Protein Counting in Single Cancer Cells

The cell is the basic unit of biology and protein expression drives cellular function. Tracking protein expression in single cells enables the study of cellular pathways and behavior but requires methodologies sensitive enough to detect low numbers of protein molecules with a wide dynamic range to distinguish unique cells and quantify population distributions. This study presents an ultrasensitive and automated approach for quantifying phenotypic responses with single cell resolution using single molecule array (SiMoA) technology. We demonstrate how prostate specific antigen (PSA) expression varies over several orders of magnitude between single prostate cancer cells and how PSA expression shifts with genetic drift. Single cell SiMoA introduces a straightforward process that is capable of detecting both high and low protein expression levels. This technique could be useful for understanding fundamental biology and may eventually enable both earlier disease detection and targeted therapy

Arylsilanated SiO_<i>x</i> Surfaces for Mild and Simple Two-Step Click Functionalization with Small Molecules and Oligonucleotides

The conversion of surface-bound aminophenyl groups to azidophenyl moieties on SiO_<i>x</i> surfaces was investigated as part of a mild, simple two-step strategy for “click”-based” surface functionalization with acetylene-functionalized reagents. Small terminal alkynes (phenylacetylene, 1-hexyne) and acetylene-modified single-stranded DNA 20-mers (T₂₀) were then used as model compounds to test the efficiency of the 1,3-dipolar cycloaddition reaction. The identities of surface species were verified, and their coverages were quantified using X-ray photoelectron spectroscopy in the C 1s, N 1s, F 1s, Cl 2p, and P 2p regions. Depending on conditions, the yield of the azidification was in the 30–90% range, and the efficiency of triazole formation depended significantly on the rigidity of the acetylene reactant. Vibrational sum frequency generation was applied to probe the C–H stretching region and test the platform’s viability for minimizing spectral interference in the C–H stretching region. Fluorescence spectroscopy was also performed to verify the presence of fluorescein-tagged DNA single strands that have been coupled to the surface, while label-free DNA hybridization studies by vibrational sum frequency generation spectroscopy readily show the occurrence of duplex formation. Our results suggest that the two-step azidification–click sequence is a viable strategy for readily functionalizing silica and glass surfaces with molecules spanning a wide range of chemical complexity, including biopolymers

Direct Probes of 4 nm Diameter Gold Nanoparticles Interacting with Supported Lipid Bilayers

This work presents molecular-level investigations of how well-characterized silica-supported phospholipid bilayers formed from either pure DOPC or a 9:1 mixture of DOPC:DOTAP interact with positively and negatively charged 4 nm gold metal nanoparticles at pH 7.4 and NaCl concentrations ranging from 0.001 to 0.1 M. Second harmonic generation (SHG) charge screening measurements indicate the supported bilayers carry a negative interfacial potential. Resonantly enhanced SHG measurements probing electronic transitions within the gold core of the nanoparticles show the particles interact irreversibly with the supported bilayers at a range of concentrations. At 0.1 M NaCl, surface coverages for the particles functionalized with the negatively charged ligand mercaptopropionic acid (MPA) or wrapped in the cationic polyelectrolyte poly­(allylamine) hydrochloride (PAH) are estimated from a joint analysis of QCM-D, XPS, AFM, and ToF-SIMS to be roughly 1 × 10⁷ and 1 × 10¹¹ particles cm^–2, respectively. Results from complementary SHG charge screening experiments point to the possibility that the surface coverage of the MPA-coated particles is more limited by interparticle Coulomb repulsion due to the charges within their hydrodynamic volumes than with the PAH-wrapped particles. Yet, SHG adsorption isotherms indicate that the interaction strength per particle is independent of ionic strength and particle coating, highlighting the importance of multivalent interactions. ¹H NMR spectra of the lipids within vesicles suspended in solution show little change upon interaction with either particle type but indicate loosening of the gold-bound PAH polymer wrapping upon attachment to the vesicles. The thermodynamic, spectroscopic, and electrostatic data presented here may serve to benchmark experimental and computational studies of nanoparticle attachment processes at the nano–bio interface

Lipid Corona Formation from Nanoparticle Interactions with Bilayers and Membrane-Specific Biological Outcomes

<a></a><a>While mixing nanoparticles with certain biological molecules can result in coronas that afford some control over how engineered nanomaterials interact with living systems, corona formation mechanisms remain enigmatic. Here, we report spontaneous lipid corona formation, i.e. without active mixing, upon attachment to stationary lipid bilayer model membranes and bacterial cell envelopes, and present ribosome-specific outcomes for multi-cellular organisms. Experiments show that polycation-wrapped particles disrupt the tails of zwitterionic lipids, increase bilayer fluidity, and leave the membrane with reduced ζ-potentials. Computer simulations show contact ion pairing between the lipid headgroups and the polycations’ ammonium groups leads to the formation of stable, albeit fragmented, lipid bilayer coronas, while microscopy shows fragmented bilayers around nanoparticles after interacting with <i>Shewanella oneidensis</i>. Our mechanistic insight can be used to improve control over nano-bio interactions and to help understand why some nanomaterial/ligand combinations are detrimental to organisms, like <i>Daphnia magna</i>, while others are not. </a