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Condeetal. Biofunctionalizationandsurfacechemistryof inorganicnanoparticles with a platinum(IV) compound capable of being tethered to an amine functionalizedDNA-AuNP surface via amide linkages (Dharetal., 2009). Similarly, conjugation of biomolecules toQDs through cou- plingreactionswithreactive functionalgroupspresentedonQDs surfaceisanothercommonstrategytoprepareQDbioconjugates. Actually, carboxylic acid groups can also be added toQDs’ sur- face and subsequently conjugated to biomoleculeswith primary amine groups throughEDCcoupling reactions (Cai et al., 2006; Hua et al., 2006; Choi et al., 2009;Wu et al., 2009a; East et al., 2011). One disadvantage of this type of chemistry is that the pres- enceofbothcarboxylatesandaminesononeof thebiomolecules tobeconjugatedwithEDCcanresult in self-polymerizationand consequently, loss of effectiveness. For instance, peptides usually contain both types of groups, so if EDC is added in the pres- ence of them, peptides canpolymerize.However, Bartczak et al. haveusedthisstrategytocouplingofpeptides toAuNPsinaone- potway.Theauthorshaveshownthattheconcentration,reaction time, and chemical environment are all critical to achieving the formationofrobust,peptide-coatedcolloidalnanoparticleswith- out aggregation (Bartczak and Kanaras, 2011). Another way to avoidpolymerizationofthebiomolecule is toeliminatetheexcess of EDC before adding the biomolecule to the NPs solution by magnetic separation in the case of MNPs or gel-filtration for otherNPs. Despite the simplicityof this technique, that doesnot require priorchemicalmodificationof thebiomolecule, itdoesnotguar- antee an oriented immobilization in the case of biomolecules withgreater structural complexity, suchasantibodies.Dueto the poorstabilityof thereactiveester,neutralpHistraditionallyused to link covalently antibodies to carboxylated-NPs. At this pH, immobilizationmainlyoccurs throughdirectcovalentbindingof themost reactive amine groups of the antibody. Unfortunately, theseare the terminal aminemoietiesof the fourAbpolypeptide chains (pKa around 7–8), which are all located in the antigen- binding domain (Puertas et al., 2011). Recently, Puertas et al. described a smart approach that takes advantage of the exist- ing kinetic differences among ionic adsorption processes and covalentreactions inorder toassure theorientedcovalentattach- ment of the Ab using EDC chemistry. Briefly, it requires the selectionof thebest incubations conditions (pH, ionic strength) to promote a fast ionic adsorption of the Ab due to the neg- ative charges of the carboxylic groups of the NP. This ionic adsorptionmakes possible the orientation of the Ab on theNP surface before irreversible covalent bond formation (Figure9) (Puertas et al., 2011). Initially, the authors optimized this two- step strategy formagnetic NPs but they have recently extended FIGURE9 |Highlyactivemagneticnanoparticle-antibodyconjugates. (A)Two-step immobilizationmechanismproposedwhenusingAbthatbindto theMNPsthroughthemostreactiveamines—randomimmobilization. (B)Covalentattachmentviathepolysaccharidemoietiesof theantibodytothe MNPs—oriented immobilization. (C)CapacitytocaptureHRPoftheanti-HRP anchoredtoMNPsbydifferentorientations.Theproteincontentofall anti-HRP-functionalizedMNPswassimilar(2μgAbpermgMNP)(Puertasetal., 2011).ReproducedwithpermissionfromPuertasetal. (2011),Copyright2013. www.frontiersin.org July2014 |Volume2 |Article48 | 22