The World Health Organization has declared the outbreak of a novel coronavirus (SARS-CoV-2 or 2019-nCoV) as a global pandemic. However, the mechanisms behind the coronavirus infection are not yet fully understood, nor are there any targeted treatments or vaccines. In this study, we identified high-binding-affinity aptamers targeting SARS-CoV-2 RBD, using an ACE2 competition-based aptamer selection strategy and a machine learning screening algorithm. The K d values of the optimized CoV2-RBD-1C and CoV2-RBD-4C aptamers against RBD were 5.8 nM and 19.9 nM, respectively. Simulated interaction modeling, along with competitive experiments, suggests that two aptamers may have partially identical binding sites at ACE2 on SARS-CoV-2 RBD. These aptamers present an opportunity for generating new probes for recognition of SARS-CoV-2 and could provide assistance in the diagnosis and treatment of SARS-CoV-2 while providing a new tool for in-depth study of the mechanisms behind the coronavirus infection.

The past decade has witnessed ongoing progress in precision medicine to improve human health. As an emerging diagnostic technique, liquid biopsy can provide realtime, comprehensive, dynamic physiological and pathological information in a noninvasive manner, opening a new window for precision medicine. Liquid biopsy depends on the sensitive and reliable detection of circulating targets (e.g., cells, extracellular vesicles, proteins, microRNAs) from body fluids, the performance of which is largely governed by recognition ligands. Aptamers are single-stranded functional oligonucleotides, capable of folding into unique tertiary structures to bind to their targets with superior specificity and affinity. Their mature evolution procedure, facile modification, and affinity regulation, as well as versatile structural design and engineering, make aptamers ideal recognition ligands for liquid biopsy. In this review, we present a broad overview of aptamer-based liquid biopsy techniques for precision medicine. We begin with recent advances in aptamer selection, followed by a summary of state-of-the-art strategies for multivalent aptamer assembly and aptamer interface modification. We will further describe aptamer-based micro-/nanoisolation platforms, aptamer-enabled release methods, and aptamer-assisted signal amplification and detection strategies. Finally, we present our perspectives regarding the opportunities and challenges of aptamer-based liquid biopsy for precision medicine.

Tumor-derived exosomal proteins have emerged as promising biomarkers for cancer diagnosis, but the quantitation accuracy is hindered by large numbers of normal cellderived exosomes. Herein, we developed a dual-target-specific aptamer recognition activated in situ connection system on exosome membrane combined with droplet digital PCR (ddPCR) (TRACER) for quantitation of tumor-derived exosomal PD-L1 (Exo-PD-L1). Leveraging the high binding affinity of aptamers, excellent selectivity of dual-aptamer recognition, and the high sensitivity of ddPCR, this method exhibits significant sensitivity and selectivity for tracing tumor-derived Exo-PD-L1 in a wash-free manner. Due to the excellent sensitivity, the level of tumor-derived Exo-PD-L1 detected by TRACER can distinguish cancer patients from healthy donors, and for the first time was identified as a more reliable tumor diagnostic marker than total Exo-PD-L1. The TRACER strategy holds great potential for converting exosomes into reliable clinical indicators and exploring the biological functions of exosomes.

Microfluidic chips with nano-scale structures have shown great potential, but the fabrication and cost issues restrict their application. Herein, we propose a conceptually new "DNA nanolithography in a microfluidic chip" by using sub-10 nm three-dimensional DNA structures (TDNs) as frameworks with a pendant aptamer at the top vertex (ApTDN-Chip). The nano-scale framework ensures that the aptamer is in a highly ordered upright orientation, avoiding the undesired orientation or crowding effects caused by conventional microfluidic interface fabrication processes. Compared with a monovalent aptamer modified chip, the capture efficiency of ApTDN-Chip was enhanced nearly 60 % due to the highly precise dimension and rigid framework of TDNs. In addition, the scaffolds make DNase I more accessible to the aptamer with up to 83 % release efficiency and 91 % cell viability, which is fully compatible with downstream molecular analysis. Overall, this strategy provides a novel perspective on engineering nano-scaffolds to achieve a more ordered nanotopography of microfluidic chips.

Transport of PEGylated silica nanoparticles (PSiNPs) with diameters of 100, 50, and 25 nm across the blood-brain barrier (BBB) was evaluated using an in vitro BBB model based on mouse cerebral endothelial cells (bEnd.3) cultured on transwell inserts within a chamber. In vivo animal experiments were further performed by noninvasive in vivo imaging and ex vivo optical imaging after injection via carotid artery. Confocal fluorescence studies were carried out to evaluate the uptake of PSiNPs by brain endothelial cells. The results showed that PSiNPs can traverse the BBB in vitro and in vivo. The transport efficiency of PSiNPs across BBB was found to be size-dependent, with increased particle size resulting in decreased efficiency. This work points to the potential application of small sized silica nanoparticles in brain imaging or drug delivery.

Due to its large enhancement effect, nanostructure-based surface-enhanced Raman scattering (SERS) technology had been widely applied for bioanalysis and cell imaging. However, most SERS nanostructures suffer from poor signal reproducibility, which hinders the application of SERS nanostructures in quantitative detection. We report an etching-assisted approach to synthesize SERS-active plasmonic nanoparticles with 1 nm interior nanogap for multiplex quantitative detection and cancer cell imaging. Raman dyes and methoxy poly(ethylene glycol) thiol (mPEG-SH) were attached to gold nanoparticles (AuNPs) to prepare gold cores. Next, Ag atoms were deposited on gold cores in the presence of Pluronic F127 to form a Ag shell. HAuCl4 was used to etch the Ag shell and form an interior nanogap in Au@AgAuNPs, leading to increased Raman intensity of dyes. SERS intensity distribution of Au@AgAuNPs was found to be more uniform than that of aggregated AuNPs. Finally, Au@AgAuNPs were used for multiplex quantitative detection and cancer cell imaging. With the advantages of simple and rapid preparation of Au@AgAuNPs with highly uniform, stable, and reproducible Raman intensity, the method reported here will widen the applications of SERS-active nanoparticles in diagnostics and imaging.

Exosomal glycoproteins play important roles in many physiological and pathological functions.H erein, we developed ad ual labeling strategy based on ap rotein-specific aptamer tagging and metabolic glycan labeling for visualizing glycosylation of specific proteins on exosomes.T he glycosylation of exosomal PD-L1 (exoPD-L1) was imaged in situ using intramolecular fluorescence resonance energy transfer (FRET) between fluorescent PD-L1 aptamers bound on exoPD-L1 and fluorescent tags on glycans introduced via metabolic glycan labeling.T his method enables in situ visualization and biological function study of exosomal protein glycosylation. Exosomal PD-L1 glycosylation was confirmed to be required in interaction with PD-1 and participated in inhibiting of CD8 + Tcell proliferation. This is an efficient and non-destructive method to study the presence and function of exosomal protein-specific glycosylation in situ, which provides apowerful tool for exosomal glycoproteomics research.

Liquid biopsy offers non‐invasive and real‐time molecular profiling of individual patients, and is thus considered a revolutionary technology in precision medicine. Exosomes have been acknowledged as significant biomarkers in liquid biopsy, as they play a central role in cell–cell communication and are closely related to the pathogenesis of most human malignancies. Nevertheless, in biofluids exosomes always co‐exist with other particles, and the cargo components of exosomes are highly heterogeneous. Thus, the isolation and molecular characterization of exosomes are still technically challenging. Microfluidics technology effectively addresses this challenge by virtue of its inherent advantages, such as precise manipulation of fluids, low consumption of samples and reagents, and a high level of integration. Recent advances in microfluidics allow in situ exosome capture and molecular detection with unprecedented selectivity and sensitivity. In this review, the state‐of‐the‐art developments in microfluidics‐based exosome research, including exosome isolation approaches and molecular detection strategies, with highlights of the characterization of exosomal biomarkers in cancer liquid biopsy is summarized. The major challenges are also discussed and some perspectives for the future directions of exosome‐based liquid biopsy in microfluidic systems are presented.