Biomaterial Chemistry: Methods of Modification and Immobilization

Biomaterial Chemistry: Methods of Modification and ImmobilizationMethods of Modification and Immobilization

Reporter: Lily Ou Instructor: Hsien-Chang Chang Nov. 30, 2011

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1. Surface Functionalization

2. Layer Coating

Surface Activation

(1)

(2)

Blocking

Immobilizing

MotivationsMotivations

• Therapeutic uses– Drug delivery

• Medical uses– Artificial organs

– Tissue engineering

• Diagnostic uses

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Ratner, B. D.; Hoffman, A. S.; Schoen, F. J.; Lemons, J. E. Biomaterials Science. 2nd ed. San Diego: Elsevier Academic Press, 2004.

Surface Modification & ActivationSurface Modification & Activation

• Want to modify materials in such a way that only the outermost surface is modified.– Functionalize materials for further steps while

retaining the physical properties of the surface.• What materials?

– Polymers, metals, ceramics, and glasses• What biomolecules?

– Enzymes, antibodies, and affinity proteins

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Ratner, B. D.; Hoffman, A. S.; Schoen, F. J.; Lemons, J. E. Biomaterials Science. 2nd ed. San Diego: Elsevier Academic Press, 2004.

• Can be divided into 3 categories:

(1) Physical alteration

- Electrostatic

- Affinity

(2) Chemical alteration

- EDC/NHS Coupling

- Self-Assembled Monolayer (SAM)

(3) Physicochemical alteration

- Plasma coating

4Ratner, B. D.; Hoffman, A. S.; Schoen, F. J.; Lemons, J. E. Biomaterials Science. 2nd ed. San Diego: Elsevier Academic Press, 2004.

Surface Modification & ActivationSurface Modification & Activation

- - - -

Metal Oxide

- - - -

+

biomolecule

+

biomolecule

+

biomolecule

5VandeVondele et al. Biotechnol. Bioeng. 2003, 82, 784.

Most metal oxides are negatively charged in an aqueous environment at a pH of

7.4.

(1) Physical Adsorption – (1) Physical Adsorption – Electrostatic InteractionElectrostatic Interaction

VandeVondele et al. Biotechnol. Bioeng. 2003, 82, 784.6

Nb2O5

PLL – PEG - Peptide

Nb2O5

PLL

– PE

G -

Pept

ide

PLL

– PE

G -

Pept

ide

PLL

– PE

G -

Pept

ide

1 mg/mL in 10 mM HEPES, pH 7.4

PLL = poly-(L-lysine) PEG = poly(ethylene glycol)

pKa = 10.5

(1) Physical Adsorption – (1) Physical Adsorption – Electrostatic InteractionElectrostatic Interaction

Lysine Ethylene glycol

VandeVondele et al. Biotechnol. Bioeng. 2003, 82, 784.

(1) Physical (1) Physical Adsorption – Adsorption – Electrostatic Electrostatic InteractionInteraction

Nb2O5

PLL Anchor

PEG Spacer

Peptide

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• Affinity of one molecule for another

– Example: Biotin and Avidin

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(1) Physical Adsorption –(1) Physical Adsorption – Affinity

Biotin (also known as vitamin H) is a small and trace organic molecule found in every cell.

MW = 244.3 DaBiotin

Avidin is a much larger protein that binds biotin with a very high affinity. The two molecules will bind to each other with such a high affinity that the binding is essentially irreversible.

Biotin • Biontin, 中文稱「生物素」，又名「 Vitamin H 」，是一普遍存於細胞中的微量物質，分子相當小，分子量只有 244.3 Dalton 。

• Biotin 有三個相親相愛的好兄弟，分別為 Avidin 、 Strepavidin 以及 NeutrAvidin 。 Biotin 與 Avidin 的親和力相當高，能形成非常強的非共價性鍵結。此鍵結的形成速度很快，而且一旦形成後，便不易受到極端的 pH, 溫度、有機溶劑或變性劑的影響而分開。 Biontin-Avidin 複合物能夠承受 3 M guanidine HCl 的環境，要一直到 8 M guanidine HCl pH 1.5 的環境下，或是用 autoclaving 方法才能將 biotin 由此複合物中給釋出。

• Avidin ：是一種醣蛋白，被發現存在於雞蛋卵白中，所以中文稱之為「卵白素」，鳥類、爬蟲類及兩棲類的組織中亦存在。 Avidin 總分子量為 67,000 Daltons ，由四個完全相同的次體 (subunit) 所組成，每個次體皆能與一分子的 biotin 結合。 Avidin 含醣比例頗高，約佔了總分子量的 10 ％。其 pI ＝ 10-10.5 ，溶於水及含鹽溶液中。 Avidin 在相當廣的溫度及 pH 範圍下皆很穩定。

AvidinAvidin

• Avidin, synthesized in the hen oviduct ( 輸卵管 ), is a glycoprotein of MW 68,000 daltons which occupies about 0.05% (w/w) of the total protein content of the hen egg white.

• The great affinity of avidin for biotin (kd= 10-15M), results in a great number of applications in biochemistry.

http://en.wikipedia.org/wiki/Avidin 10

• Native avidin is a tetrameric protein composed of four identical subunits, and each subunit can bind a biotin molecule

• The isoelectric point of native avidin is 10.5.• The basic glycoprotein nature of native

avidin results in some non-specific binding observed in different studies.

AvidinAvidin

• Biotin has 3 binding partners: Avidin, Streptavidin, and NeutrAvidin.• Avidin

– The affinity of avidin for biotin is very strong. – This non-covalent interaction forms rapidly and does

not break easily once formed. • Not even extreme pH values, temperatures, organic

solvents can break the bond. – The only two ways to break the bond are in the

environment of 8 M guanidine HCl, pH 1.5 and using autoclaving.

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Strepavidin & NeutrAvidin• NeutrAvidin: 是去除了”醣”以後的 Avidin ，分子量為

60,000 daltons 。其優點為 pI 值為中性，且其與 biotin-binding protein 之間會造成的非專一性結合為最低。

• Streptavidin: 由 Steptomyces Avidinii 中純化出的蛋白質，同樣能與 Biotin 做專一性結合，分子量為 60,000 daltons 。 Streptavidin 也是個由四個相同次體 (subunit) 所組成的蛋白質，每個次體皆能與一分子的 biotin 結合，且其與 biotin 之鍵結親和力相當於 Avidin-biotin 之親和力。不過 Biotin-Streptavidin 複合物對於 guanidine.HCl 將兩者分開的抗拒能力較 Biotin-Avidin 還要強。此外， Streptavidin 不含醣且具有酸性的 pI = 5 ，正好與 Avidin 相反。

StreptavidinStreptavidin

• Streptavidin (SAv)– Isolated from a bacterial strain Steptomyces Avidinii.– Tetramer and each subunit interacts specifically with a

biotin molecule. – MW = 60,000 Daltons.– The binding affinity between streptavidin and biotin is

similar to the affinity between avidin and biotin.– Biotin-streptavidin complex is more resistant to

guanine HCl than biotin-avidin is.– Streptavidin has a pI value of 5.– Not a glycoprotein.

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StreptavidinStreptavidin

http://arrayit.com/Products/Microarray_Slides/Microarray_Slide_SuperStreptav/microarray_slide_superstreptav.html

Biotinylation of EnzymesBiotinylation of Enzymes

• Because of the rapid and strong interaction between biotin and streptavidin and the small size of biotin, biotin can be attached to different biomolecules. With the use of streptavidin conjugated to detecting molecules such as enzymes, the biomolecules can be isolated and extracted for detection.

15NHS-Biotin, Wt.: 341

1 2 3 4

-OOC-OOC-OOC-OOC

BiotinylationBiotinylation• Immunoblotting

– Attach biotin to specific antibody– Use enzymes such as HRP linked to streptavidin and the

enzyme substrate for blot detection• ELISA

– Immobilize streptavidin on the well– Introduce antibodies conjugated to biotin for detection

• IP/Co-IP– Use streptavidin-immobilized beads and biotin-conjugated

protein sample. – Spin down the complex.– Remove the biotin for purification or further steps.

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Biotin-Streptavidin InteractionBiotin-Streptavidin Interaction

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Further Reactions

Hasegawa et al. Sensors. 2008, 8, 1090.

20 nM Biotinylated Anti-Thrombin Aptamer

in Binding Buffer

Binding Buffer: 50 mM Tris-HCl, 300 mM NaCl, 5 mM KCl, pH 8.0

Avi Avi Avi Avi Avi AviB B BB

EDC/NHS CouplingEDC/NHS Coupling

• In the reaction, 1-ethyl-3-(3’-dimethylaminopropyl) carbodiimide (EDC) converts a carboxylic acid group into a reactive intermediate which is susceptible to amine attacks.

• In some cases EDC and N-hydroxysuccinimide (NHS) are used as the NHS produces a more stable reactive intermediate which has been shown to give a greater reaction yield.

• The carbodiimide coupling in the presence of EDC (sometimes also in the presence of NHS) is usually performed in HEPES-buffer solution at pH = 7.2-7.5

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(EDC)

Joos et al. Anal. Biochem. 1997, 247, 96.

EDC/NHS CouplingEDC/NHS Coupling

Cl-Cl-

• EDC catalyzes the formation of amide bonds between carboxylic acids or phosphates and amines by activating carboxyl or phosphate to form an O-urea derivative

• This intermediate is unstable. Random reactions can result in undesired products.

1-ethyl-3-(3’-dimethylaminopropyl) carbodiimide

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NHS

• N-Hydroxysuccinimide (NHS) is often used to assist the carbodiimide coupling in the presence of EDC.

• The reaction includes the formation of an intermediate ester (the product of condensation of the carboxylic group and N-hydroxysuccinimide)

EDC/NHS CouplingEDC/NHS Coupling

Cl- N

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This active ester intermediate further reacts with the amine functional group on the desired biomolecule to yield the final amide bond.

EDC/NHS CouplingEDC/NHS Coupling

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EDC/NHS CouplingEDC/NHS CouplingHA = Hyaluronic AcidReaction Buffer = 10 mM HEPES, pH 7.0EDC = 200 mMSulfo-NHS = 50 mM

Stile et al. J. Biomed. Mater. Res. 2002, 61, 391.

Glutaraldehyde (戊二醛 )

1,5-Pentanedial C5H8O2

分子量 : 100.13

GA MethodGA Method

GA 不論是單分子 (Monomer) 或多分子 (Multimer) 結構，基本上都是利用兩端的醛基 (-CHO) 與生化分子上 peptide 鍵上 (-NH2)官能基進行鍵結，以達到固定的目的。

Schiff baseSchiff base

• A Schiff base (or azomethine), named after Hugo Schiff, is a functional group that contains a carbon-nitrogen double bond with the nitrogen atom connected to an aryl or alkyl group—but not hydrogen. Schiff bases are of the general formula R1R2C=N-R3, where R3 is an aryl or alkyl group that makes the Schiff base a stable imine. A Schiff base derived from an aniline, where R3 is a phenyl or substituted phenyl, can be called an anil.

• Schiff bases can be synthesized from an aromatic amine and a carbonyl compound by nucleophilic addition forming a hemiaminal, followed by a dehydration to generate an imine.

http://en.wikipedia.org/wiki/Image:Schiffbase.png

Self-Assembled Monolayer (SAM)Self-Assembled Monolayer (SAM)

• A single layer of molecules on a substrate

• Introduce the specific molecules to the substrate. After washing, the single layer is formed spontaneously.

26http://en.wikipedia.org/wiki/Self_assembled_monolayer

• Example: alkane thiol on gold substrate

– Based on the affinity of sulfur for gold

– Binding energy:

85-145 kJ/mol

Attachment Group (-COOH, silane, -SH, -PO4)

Assembling Structure (Alkyl Groups)

Functional Head Group (-CF3, -OH, -CH=O)

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Substrate (ex. gold, silica, Al2O3)

Strong Interactions

Van der Waals Forces

Surface Interactions

Self-Assembled Monolayer (SAM)Self-Assembled Monolayer (SAM)

單層自組法之基材、配位體與鍵結後之結構

HEPES (Biological Buffer),C8H18O4N2S F.W. 238.30, pKa = 7.55 (at 20°C), pH (Working Range) = 7.0-8.0

A buffer solution of HEPES can be prepared by two methods:

(1)The free acid can be added to water, then titrated with approximately one-half mole equivalent of sodium hydroxide or potassium hydroxide to the precise pH desired, with adjustments made for final temperature and volume.

(2)Alternatively, equimolar concentrations of HEPES and of sodium HEPES can be mixed in approximately equal volumes, back-titrating with either solution to the appropriate pH.

(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)

+ -

• Both the anode and cathode are water cooled.• The process is initiated by a high voltage discharge which

causes localized ionization of the gas and a conductive path for a DC arc to form between cathode and anode.

Plasma SprayPlasma Spray

http://www.gordonengland.co.uk/ps.htm

copper

tungsten

argon, nitrogen, hydrogen, helium

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Text p. 205-7

• The resistance heating from the arc causes the gas to reach extreme temperatures, dissociate and ionize to form a plasma.

• The plasma flows around the cathode and through the anode which is shaped as a constricting nozzle. It then exits the anode nozzle as a free or neutral plasma flame.

Plasma SprayPlasma Spray

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• Powder is fed into the plasma flame most commonly via an external powder port mounted near the anode nozzle exit.

• The melted or partially melted particles impact the surface and rapidly solidify. The powder is so rapidly heated and accelerated that spray distances can be in the order of 25 to 150 mm.

Plasma SprayPlasma Spray

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Adsorption of dithiobis(Adsorption of dithiobis(NN-succinimidyl -succinimidyl propionatepropionate) (DTSP) on a Au-electrode) (DTSP) on a Au-electrode

Fig. 3 Adsorption of dithiobis(N-succinimidyl propionate) (DTSP) on a Au-electrode surface and application of the self-assembled monolayer containing active ester groups for covalent coupling of HRP.  (E. Katz, J. Electroanal. Chem., 1990, 291, 257)

丙酸鹽

Horseradish Peroxidase山葵過氧化氫酶

ApplicationsApplications

AgAg++ (aq) (aq)Ag (s)Ag (s)

CD

RP215

RP215

BiotinStreptavidin

Gold Nanoparticle

Silver Particles

EDC/NHS Coupling

Affinity Coupling

EDC/NHS Coupling