Quantitative considerations about the size dependence of cellular entry and excretion of colloidal nanoparticles for different cell types.

Most studies about the interaction of nanoparticles (NPs) with cells have focused on how the physicochemical properties of NPs will influence their uptake by cells. However, much less is known about their potential excretion from cells. However, to control and manipulate the number of NPs in a cell, both cellular uptake and excretion must be studied quantitatively. Monitoring the intracellular and extracellular amount of NPs over time (after residual noninternalized NPs have been removed) enables one to disentangle the influences of cell proliferation and exocytosis, the major pathways for the reduction of NPs per cell. Proliferation depends on the type of cells, while exocytosis depends in addition on properties of the NPs, such as their size. Examples are given herein on the role of these two different processes for different cells and NPs.

Design of micromagnetic arrays for on-chip separation of superparamagnetic bead aggregates and detection of a model protein and double-stranded DNA analytes.

Magnetically actuated lab-on-a-chip (LOC) technologies have enabled rapid, highly efficient separation of specific biomarkers and cells from complex biological samples. Nonlinear magnetophoresis (NLM) is a technique that uses a microfabricated magnet array (MMA) and a time varying external magnetic field to precisely control the transport of superparamagnetic (SPM) beads on the surface of a chip based on their size and magnetization. We analyze the transport and separation behavior of SPM monomers and dimers on four MMA geometries, i.e., circular, triangular, square and rectangular shaped micromagnets, across a range of external magnetic field rotation frequencies. The measured critical frequency of the SPM beads on an MMA, i.e., the velocity for which the hydrodynamic drag on a bead exceeds the magnetic force, is closely related to the local magnetic flux density landscape on a micromagnet in the presence of an external magnetic field. A set of design criteria has been established for the optimization of MMAs for NLM separation, with particular focus on the shape of the micromagnets forming the array. The square MMA was used to detect a model protein biomarker and gene fragment based on a magnetic bead assembly (MBA) assay. This assay uses ligand functionalized SPM beads to capture and directly detect an analyte through the formation of SPM bead aggregates. These beads aggregates were detected through NLM separation and microscopic analysis resulting in a highly sensitive assay that did not use carrier fluid.

Direct identification of the herpes simplex virus UL27 gene through single particle manipulation and optical detection using a micromagnetic array.

Magnetophoretic lab on a chip technologies are rapidly evolving into integrated systems for the identification of biomarkers and cells with ultra-high sensitivity. We demonstrate the highly efficient detection of the Human herpes simplex virus type 1 (HSV) UL27 gene through the programmed assembly of superparamagnetic (SPM) nanoparticles based on oligonucleotide hybridization. The state of assembly of the SPM nanoparticles was determined by optical signature of the synchronized motion on the beads on a micromagnetic array (MMA). This technique has been used to identify <200 copies of the HSV UL27 gene without amplification in less than 20 minutes. The MAA can also be used to separate gene-SPM bead aggregates from millions of unreacted SPM beads based on nonlinear magnetophoresis (NLM). The MMA-optical detection system promises to enable highly sensitive, nucleic acid analysis to be performed without amplification and with the consumption of minimal amounts of reagent.

Nonradioactive Cell Assay for the Evaluation of Modular Prostate-Specific Membrane Antigen Targeting Ligands via Inductively Coupled Plasma Mass Spectrometry.

The development of novel prostate-specific membrane antigen (PSMA)-targeted radioactive theranostic agents is currently limited to facilities capable of working with high-energy radioisotopes. Even preselection of lead structures in vitro relies mostly on radioactive assays with PSMA(+) LNCaP and PSMA(-) PC-3 cells. Assays utilizing radioisotopes are time consuming, costly, and limit discovery to a small group of scientists with special facilities. Nonradioactive alternatives are therefore needed in the field. In this paper, we describe an inductively coupled plasma mass spectrometry (ICP-MS)-based method for the evaluation of PSMA-targeting ligands conjugated to DOTA-chelates of Europium. This method is based on LNCaP and PC-3 cells and has been validated with the well-established targeting ligand PSMA-617.

Specific binding and internalization: an investigation of fluorescent aptamer-gold nanoclusters and cells with fluorescence lifetime imaging microscopy.

Fluorescent gold nanoclusters show promising properties for biological applications. We biofunctionalized fluorescent 11-mercaptoundecanoic-acid stabilized gold nanoclusters (AuNCs) with an aptamer to target the interleukin-6-receptor expressed on BaF3 cells specifically. Although the fluorescence emission of the AuNCs (535 nm) is in the same wavelength region as the autofluorescence of the cell, we are able to distinguish between nanoclusters and cells using the fluorescence decay time, which is much longer for the AuNCs (100 ns) than for the autofluorescence. After a first short incubation period we detected AuNCs specifically bound to the cell membrane by using two fluorescence lifetime imaging microscopy (FLIM) methods: gated and direct FLIM. After a second incubation period the previously bound AuNCs are internalized by the cells, as could be resolved solely by the direct FLIM. This proves the superior sensitivity of this method compared to gated FLIM. We find that the optical properties of AuNCs do not change upon binding to the cells, but exhibit a change when internalized into the cells, induced by an interaction between the AuNCs and cells.