of various liquidsis measured for aspecific material, and aplot of contact angleversus γ
LV is generated.
The extrapolated valueof γ LV at θ= 0 is γ c
• Instrumentation1. Holder for solid sample
2 H ld f li id
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2. Holder for liquid3. Means to determine contact angle (may be automated)
– Unlike many other characterization techniques, the output of
contact angle analysis is a single number (θ or γ c ).
– because the contact angle measurement is not always fullyautomated, user bias can have a substantial effect on the results.
Fig. Different experimental setupsfor determining the contact angle: (a)sessile drop, (b) captive air bubble,
(c) capillary rise method, and (d)Wilhelmy plate method. The circlesindicate where the contact angle ismeasured.
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KSV Instruments Ltd
• Although contact angle data is widely available for most biomedicalmaterials, this technique cannot provide detailed information about thechemical composition of the surface, so it is often used as a first step insurface characterization.
D i t t l t l d t t t
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• Dynamic contact angle measurements are also used to measure contactangle hysteresis. In the most common of these measurements, water isslowly added to an area of the surface with a syringe and the advancingcontact angle is measured. The water is then removed via the samemechanism and the receding contact angle is recorded. The difference
between these two values represents the contact angle hysteresis for thatmaterial and describes how the surface tension of the material changesbefore and after it has been exposed to an aqueous environment.
• Hysteresis can occur for a variety of reasons.For example, hydrophilic domains within thematerial may become reorientated outwardfrom the surface after contact with water,whereas they may be "hidden" within thebulk when exposed to hydrophobicenvironments such as air.
Light Microscopy
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• Light microscopy is a relatively simple techniquethat is used as a first approach to gain primarily
qualitative information about surface topography,or to view thin sections of a sample.
• For opaque samples, the light source can belocated above rather than below the sample.The resolution of such instruments is limited by
the wavelength of white light, and in most casesis around 0.2 μm.
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Fig. (a) Gross image of a compound microscope. On a compoundmicroscope, the light source is placed below the specimen and thespecimen is viewed from above. (b) Light path of a compound microscope.The objective forms a magnified image of the object that is larger than theoriginal object. This image is then magnified many times by the eyepiece toform the large (inverted) virtual image.
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Olympus America INC.
Instrumentation – 1. Source-produces white light. – 2. Lenses-glass lenses focus light beam and/or magnify image of sample.– 3. Sample stage-holds sample securely.
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3. Sample stage holds sample securely. – 4. Detector (camera or human eye)-views and captures resulting image.
Information Provided
• Light microscopy is used exclusively for imaging, and provides initself only qualitative assessment. Images can be further analyzedusing specialized software to obtain semi-quantitative measurements.
• In addition to imaging surface topography, this instrument is extremely
important in histological analysis (Ex: the extent of the inflammatoryresponse).
• While light microscopy has the advantage that vacuum is not requiredto view samples, it remains difficult to see thick or hydrated samples.
Fig. Sample of a light microscopyimage, in this case, a fibroblastattached to a substrate.
XPS-ESCA
I thi t f t X b ti th l f
X-ray Photoelectron Spectroscopy (XPS)
Electron Spectroscopy for Chemical Analysis (ESCA)
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• In this type of spectroscopy, X-ray absorption causes the removal ofan electron from one of the innermost atomic orbitals (not thevalence shell). The kinetic energy of the emitted electron is thenrecorded.
• The binding energy gives an idea of how tightly bound the electron isto the nucleus, and it changes in accordance with the type of atom aswell as interactions with the nuclei of neighboring atoms.
• ESCA is often used to identify elements present at the surface of amaterial. Its analytical capabilities are limited to the uppermost ~100 Åof a sample because the energy of the emitted electrons allowsescape from only the first few atomic layers.
E k : the kinetic energy of the electron
E b: the binding energy of the electronν is the frequency
h is the Planck's constant (6.6 X 10-34 J-s)
化學分析電子能譜儀其基本原理乃根據光電效應,當足夠能量的電磁波
(X-Ray)照射在材料表面
時,原子內電子會經由吸收電磁波的能量而被游離出來成為所謂
”光電子
(photoelectron)” 光
電子的動能為入射電磁波的能量減去該電子在原子內特定軌道的束縛能,因此不同元素之光電
子具有特定動能而可作為判定材料表面元素成份的根據
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Fig. (a) A typical ESCA spectrumof poly(dimethyl siloxane) (PDMS),plotted as electron count (y-axis) as afunction of binding energy (x-axis). (b)Chemical structure of PDMS.
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• Fig. Schematic of ESCA equipment. First, the sample is bombarded with X-rays. The resulting emitted electrons then enter the analyzer chamber.Because of the difference in voltage between the two walls and the
geometry of the analyzer, only electrons with a certain kinetic energycan be collected by the detector , with the remaining electrons strikingnon-detectable areas. By altering the voltage difference between the wallsin a controlled manner, the electrostatic field is altered to permit the detectorto record the amount of electrons having various kinetic energies. At theend of the voltage sweep, the entire spectrum is plotted for that sample.
Instrumentation – 1. Source-produces X-rays with known wavelength.
– 2. Electron analyzer-uses an electrostatic field to separate
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ect o a a y e uses a e ect ostat c e d to sepa ateelectrons based on kinetic energy.
– 3. Detector-converts impact by separated electrons into an
electrical signal. – 4. Processor (computer)-translates the signal from the
detector into the appropriate spectrum.
Information Provided – ESCA methods are extremely sensitive and can detect all
elements except hydrogen and helium in the outermost~100 Å of an organic or inorganic material atconcentrations down to 0.1 atomic percent.
ATR-FTIR• When a beam of electromagnetic radiation passes from a
Attenuated Total Internal Reflectance Fourier Transform-Infrared Spectroscopy (ATR-FTIR)
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When a beam of electromagnetic radiation passes from amedium that is more dense to one that is less dense,reflection occurs. Upon reflection, the beam penetrates asmall distance into the less dense medium and this
penetrating radiation is called an evanescent wave. Likefor other IR methods, the sample may absorb theevanescent beam due to the vibration frequency ofbonds found within the material. Absorption of certain
wavelengths causes their attenuation (thus giving themethod its name) and provides information about thechemical structure of the material.
漸逝波
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• Fig. Typical ATR-FTIR spectrum
of poly(dimethyl siloxane).Characteristic peaks include theC-H bond in Si-CH3 at 800 cm
-1(A),the Si-O-Si bonds at 1020 cm-1(B),and the C-H bond in Si-CH3 at1260 cm-1 (C).
Ewing G.E. (2004): Thin film water. J. Phys. Chem. B. 108, 15953-15961
• The basic instrumentation for ATR-FTIR spectroscopy isvery similar to that described preciously for other FTIRmethods and includes an IR source, an interferometer, a
detector and a processor. Fourier-transform techniquesare required for this application because they significantly
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p qare required for this application because they significantlyincrease the signal strength (signal to noise ratio).
• A common use for this technique is to follow the relativechange over time of certain peaks that correspond toa specific type of bond. Examining the appearance ofamide bonds, for example, allows the kinetics of proteinadsorption to a biomaterial to be sensitively monitored.The development of sample cells that permit theintroduction of a liquid/water interface has greatlyfacilitated these types of experiments.
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• Fig. Two types of ATR sample cells, (a) onefor solids and (b) one for the solid/liquidinterface.
SIMSSecondary Ion Mass Spectrometry (SIMS)
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• The theory behind SIMS is identical to that described for massspectrometry and centers on separation of ionic species by mass.
• A minor difference in the surface version is the method of sampleionization, which involves the use of primary and secondary ions(thus giving SIMS its name).
• Ionization begins when primary ions, such as O2+
, Ar
+
, Xe
+
, or Cs
+
,are ejected from an ion gun and strike the sample surface. Thiscauses the surface layer of atoms to be stripped off, or sputtered,both as neutral species and as ions.
• These emitted ions are called secondary ions and are drawn intothe analyzer for separation by mass in a similar manner to bulkmass spectroscopy.
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Fig. Block diagram of instrumentationfor SIMS, which includes four maincomponents: an ionization chambercontaining the sample and ion gun, amass analyzer (mass filter), an ion
detector, and a processor/computer totranslate signals from the detector intothe appropriate spectrum. SIMSanalysis is conducted under ultra-high vacuum (UHV).
(Leibniz Institute for Solid State and Materials Research, Dresden, Germany)
SIMS is considered a surface analysis technique since
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• SIMS is considered a surface analysis technique sincethe energy of the incident ions generates collisioncascades only in the surface region of the sample, and,
of these collisions, only those that occur in the outermostlayers produce secondary ions with sufficient energy toescape from the surface.
• Both static and dynamic SIMS methods have beendeveloped. Static SIMS uses a relatively low ion dose(less than 1013 ions/cm2) and induces minimal surfacedamage. In contrast, dynamic SIMS bombards thesample with a much larger ion dose. In this case, so
much material is sputtered that the surface erodes whilethe experiment is being performed. This allows depthprofiling of specimens (monitoring the intensity of a peakof interest as the surface erodes).
I f ti P id d
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Information ProvidedSIMS provides information about thestructure and composition of theoutermost few Å of both inorganic andorganic materials, although the accuracyof quantitative methods for this type ofspectroscopy is limited. With dynamicSIMS, composition as a function of depthcan also be recorded.
• Fig. A spectrum
produced by a type ofSIMS for fibronectinadsorbed on a
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adsorbed on apoly(styrene) surface.The various peakscorrespond to different
amino acids found inthe fibronectin protein.By comparing therelative intensities ofcertain peaks as the
protein is adsorbed ondifferent surfaces, thebiomaterialist canobtain informationabout the orientationof the protein on eachsurface.
TEM and SEM
• The principles of quantum mechanics predict that
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• The principles of quantum mechanics predict thataccelerated electrons have wave-like properties.Electron microscopy techniques utilize this property of
electrons to form images.
• Because the wavelength associated with electrons isshorter than that for white light, the resolving power of
the TEM is greater, and much more detailed images canbe obtained.
• TEM requires very thin samples (20-200 μm thick)because electron beams are completely absorbed bythicker samples and therefore are unavailable to createan image. Due to the stringent preparation techniquesinvolved in making these thin sections, TEM is less oftenused in biomaterials research than SEM.
• Fig. (a) Image ofsperm cells under awhite lightmicroscope (scale
bar 10 μm). (b)Image of a sperm cellunder transmission
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under transmissionelectron microscopy(scale bar 2 μm).Because the
wavelengthassociated withelectrons is shorterthan that for whitelight, the resolvingpower of the TEM is
greater, and muchmore detailedimages can beobtained.
Internet Health Resources, CA
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• Fig. Location of cadherins in epithelial cells. Transmembrane cadherins areresponsible for cell-cell contacts in the form of desmosomes (胞橋小體). Theyundergo calcium dependent homophilic binding (cadherin molecules on the two cellsbind to each other), while their cytoplasmic regions attach to intermediate filaments,
thus linking the extracellular and intracellular environments.
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Keratinocytes
J Biol Chem 2003 Jan 10;278(2):1220-8
http://virtuallaboratory.net/OmniaCellula/Contents/Topic6-4_Signaling.htm
• In SEM, the surface of a sample is scanned with anelectron beam. The electrons from the beam undergo
elastic and inelastic scattering as they collide withatoms in the sample.
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• Elastic scattering results in alteration of the trajectory ofthe electron, but not its energy. In many cases, after anumber of elastic collisions, the electron will exit thesample as a back scattered electron. On the other hand,inelastic scattering occurs when the electron transferspart or all of its energy to a sample atom. The atom then
emits secondary electrons, Auger electrons or X-rays asa means to release this excess energy. SEM imagesare produced by recording the production ofsecondary electrons after an area is bombarded withthe primary electron beam.
• Since the intensity of these electrons is dependent onthe surface topography of the sample, SEM isconsidered a surface imaging technique.
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Fig. Comparison of Energy path in (a) lightmicroscopy and (b) transmission electronmicroscopy, which is designed very similarly to acompound microscope but uses magnetic
lenses, as opposed to glass. Iowa State University SEM Homepage
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Fig. SEM image of osteoblasts cultured ona titanium mesh. This image shows theinteraction of cells and a biomaterialscaffold for bone tissue engineering. Theindividual cells can be seen, particularly on
the fiber in the back of the pore (in thecenter of the image). Scale bar 100 μm
• For optimal imaging, nonconductive samples, such as polymers,must be precoated with a thin layer of conductive material (metal) to
reduce charge build-up during scanning. This is accomplished viaphysical sputtering from a metallic target onto the sample beforeimaging In reality the secondary electrons are produced from
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imaging. In reality, the secondary electrons are produced fromthe coating only, so a high-fidelity image of the surfacetopography is produced only if the coating is sufficiently thin
and conformational.• Because electrons readily interact with atoms in the air, all electron
microscopy must be completed under vacuum.
• SEM is commonly used to visualize the surface topography of abiomaterial, or a biomaterial with attached tissue or cells. Although the requirement of imaging in vacuum prevents fullanalysis of biomaterials as they would be found in vivo, recentdevelopment of the environmental SEM (ESEM) allows imagingof partially hydrated samples. The combination of SEM and EDXAprovides information about chemical composition of the sample,although the ability to distinguish surface chemistry is limited.
• Fig The (coated) sample is placed
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Fig. The (coated) sample is placedinto the holder and the electron beamis scanned over the surface in a rastermotion. (The beam scans each line
from side to side and all the lines fromtop to bottom.) The secondaryelectron detector is positioned so thatthe locations of the emitted electronsare recorded. The signal from thedetector (indicating electron impactintensity and position) is then
processed using appropriate softwareto produce a three-dimensional image.If emitted X-rays are also to beexamined, a specialized detectorsystem called energy-dispersive X-ray analysis (EDXA) is included tocollect and analyze this radiation.
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Blackburn MicroTech Solutions Ltd
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Schematic view of an STM
STM: Scanning Tunneling Microscope
AFM
• Scanning probe microscopy is a general term that refers
AFM: Atomic Force Microscope
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• Scanning probe microscopy is a general term that refersto any number of techniques producing a three-
dimensional image due to the interactions between asmall probe and atomic constituents of the samplesurface.
• AFM can provide three-dimensional images of materialsurfaces with Å to nm spatial resolution.
• The analytical capabilities of AFM are limited to the
uppermost atomic layer of a sample because itsoperation is based on interactions with the electronclouds of atoms at the surface.
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• There are four basic components to an AFM: – 1. Cantilever/tip-bends in response to forces between tip and
sample.
– 2. Laser/detector-a laser beam is bounced off the cantilever anddirected toward a photodiode detector to record the deflection of thecantilever in response to the surface.
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p – 3. Sample stage-holds sample securely. Uses a piezoelectric driver
to alter position up or down to maintain contact between tip andsample.
– 4. Computer-translates the signal from the photodiode detector andprovides feedback to control stage position. Records stage positionand produces image.
Dr. Antonio Siber, Institute of Physics, Zagreb, Croatia
懸臂
• In AFM, a small tip is attached to a cantilever. When thetip encounters the material surface, van der Waals and
electrostatic interactions between atoms in the tip andthose on the surface create a characteristic force profileand cause eventual attraction of the tip to the surface,
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pthus bending the cantilever.
• To obtain an image, the cantilever/tip assembly israstered across the material in a controlled manner andthe stage containing the sample is moved up or down inresponse to the bending of the cantilever so that the tip
contacts the surface at all times. The record of thechange in stage position required to achieve thisconstant contact forms the basis of the height datadisplayed in the three-dimensional image.
Fig. Tip used for atomic force microscopy.The tip is attached to the end of a cantilever beam.
• AFM images are usually obtained via either
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contact or tapping mode.
• For proteins or polymeric samples, in contactmode, the pressure applied due to the small
area at the tip may cause damage to the sample.Thus, tapping mode is more often employed formore delicate specimens. Both modes can beused with a specially designed fluid cell to allow
imaging of biomaterials in aqueous conditionslike those found in vivo.
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• AFM is most often used to visualize surface topography,either of a biomaterial or a layer of adsorbed proteins.
• The number of interacting atoms increases as the widthof the tip increases, thus decreasing the spatial
l ti Th f f ti l lt th idth f th
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resolution. Therefore, for optimal results, the width of theend of the tip should be smaller than the smallest featureto be imaged.
• This modality has been exploited to determine bindingforces between biological molecules. In theseexperiments, a molecule is covalently bonded to the tipand the adhesive force is recorded between the tip and asecond molecule of interest that is attached to thesample surface.
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• Fig. Schematic of AFM instrumentation. Imaging begins when the sample is placed on a stageand the tip is lowered until it contacts the surface. When this occurs, reflection of a laser off thecantilever indicates that the cantilever is bent toward the sample. The tip/cantilever are thenmoved across the sample surface and the cantilever deflection is monitored. In response tochanges in defection, the stage is moved up and down to maintain contact between the sampleand the tip. Alterations in stage position are recorded and processed by the appropriate softwareto form a three-dimensional image.
Tapping mode
Dr. Mauro Ciappa, Integrated Sysyems Lab, Zurich
AFM Scanning Mode
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http://niufood.niu.edu.tw/img.php?img=662_51121f09.png&dir=users_sharing/1
AFM Applications-DPN
• Dip Pen Nanolithography
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• Dip-Pen Nanolithography
Nanografting
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http://www.sciencedirect.com/science/article/pii/S0304416510001133#gr6
Direct DPN
Indirect DPN
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• Fig. Surface topography (via AFM) of poly(D,L-lactic acid)-poly(ethyleneglycol)-monomethyl ether diblock copolymer (Me.PEG-PLAs-numbers
indicate the weight of the corresponding polymer blocks in kDa) filmscompared to a PLA film: (a) PLA, (b) Me.PEGS-PLA45, (c) Me.PEGS-PLA20, (d) Me.PEGS-PLA10 as observed by AFM. The PLA film has analmost smooth, nonstructured surface. With increasing Me.PEG content ofthe polymers, the density of particulate structures (predominantlycrystallized PEG) increases tremendously.