title: Nanobarcoding: detecting nanoparticles in biological samples using in situ polymerase chain reaction.
abstract: BACKGROUND
  Determination of the fate of nanoparticles (NPs) in a biological system, or NP biodistribution, is critical in evaluating an NP formulation for nanomedicine. Current methods to determine NP biodistribution are greatly inadequate, due to their limited detection thresholds. Herein, proof of concept of a novel method for improved NP detection based on in situ polymerase chain reaction (ISPCR), coined "nanobarcoding," is demonstrated.
  
  
  METHODS
  Nanobarcoded superparamagnetic iron oxide nanoparticles (NB-SPIONs) were characterized by dynamic light scattering, zeta potential, and hyperspectral imaging measurements. Cellular uptake of Cy5-labeled NB-SPIONs (Cy5-NB-SPIONs) was imaged by confocal microscopy. The feasibility of the nanobarcoding method was first validated by solution-phase PCR and "pseudo"-ISPCR before implementation in the model in vitro system of HeLa human cervical adenocarcinoma cells, a cell line commonly used for ISPCR-mediated detection of human papilloma virus (HPV).
  
  
  RESULTS
  Dynamic light-scattering measurements showed that NB conjugation stabilized SPION size in different dispersion media compared to that of its precursor, carboxylated SPIONs (COOH-SPIONs), while the zeta potential became more positive after NB conjugation. Hyperspectral imaging confirmed NB conjugation and showed that the NB completely covered the SPION surface. Solution-phase PCR and pseudo-ISPCR showed that the expected amplicons were exclusively generated from the NB-SPIONs in a dose-dependent manner. Although confocal microscopy revealed minimal cellular uptake of Cy5-NB-SPIONs at 50 nM over 24 hours in individual cells, ISPCR detected definitive NB-SPION signals inside HeLa cells over large sample areas.
  
  
  CONCLUSIONS
  Proof of concept of the nanobarcoding method has been demonstrated in in vitro systems, but the technique needs further development before its widespread use as a standardized assay.
authors.affiliation: Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Trisha
authors.initials: T
authors.lastname: Eustaquio
authors.forename: James F
authors.initials: JF
authors.lastname: Leary
mesh_terms: D002232:Carbocyanines / Q000032:analysis / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D058185:Magnetite Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure*; D008856:Microscopy, Fluorescence / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling
chemical_list: D002232:Carbocyanines; D058185:Magnetite Nanoparticles; C085321:cyanine dye 5
keywords: DNA-functionalized nanoparticles; in situ PCR; nanomedicine; single cell