Circulating cancer-derived small extracellular vesicles (EVs) are nanoscale membranous vesicles shed from cancer cells that are released into surrounding body fluids. Small EVs contain biomolecules associated with cancer such as DNA and proteins for cell-to-cell communication. Therefore, small EVs have been regarded as important cancer biomarkers for liquid biopsy-based cancer diagnosis and drug treatment monitoring. However, because of the high heterogeneity and low level of small EVs in body fluids, there is a high demand for sensitive detection and characterization of such vesicles at a molecular level. In this study, we have developed a sensitive and effective approach to simultaneously profile multiple protein biomarkers expressed on cancerderived small EVs using surface-enhanced Raman spectroscopy (SERS) nanotags in a single test, without complex isolation steps. Rapid and multiplexed phenotypic profiling of small EVs is achieved by mixing specific detection antibody-coated SERS nanotags, filtered conditioned EV-suspended medium (conditioned EVs), and capture antibody (CD63)-conjugated magnetic beads to form a sandwich immunoassay. As a proof-ofconcept demonstration, we applied this approach to characterize pancreatic cancer-derived EVs by simultaneously detecting three specific EV surface receptors including Glypican-1, epithelial cell adhesion molecules (EpCAMs), and CD44 variant isoform 6 (CD44V6). The sensitivity of this method was measured down to 2.3 × 10 6 particles/mL, which is more sensitive and shows higher multiplexing capability than most other reported EV profiling techniques, such as western blot, enzyme-linked immunosorbent assay, and flow cytometry. Furthermore, phenotypic profiling of small EVs from colorectal cancer and bladder cancer cell lines (SW480 and C3) was conducted and compared to those derived from pancreatic cancer (Panc-1), highlighting the significant difference in EV phenotypes for various cancer cell types suspended in both phosphate-buffered saline and plasma. Thus, we believe that this technology enables a comprehensive evaluation of small secreted EV heterogeneity with high sensitivity, offering strong potential for accurate noninvasive cancer diagnosis and monitoring of drug treatment. In addition, this assay provides point-of-care use because of the easy sample preparation and portable nature of the Raman spectrometer.

No abstract

The sensitive and simultaneous detection of cytokines will provide new insights into the physiological process and disease pathways due to the complex nature of cytokine networks. However, the key challenge is the lack of probes that can simultaneously detect multiple cytokines in a single sample. In this contribution, we proposed an alternative approach for sensitive cytokine detection in a multiplex manner by the use of a new set of surface-enhanced Raman spectroscopy (SERS) nanotags. Typically, the newly designed SERS nanotags are composed of gold nanoparticles as the core, tuneable Raman molecules as the reporters, and a thin silver layer as the shell. As demonstrated through rigorous numerical simulations, enhanced Raman signal is achieved due to a strong localization of light in the 0.2 nm thin, optically deep-subwavelength region between the Au core and the Ag shell. Sensitive detection of cytokines is realized by forming a sandwich immunoassay. The detection limit is down to 4.5 pg mL −1 (S/N = 3). The specificity of the assay is proved as negligible signals were detected for the false targets. Furthermore, multiple cytokines are simultaneously detected in a single assay from the secretion of Blymphocyte cell line (Raji) after concanavalin A (Con A) stimulation. The results indicate that our method holds a significant potential for sensitive and multiplexed detection of cytokines and offers the opportunity for future applications in clinical settings.

Although droplet-based microfluidics has been broadly used as a versatile tool in biology, chemistry, and nanotechnology, its rather complicated microfabrication process and the requirement of specialized hardware and operating skills hinder researchers fully unleashing the potential of this powerful platform. Here, we develop an integrated microdroplet generator enabled by a spinning conical frustum for the versatile production of near-monodisperse microdroplets in a highthroughput and off-chip manner. The construction and operation of this generator are simple and straightforward without the need of microfabrication, and we demonstrate that the generator is able to passively and actively control the size of the produced microdroplets. In addition to water microdroplets, this generator can produce microdroplets of liquid metal that would be difficult to produce in conventional microfluidic platforms as liquid metal has high surface tension. Moreover, we demonstrate that this generator can produce solid hydrogel microparticles and fibers using integrated ultraviolet (UV) light. In the end, we further explore the ability of this generator for forming double emulsions by coflowing two immiscible liquids. Given the remarkable abilities demonstrated by this platform and the tremendous potential of microdroplets, this user-friendly method may revolutionize the future of dropletbased chemical synthesis and biological analysis.

We present the MilliDrop Analyzer (MDA), a droplet-based millifluidic system for digital antimicrobial susceptibility testing (D-AST), which enables us to determine minimum inhibitory concentrations (MICs) precisely and accurately. The MilliDrop technology was validated by using resazurin for fluorescence readout, for comparison with standard methodology, and for conducting reproducibility studies. In this first assessment, the susceptibility of a reference Gram-negative strain Escherichia coli ATCC 25922 to gentamicin, chloramphenicol, and nalidixic acid were tested by the MDA, VITEK®2, and broth microdilution as a reference standard. We measured the susceptibility of clinically relevant Gram-positive strains of Staphylococcus aureus to vancomycin, including vancomycin-intermediate S. aureus (VISA), heterogeneous vancomycin-intermediate S. aureus (hVISA), and vancomycin-susceptible S. aureus (VSSA) strains. The MDA provided results which were much more accurate than those of VITEK®2 and standard broth microdilution. The enhanced accuracy enabled us to reliably discriminate between VSSA and hVISA strains.