Live Organoid Cyclic Imaging.

Organoids are becoming increasingly relevant in biology and medicine for their physiological complexity and accuracy in modeling human disease. To fully assess their biological profile while preserving their spatial information, spatiotemporal imaging tools are warranted. While previously developed imaging techniques, such as four-dimensional (4D) live imaging and light-sheet imaging have yielded important clinical insights, these technologies lack the combination of cyclic and multiplexed analysis. To address these challenges, bioorthogonal click chemistry is applied to display the first demonstration of multiplexed cyclic imaging of live and fixed patient-derived glioblastoma tumor organoids. This technology exploits bioorthogonal click chemistry to quench fluorescent signals from the surface and intracellular of labeled cells across multiple cycles, allowing for more accurate and efficient molecular profiling of their complex phenotypes. Herein, the versatility of this technology is demonstrated for the screening of glioblastoma markers in patient-derived human glioblastoma organoids while conserving their viability. It is anticipated that the findings and applications of this work can be broadly translated into investigating physiological developments in other organoid systems.

Double Digital Assay for Single Extracellular Vesicle and Single Molecule Detection.

Extracellular vesicles (EVs) have emerged as a promising source of biomarkers for disease diagnosis. However, current diagnostic methods for EVs present formidable challenges, given the low expression levels of biomarkers carried by EV samples, as well as their complex physical and biological properties. Herein, a highly sensitive double digital assay is developed that allows for the absolute quantification of individual molecules from a single EV. Because the relative abundance of proteins is low for a single EV, tyramide signal amplification (TSA) is integrated to increase the fluorescent signal readout for evaluation. With the integrative microfluidic technology, the technology's ability to compartmentalize single EVs is successfully demonstrated, proving the technology's digital partitioning capacity. Then the device is applied to detect single PD-L1 proteins from single EVs derived from a melanoma cell line and it is discovered that there are ≈2.7 molecules expressed per EV, demonstrating the applicability of the system for profiling important prognostic and diagnostic cancer biomarkers for therapy response, metastatic status, and tumor progression. The ability to accurately quantify protein molecules of rare abundance from individual EVs will shed light on the understanding of EV heterogeneity and discovery of EV subtypes as new biomarkers.

Gold Nanoparticles Mineralized by Peptide Liquid Crystals with Dual-Functional Enzyme-like Activities: An Automatic Membrane Reactor for Glucose Detection.

The development of stable multifunctional enzyme mimics with tandem catalytic effects provides a great opportunity to construct economical and convenient bioassays. Inspired by biomineralization, in this work self-assembled N-(9-fluorenylmethoxycarbonyl)-protected tripeptide (Fmoc-FWK-NH2) liquid crystals were used as templates to in situ mineralize Au nanoparticles (AuNPs), and then a dual-functional enzyme-mimicking membrane reactor based on AuNPs and peptide-based hybrids was constructed. AuNPs with a uniform particle size and good dispersion were in situ reduced on the surface of the peptide liquid crystal due to the reduction of the indole group on the tryptophan residue, which exhibited excellent peroxidase-like and glucose oxidase-like activities simultaneously. Meanwhile, the oriented nanofibers aggregated into a three-dimensional network, which was further immobilized on the mixed cellulose membrane to form a membrane reactor. A biosensor was made to realize fast, low-cost, and automatic detection for glucose. This work represents a promising platform for the design and construction of novel multifunctional materials based on the biomineralization strategy.