Letter to the Editor Concerning Simultaneous, Single-Particle Measurements of Size and Loading Give Insights into the Structure of Drug-Delivery Nanoparticles

The vexing error of excess variance in the sizing of single particles degrades accuracy in applications ranging from quality control of nanoparticle products to hazard assessment of nanoplastic byproducts. The particular importance of lipid nanoparticles for vaccine and medicine delivery motivates this comment on a publication$^{\textrm{1}}$ in ACS Nano. In ref 1, the benchmark measurements of a nanoparticle standard manifest large errors of the size distribution that contradict the claim of validation. Such errors can bias the correlation of fluorescence intensity as an optical proxy for the molecular loading of lipid nanoparticles and give misleading insights from power-law models of intensity$-$size data. Looking forward, measurement error models have the potential to address this widespread issue.Comment: Peer reviewed and pending acceptance by ACS Nan

Subnanometer traceability of localization microscopy

In localization microscopy, subnanometer precision is possible but supporting accuracy is challenging, and no study has demonstrated reliable traceability to the International System of Units (SI). To do so, we measure the positions of nanoscale apertures in a reference array by traceable atomic-force microscopy, creating a master standard. We perform correlative measurements of this standard by optical microscopy, correcting position errors from optical aberrations by a Zernike calibration. We establish an uncertainty field due to localization errors and scale uncertainty, with regions of position traceability to within a 68 % coverage interval of +/- 1.0 nm. These results enable localization metrology with high throughput, which we apply to measure working standards, validating the subnanometer accuracy of lithographic pitch

A lateral nanoflow assay reveals nanoplastic fluorescence heterogeneity

Colloidal nanoplastics present technological opportunities, environmental concerns, and measurement challenges. To meet these challenges, we develop a lateral nanoflow assay from sample-in to answer-out. Our measurement system integrates complex nanofluidic replicas, super-resolution optical microscopy, and comprehensive statistical analyses to measure polystyrene nanoparticles that sorb and carry hydrophobic fluorophores. An elegant scaling of surface forces within our silicone devices hydrodynamically automates the advection and dominates the diffusion of the nanoparticles. Through steric interaction with the replica structure, the particle size distribution reciprocally probes the unknown limits of replica function. Multiple innovations in the integration and calibration of device and microscope improve the accuracy of identifying single nanoparticles and quantifying their diameters and fluorescence intensities. A statistical model of the measurement approaches the information limit of the system, discriminates size exclusion from surface adsorption, and reduces nonideal data to return the particle size distribution with nanometer resolution. A Bayesian statistical analysis of the dimensional and optical properties of single nanoparticles reveals their fundamental structure-property relationship. Fluorescence intensity shows a super-volumetric dependence, scaling with nanoparticle diameter to nearly the fourth power and confounding basic concepts of chemical sorption. Distributions of fluorescivity - the product of the number density, absorption cross section, and quantum yield of an ensemble of fluorophores - are ultrabroad and asymmetric, limiting ensemble analysis and dimensional or chemical inference from fluorescence intensity. These results reset expectations for optimizing nanoplastic products, understanding nanoplastic byproducts, and applying nanoplastic standards

The Nanolithography Toolbox

This article introduces in archival form the Nanolithography Toolbox, a platform-independent software package for scripted lithography pattern layout generation. The Center for Nanoscale Science and Technology (CNST) at the National Institute of Standards and Technology (NIST) developed the Nanolithography Toolbox to help users of the CNST NanoFab design devices with complex curves and aggressive critical dimensions. Using parameterized shapes as building blocks, the Nanolithography Toolbox allows users to rapidly design and layout nanoscale devices of arbitrary complexity through scripting and programming. The Toolbox offers many parameterized shapes, including structure libraries for micro- and nanoelectromechanical systems (MEMS and NEMS) and nanophotonic devices. Furthermore, the Toolbox allows users to precisely define the number of vertices for each shape or create vectorized shapes using Bezier curves. Parameterized control allows users to design smooth curves with complex shapes. The Toolbox is applicable to a broad range of design tasks in the fabrication of microscale and nanoscale devices

A localized transition in the size variation of circular DNA in nanofluidic slitlike confinement

We report strong evidence for a localized transition in the size variation of circular DNA between strong and moderate regimes of slitlike confinement. A novel and rigorous statistical analysis was applied to our recent experimental measurements of DNA size for linear and circular topologies in nanofluidic slits with depths around ≈ 2p, where p is the persistence length. This empirical approach revealed a localized transition between confinement regimes for circular DNA at a slit depth of ≈ 3p but neither detected nor ruled out the possibility for such a transition for linear DNA. These unexpected results provide the first indication of the localized influence of polymer topology on size variation in slitlike confinement. Improved understanding of differences in polymer behavior related to topology in this controversial and relevant system is of fundamental importance in polymer science and will inform nanofluidic methods for biopolymer analysis

Single molecule analysis of bacterial polymerase chain reaction products in submicrometer fluidic channels

Laser induced fluorescence in submicrometer fluidic channels was used to characterize the synthesis of polymerase chain reaction (PCR) products from a model bacterial system in order to explore the advantages and limitations of on chip real time single molecule PCR analysis. Single oligonucleotide universal bacterial primers and PCR amplicons from the 16S rDNA of Thermobifida fusca (325 bp) were directly detected at all phases of the reaction with low sample consumption and without post-amplification purification or size screening. Primers were fluorescently labeled with single Alexa Fluor 488 or Alexa Fluor 594 fluorophores, resulting in double labeled, two color amplicons. PCR products were driven electrokinetically through a fused silica channel with a 250 nm by 500 nm rectangular cross section. Lasers with 488 nm and 568 nm wavelengths were focused and overlapped on the channel for fluorescence excitation. All molecules entering the channel were rapidly and uniformly analyzed. Photon burst analysis was used to detect and identify individual primers and amplicons, and fluorescence correlation and cross-correlation spectroscopy were used to account for analyte flow speed. Conventional gel and capillary electrophoresis were also used to characterize the PCR amplification, and the results of differences in detection sensitivity and analyte discrimination were examined. Limits were imposed by the purity and labeling efficiency of the PCR reagents, which must be improved in parallel with increases in detection sensitivity

Conformation, Length, and Speed Measurements of Electrodynamically Stretched DNA in Nanochannels

A method is presented to rapidly and precisely measure the conformation, length, speed, and fluorescence intensity of single DNA molecules constrained by a nanochannel. DNA molecules were driven electrophoretically from a nanoslit into a nanochannel to confine and dynamically elongate them beyond their equilibrium length for repeated detection via laser-induced fluorescence spectroscopy. A single-molecule analysis algorithm was developed to analytically model bursts of fluorescence and determine the folding conformation of each stretched molecule. This technique achieved a molecular length resolution of 114 nm and an analysis time of around 20 ms per molecule, which enabled the sensitive investigation of several aspects of the physical behavior of DNA in a nanochannel. λ-bacteriophage DNA was used to study the dependence of stretching on the applied device bias, the effect of conformation on speed, and the amount of DNA fragmentation in the device. A mixture of λ-bacteriophage with the fragments of its own HindIII digest, a standard DNA ladder, was sized by length as well as by fluorescence intensity, which also allowed the characterization of DNA speed in a nanochannel as a function of length over two and a half orders of magnitude