Detection of aldehydes from degradation of lipid nanoparticle formulations using a hierarchically-organized nanopore electrochemical biosensor.

Degradation of ionizable lipids in mRNA-based vaccines was recently found to deactivate the payload, demanding rigorous monitoring of impurities in lipid nanoparticle (LNP) formulations. However, parallel screening for lipid degradation in customized delivery systems for next-generation therapeutics maintains a challenging and unsolved problem. Here, we describe a nanopore electrochemical sensor to detect ppb-levels of aldehydes arising from lipid degradation in LNP formulations that can be deployed in massively parallel fashion. Specifically, we combine nanopore electrodes with a block copolymer (BCP) membrane capable of hydrophobic gating of analyte transport between the bulk solution and the nanopore volume. By incorporating aldehyde dehydrogenase (ALDH), enzymatic oxidation of aldehydes generates NADH to enable ultrasensitive voltammetric detection with limits-of-detection (LOD) down to 1.2 ppb. Sensor utility was demonstrated by detecting degradation of N-oxidized SM-102, the ionizable lipid in Moderna's SpikeVax™ vaccine, in mRNA-1273 LNP formulation. This work should be of significant use in the pharmaceutical industry, paving the way for automated on-line quality assessments of next-generation therapeutics.

Nanopore-Enabled Dark-Field Digital Sensing of Nanoparticles.

In this study, we use nanopore arrays as a platform for detecting and characterizing individual nanoparticles (NPs) in real time. Dark-field imaging of nanopores with dimensions smaller than the wavelength of light occurs under conditions where trans-illumination is blocked, while the scattered light propagates to the far-field, making it possible to identify nanopores. The intensity of scattering increases dramatically during insertion of AgNPs into empty nanopores, owing to their plasmonic properties. Thus, momentary occupation of a nanopore by a AgNP produces intensity transients that can be analyzed to reveal the following characteristics: (1) NP scattering intensity, which scales with the sixth power of the AgNP radius, shows a normal distribution arising from the heterogeneity in NP size, (2) the nanopore residence time of NPs, which was observed to be stochastic with no permselective effects, and (3) the frequency of AgNP capture events on a 21 × 21 nanopore array, which varies linearly with the concentration of the NPs, agreeing with the frequency calculated from theory. The lower limit of detection (LOD) for NPs was 130 fM, indicating that the measurement can be used in applications in which ultrasensitive detection is required. The results presented here provide valuable insights into the dynamics of NP transport into and out of nanopores and highlight the potential of nanopore arrays as powerful, massively parallel tools for nanoparticle characterization and detection.

Potential Dependent Spectroelectrochemistry of Electrofluorogenic Dyes on Indium-Tin Oxide.

Indium-tin oxide (ITO) is used in a variety of applications due to its electrical conductivity and optical transparency. Moreover, ITO coated glass is a common working electrode for spectroelectrochemistry. Thus, the ITO substrates should exhibit well-understood spectroscopic characteristics. Here, we report anomalous potential-dependent luminescence emission from three structurally-dissimilar electrofluorogenic probe on ITO coated glass. The three probes, flavin mononucleotide, resorufin, and Nile blue, show the expected fluorescence modulation between their oxidized, emissive forms and their reduced, non-fluorescent forms at low laser irradiance and/or high concentrations. However, at high irradiance and/or low concentration, the emission intensity increases at reducing potentials, contrary to expectations. In addition, a strong interplay between probe molecule concentration and laser irradiance is observed. We attribute the anomalous behavior to a combination of (1) irradiance-dependent ITO carrier dynamics, and (2) interaction of the fluorescent probe with ITO at reducing potentials resulting in a charge transfer state with altered emission behavior. Thus, the potential- and irradiance-dependent behavior of ITO and the resulting charge transfer state may not only interfere with the observation of potential-dependent fluorescence from redox probes but can completely reverse the polarity of the potential-dependent luminescence, especially at high irradiance and low concentration.