the critical need for rapid and sensitive molecular diagnostics to combat current and future pandemics. Sensitive polymerase chain reaction (PCR) based tests are the gold standard for molecular diagnostics but rely on bulky and expensive instruments. Thus, they are not suitable for self-diagnosis or point-of-care (POC) settings. [2] High-throughput sequencing can decipher the entire genomic landscape of the pathogens but is time consuming and requires bioinformatics for data interpretation. [3] Immunoassays, such as rapid antigen tests, are simple and rapid diagnostic methods but normally lack the sensitivity to reporting the low concentration biomarkers. [4] Assays with high limits of detection and low accuracy have been acceptable out of necessity, but there are many scenarios where a rapid, sensitive, POC device would be beneficial. Therefore, developing a simple to use, portable, and sensitive diagnostic platform is one important key to addressing the current challenges for molecular diagnostics.Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are bacterial systems evolved to combat bacteriophage infections by recognizing specific nucleic acid sequences to activate nucleolytic cleavage activities. The trans-cleavage of A gold nanoparticle (AuNP)-labeled CRISPR-Cas13a nucleic acid assay is developed for sensitive solid-state nanopore sensing. Instead of directly detecting the translocation of RNA through a nanopore, the system utilizes non-covalent conjugates of AuNPs and RNA targets. Upon CRISPR activation, the AuNPs are liberated from the RNA, isolated, and passed through a nanopore sensor. Detection of the AuNPs can be observed as increasing ionic current in the chip. Each AuNP that is detected is enumerated as an event, leading to quantitative of molecular targets. Leveraging the high signal-to-noise ratio enabled by the AuNPs, a detection limit of 50 fM before front-end target amplification is achieved using SARS-CoV-2 RNA segments as a Cas13 target. Furthermore, a dynamic range of six orders of magnitude is demonstrated for quantitative RNA sensing. This simplified AuNP-based CRISPR assay is performed at the physiological temperature without relying on thermal cyclers. In addition, the nanopore reader is similar in size to a smartphone, making the assay system suitable for rapid and portable nucleic acid biomarker detection in either low-resource settings or hospitals.

Scanning Electron Microscope/Focused Ion Beam (SEM/FIB) system has become a valuable and popular tool for the analysis of biological materials such as dentine structures. According to physiological and anatomical studies, dentine structures are a complicated system containing collagen fibers, nanocrystalline hydroxyapatite, and numerous networks of tubular pores. During a routine FIB milling process, collagen fibers and other organic structures are vaporized, while the number of tubular pores remaining is increased. This causes the final cross-section to be more porous than the real sample. Unfortunately, little attention has been paid to the collagen fiber loss and how to preserve them during a FIB milling process. In this work, we present a novel and simple approach to preserve the organic portions of the dentine structure through metal staining. By using this method, the porosity of the dentine structure after the FIB milling process is significantly reduced similar to the real sample. This indicates that the organic portion of the dentine structure is well protected by the metal staining. This approach enables the SEM/FIB system to generate super-high quality SEM images with less ion beam damage; and the SEM images can better reflect the original condition of the dentine structure. Further, serial energy-dispersive X-ray spectroscopy (EDS) mapping of the stained dentine structure is achieved without an additional metal coating; and three-dimensional (3-D) elemental mapping of an occluded dentine is achieved with a significantly reduced data acquisition time.

The occlusion of dentinal tubules has become a rapid and effective method for treating dentin hypersensitivity. Accurate evaluation of dentin occlusion is critical to illustrate the efficacy of oral care products and to optimize dental therapy in the clinics, which is limited by the conventional two-dimensional (2-D) characterization methods. Here, we demonstrate the visualization of the dentin occlusion via three-dimensional (3-D) characterization using a focused ion beam-scanning electron microscopy (FIB-SEM) tomography. Using the “Slice and View” approach, the material used for occluding dentin tubules is imaged with a very high-resolution voxel (10 nm × 10 nm × 20 nm) from 2-D SEM images and then reconstructed into a 3-D volume, which presents the mode of action of toothpaste for treating dentin hypersensitivity. Meanwhile, quantitative analysis of the depth of occlusion is successfully obtained. This work validates the feasibility of FIB-SEM tomography in the analysis of dentin occlusion within the complicated networks of dentine tubules at the nanoscale, and provides a novel approach to facilitate the research and development of oral care products.

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