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Peptide coated semiconductor nanocrystals for biomedical applications X Michalet F F Pinaud L A Bentolila J M Tsay S Doose J J Li G Iyer S Weiss Dpt of Chemistry Biochemistry UCLA 607 Charles E Young Drive East Los Angeles CA 90095 Applied Laserphysics Laserspectroscopy University of Bielefeld 33615 Bielefeld Germany ABSTRACT We have developed a new functionalization approach for semiconductor nanocrystals based on a single step exchange of surface ligands with custom designed peptides This peptide coating technique yield small monodisperse and very stable water soluble NCs that remain bright and photostable We have used this approach on several types of core and core shell NCs in the visible and near infrared spectrum range and used fluorescence correlation spectroscopy for rapid assessment of the colloidal and photophysical properties of the resulting particles This peptide coating strategy has several advantages it yields probes that are immediately biocompatible it is amenable to improvements of the different properties solubilization functionalization etc via rational design parallel synthesis or molecular evolution it permits the combination of several functions on individual NCs These functionalized NCs have been used for diverse biomedical applications Two are discussed here single particle tracking of membrane receptor in live cells and combined fluorescence and PET imaging of targeted delivery in live animals KEYWORDS quantum dots nanocrystal fluorescence photophysics single molecule peptide functionalization live cell FCS 1 INTRODUCTION Fluorescent semiconductor nanocrystals NCs 3 have become increasingly popular as potential replacement of fluorescent dyes for multiple biomedical applications Their broad excitation spectra associated with tunable and narrow emission spectra their photostability brightness and quantum yield make them serious contenders as ideal fluorescent probes for applications demanding high sensitivity high signal to noise or long observations4 However high quality NCs are usually synthesized in organic solvents and additional chemical modifications are required to solubilize them in aqueous buffers and functionalize them for biomedical applications This is accomplished by exchanging the hydrophobic surface ligands with amphiphilic ones Different NC solubilization strategies have been devised over the past few years including i ligand exchange with simple molecules such as mercaptoacetic acid5 dithiothreitol6 or more sophisticated ones such as oligomeric phosphines7 dendrimers8 amphiphilic polymers9 triblock copolymers10 and peptides1 ii encapsulation in silica shells11 12 phospholipid micelles13 polymer beads14 polymer shells15 amphiphilic polysaccharides16 and iii combination of several layers conferring the required colloidal stability to NCs17 19 Among these possible routes some have known limitations leading to particles that either lack long term stability5 6 have reduced QY are significantly larger than the original particles9 10 have broader size distributions11 or do not work well with all particle sizes13 Recently some groups have developed promising water based synthesis20 21 yielding particles emitting from the visible to the NIR spectrum that are natively water soluble but have yet to be tested in biological environments michalet chem ucla edu sweiss chem ucla edu 57 Solubilization is but the first step towards using NCs as biological probes unless they are used as mere non specific fluorescent stain as demonstrated in experiment involving E coli bacteria22 amoeba23 and human cell lines23 25 or Xenopus embryo13 For biological targeting some kind of biological interfacing is necessary Some applications will require having a single recognition moiety attached to the nanocrystal e g DNA oligonucleotide aptamer antibody etc A simple method consists of exchanging the solubilization ligands with the molecule of interest as was demonstrated with DNA oligonucleotides 26 More generally as most NC ligands expose either a carboxyl or an amine group standard bioconjugation reactions can be used to functionalize NCs with molecules containing a thiol group13 27 28 or an NHS ester moiety11 respectively Alternatively heterobifunctional reagents 5 7 19 29 can be used to cross link molecules to the NC ligands Avoiding functionalization chemistry altogether some researchers have used electrostatic interactions between NCs and charged adapter molecules or proteins modified to incorporate charged domains30 31 For instance biotinylated or streptavidin SAv coated NCs can be used in combination with SAv functionalized or biotinylated proteins or antibodies1 9 17 23 32 35 Using an antibody against a specific target and a biotinylated secondary antibody itself bound to a SAv coated NC any type of target can be labeled using a three layer approach 9 34 In contrast to classical fluorophores used for biological labeling the large surface area of NCs their diameter varying from a few nm36 to a few dozens nm10 increases the number of available attachment groups 10 100 In other words labeling of NCs is therefore statistical and conrolled by stoichiometry If the size of the attached moiety approaches the A B S P B R Q D X X 10 14 nm S P B R Q D 58 Fig 1 Nanocrystal peptide coating approach A Schematic representation of the surface coating chemistry of CdSe ZnS nanocrystals with phytochelatin related peptides The peptide C terminal adhesive domain binds to the ZnS shell of CdSe ZnS nanocrystals after exchange with the trioctylphosphine oxide TOPO surfactant A polar and negatively charged hydrophilic linker domain in the peptide sequence provides aqueous buffer solubility to the nanocrystals TMAOH Tetramethyl ammonium hydroxide Cha 3cyclohexylalanine From ref 1 B Peptide toolkit The light blue segment contains cysteines and hydrophobic aminoacids ensuring binding to the nanocrystal adhesive domain of Fig 1A and is common to all peptides S solubilization sequence hydrophilic linker domain of Fig 1A P PEG B biotin R recognition sequence Q quencher D DOTA 1 4 7 10tetraazacyclododecane 1 4 7 10 tetraacetic acid for radionuclide and nuclear spin label chelation X any unspecified peptideencoded function NCs solubilization is obtained by a mixture of S and P NCs can be targeted with biotin B a peptide recognition sequences R or other chemical moieties NCs fluorescence can be turned on or off by attaching a quencher Q via a cleavable peptide link In the presence of the


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