Biomod/2013/LMU/coating

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Contents

Requirements on coating strategy

In prospect of future applications of nanodiamond arrangements a coating strategy has to met several requirements. First of all it has to provide a functionalization suitable for conjugation with DNA origami which is the main goal of our project.
Conjugation to DNA origami bases on well dispersed nanoparticles at physological conditions is still a difficult task but will be solved by our surface modification [1, 2] . Above that we must link the diamond specifically to our structure since exact positioning is crucial for plasmonic field enhancement studies of nanoparticle arrangements. Medical applications such as bio-labeling and hence in-vivo use demand biocompatibility. Finally any further use of our structures needs sufficient stability with respect to both mechanical and electric forces.

Coating requirements:

  • Functionalizes nanodiamonds surface
  • Provides specific binding sites to DNA origami structure
  • Disperses nanodiamonds at near physiological conditions
  • Is biocompatible
  • Is sufficiently stable

Plan

Materials included:

Bovine serum albumin, BSA:

  • Globular plasma protein extracted from cows
  • Primary structure of 607 amino acids
  • Spherical tertiary structure with polar (hydrophilic) amino acids bounded outwards
  • Thiol, carboxylic and amino groups

UREA, CO(NH2)2

  • Widely used organic compound containing to amino groups
  • Cuts hydrogen bonds of BSA

TCEP, (tris(2-carboxyethyl)phosphine)

  • Frequently used reducing agent
  • Cuts S-S bonds of BSA

NAM, N-Acetylmuramic acid

  • Occupies thiol groups of BSA

Biotin-PEG-Mal (Polyethylene glycol)

  • Polyether of variable length
  • Functionalized with maleimide group on one side, biotin group on the other side

Acid treated nanodiamonds, short NDs

  • Surface shows hydroxilic and carboxylic groups
  • ~50nm in diameter

Neutravidin

  • Tetrameric protein with four binding sites for biotin
  • Binds with high affinity and specificity


Short explanation:

BSA will be used as the coating protein. After being denatured by use of UREA and TCEP Biotin-PEG-Mal, which binds to the thiol groups of BSA by its maleimide group, functionalizes the protein with biotin groups. The remaining amino groups on the BSA ar able to establish hydrogen bonds to the carboxylic groups on the ND surface. The BSA wraps around the ND. The coated ND disperses well at physiological conditions and its biotin groups can link to neutravidin. Neutravidin itself is bound to the used DNA origami structure.

Coating: Step by step

UREA&TCEP induced BSA denaturation

Bovine serum albumin in natural globular form
Bovine serum albumin in natural globular form

Bovine serum albumin is a globular protein extracted from cows. The family of serum albumin is the most abundant protein in the blood plasma of mammals. Its primary structure consists of 607 amino acids. The spherical tertiary structure of the protein is caused by polar and hence hydrophilic amino acids bound outwards, whereas the apolar hydorphobic amino acids are bound towards the molecule's interior. The resulting shape is a prolate ellipse[3]. Hydrogen bonds as well as S-S bonds stabilize the structure of the protein in its natural form. The protein contains several functional groups located on the hydrophilic amino acids of the chain, in particular it contains 35 thiol groups, 99 carboxyilic groups and 120 amino groups which are crucial for the coating process (see table).


No Functional group Amino acid
35 Thiol, SH Cysteine
120 Amino, NH2 Aspartic, Glutamic acid
99 Carboxyl, COOH Asparagine, Lysine, Glutamine, Arginine


With the BSA in its natural form the functional groups are not fully accessible and linking would not be possible. Therefore it has to be brought into a chain-like structure first. This allows linking of the Biotin-PEG to its thiol groups and coating of the nanodiamonds surface by use of its amino groups. First UREA is mixed together with BSA for 5min to cut the hydrogen bonds. After this, TCEP is applied for 30min cutting the S-S bonds. The BSA is now denaturated and its shape has changed to a prolate ellipse with an elongated symmetry axis in comparison to its natural form. Since denaturalized BSA is not stable under the applied conditions, Biotin-PEG-Mal is used to prohibit refolding [1, 2].

UREA breaks hydrogen bonds, TCEP breaks S-S bonds
UREA breaks hydrogen bonds, TCEP breaks S-S bonds
Denaturated BSA protein ready for further use
Denaturated BSA protein ready for further use


Biotin functionalization of BSA by PEG

Biotinylation of BSA using Biotin-PEG-Mal
Biotinylation of BSA using Biotin-PEG-Mal

As a next step we use Biotin-PEG-Mal, a polyether with linear structure and a molecular weight of 3000 Da. It is commercially available in various forms due to its widespread use from industrial manufacturing in medicine. In our case Biotin-PEG-Mal serves two purposes. On the one hand it will act as a linker to neutravidin which itself links to the DNA origami structure. On the other hand it prevents the BSA from refolding. After denaturizing the BSA, 35 thiol groups are accessible on the BSA strand, which can be linked to Biotin-PEG-Mal via maleimide-sulfhydril reaction chemistry. This chemcial process automatically runs due to the appropriate and stable pH-value of our solution. For more information about maleimide reaction chemistry visit: http://www.piercenet.com/method/sulfhydryl-reactive-crosslinker-chemistry


Not all of the BSA's thiol groups will be occupied by Biotin-PEG-Mal due to the large size of the PEG-molecule. For this reason, we use NAM to occupy the remaining thiol groups. This prohibits a re-formation of the S-S bonds to a disulfide bond again. In the case of simultaneous mixing of Biotin-PEG-Mal and NAM with the denatured BSA Biotin-PEG-Mal would have to compete with NAM for the thiol binding sites of the BSA. Due to the difference in molecular weight it would be very likely that the thiol groups would be mainly linked to NAM. Because of that, we first apply Biotin-PEG-Mal. After waiting for 3h we mix this sample with NAM. No purification is needed up to this step. The functionalized denatured BSA is now ready to be used for the coating of the nanodiamond.

Nanodiamond coating

BSA coated nanodiamond, PEG anchors with biotin groups
BSA coated nanodiamond, PEG anchors with biotin groups

Finally the nanodiamonds will be coated with the modified BSA preparing it for the conjugation with the DNA origami structure. For this we used acid treated nanodiamonds. On their surface exist hydroxylic as well as carboxylic groups which will be used as binding sites in this step. The denatured BSA offers up to 120 amino groups, which can attach to the nanodiamonds carboxylic groups by charge interaction. This is due to the static dipole moment of both groups caused by the big electron affinity of both nitrogen (amino group) and oxygen (carboxylic group). This mechanism allows BSA strands to wrap around the nanodiamonds. After mixing the denatured functionalized BSA with the nanodiamonds, the sample has to be purified to remove the remaining BSA which would interfere with the conjugation. The now biotin-functionalized nanodiamonds can link to neutravidin and thus conjugate to the DNA origami structure.


The coating also causes the nanodiamonds to disperse. Former approaches always failed to decluster the nanodiamonds and therefore could not provide specific attachment of single nanodiamonds to DNA origami. This effect of the surface modification can be easily observed by comparing TEM pictures of untreated and coated nanodiamonds (cf. figures below).

TEM image: Unmodified nanodiamonds (Scaling: 100nm)
TEM image: Unmodified nanodiamonds (Scaling: 100nm)
TEM image: BSA coated nanodiamonds (Scaling: 200nm)
TEM image: BSA coated nanodiamonds (Scaling: 200nm)


In summary the coating both functionalizes and diperses the nanodiamonds and therefore solves two crucial problems concerning the conjugation of nanodiamond with DNA origami.

Review

Finally we have to ask ourselves wheter our coating plan matches the conditions we defined at the beginning:

  • Our coating procedure functionalizes the nanodiamond with biotin
  • The linking to neutravidin is routinely used and offers both high affinity and specificity
  • After the coating nanodiamonds disperse well at physiological conditions
  • The materials included are fully biocompatible
  • The stability of the coating is still an open question but will be checked in further experiments


In summary we believe that the developed coating offers a flexible and easy way to produce nanodiamond DNA origami arrangements of any shape and thus opens new routes to both fundamental research and medical applications.

References

  1. Wu Y. et al.; "pH-Responsive Quantum Dots via an Albumin Polymer Surface Coating"; J. AM. CHEM. SOC., 2010, 132, 5012–5014 [1]
  1. Kuan S. , Wu Y. , Weil T.; "Precision Biopolymers from Protein Precursors for Biomedical Applications"; Macromol. Rapid Commun., 2013, 34, 380−392 NANO [2]
  1. Wright AK, Thompson MR; "Hydrodynamic structure of bovine serum albumin determined by transient electric birefringence"; Biophys. J., 1975, 15 (2 Pt 1): 137–41 [3]
  1. Wu Y. et al; "Convenient Approach to Polypeptide Copolymers Derived from Native Proteins"; Biomacromolecules, 2012, 13, 1890−1898 [4]
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