A variety of rapid biomolecular assays under development rely on the selective adsorption of single-stranded DNA onto unfunctionalized, negatively charged, citrate-stabilized gold nanoparticles. We investigate the adsorption mechanism with a study of the binding kinetics and find strong evidence for the dominance of hydrophobic effects including linear compensation between the activation energy and the natural log of the Arrhenius prefactor and the correlation of the adsorption rate in the presence of various salts with the Hofmeister series. These results explain the selectivity for single-stranded over double-stranded DNA adsorption and contradict previous work citing an electrostatic DLVO-like mechanism. Our understanding should facilitate improvements to the selective-adsorption-based assays and, more generally, contribute to the understanding of interactions between like-charged species in aqueous solution.

A nanopore is the ultimate analytical tool. It can be used to detect DNA, RNA, oligonucleotides, and proteins with submolecular sensitivity. This extreme sensitivity is derived from the electric signal associated with the occlusion that develops during the translocation of the analyte across a membrane through a pore immersed in electrolyte. A larger occluded volume results in an improvement in the signal-to-noise ratio, and so the pore geometry should be made comparable to the size of the target molecule. However, the pore geometry also affects the electric field, the charge density, the electro-osmotic flow, the capture volume, and the response time. Seeking an optimal pore geometry, we tracked the molecular motion in three dimensions with high resolution, visualizing with confocal microscopy the fluorescence associated with DNA translocating through nanopores with diameters comparable to the double helix, while simultaneously measuring the pore current. Measurements reveal single molecules translocating across the membrane through the pore commensurate with the observation of a current blockade. To explain the motion of the molecule near the pore, finite-element simulations were employed that account for diffusion, electrophoresis, and the electro-osmotic flow. According to this analysis, detection using a nanopore comparable in diameter to the double helix represents a compromise between sensitivity, capture volume, the minimum detectable concentration, and response time.

No abstract

We report direct, concurrent measurements of the forces and currents associated with the translocation of a single-stranded DNA molecule tethered to the tip of an atomic force microscope (AFM) cantilever through synthetic pores with topagraphies comparable to the DNA. These measurements were performed to gauge the signal available for sequencing and the electric force required to impel a single molecule through synthetic nanopores ranging from 1.0 to 3.5 nm in diameter in silicon nitride membranes 6-10 nm thick. The measurements revealed that a molecule can slide relatively frictionlessly through a pore, but regular fluctuations are observed intermittently in the force (and the current) every 0.35-0.72 nm, which are attributed to individual nucleotides translating through the nanopore in a turnstile-like motion.

It is now possible to visualize at nanometer resolution the infection of a living biological cell with virus without compromising cell viability using scanning transmission electron microscopy (STEM). To provide contrast while preserving viability, Escherichia coli and P1 bacteriophages were first positively stained with a very low concentration of uranyl acetate in minimal phosphate medium and then imaged with low-dose STEM in a microfluidic liquid flow cell. Under these conditions, it was established that the median lethal dose of electrons required to kill half the tested population was LD50 = 30 e(-)/nm(2), which coincides with the disruption of a wet biological membrane, according to prior reports. Consistent with the lateral resolution and high-contrast signal-to-noise ratio (SNR) inferred from Monte Carlo simulations, images of the E. coli membrane, flagella, and the bacteriophages were acquired with 5 nm resolution, but the cumulative dose exceeded LD50. On the other hand, with a cumulative dose below LD50 (and lower SNR), it was still possible to visualize the infection of E. coli by P1, showing the insertion of viral DNA within 3 s, with 5 nm resolution.