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.
Tumor‐derived exosomal proteins have emerged as promising biomarkers for cancer diagnosis, but the quantitation accuracy is hindered by large numbers of normal cell‐derived 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.
We developed a dual‐target‐specific aptamer recognition system combined with droplet digital PCR for precise quantitative analysis of exosomal PD‐L1 (Exo‐PD‐L1). This method can distinguish tumor‐derived from non‐tumor‐derived Exo‐PD‐L1, holding great potential for the analysis of exosome subtypes and offering unprecedented opportunities for the study of the biological functions of exosomes and their conversion into reliable clinical indicators.
Circulating tumor cell (CTC)‐enrichment by using aptamers has a number of advantages, but the issue of compromised binding affinities and stabilities in real samples hinders its wide applications. ...Inspired by the high efficiency of the prey mechanism of the octopus, we engineered a deterministic lateral displacement (DLD)‐patterned microfluidic chip modified with multivalent aptamer‐functionalized nanospheres (AP‐Octopus‐Chip) to enhance capture efficiency. The multivalent aptamer–antigen binding efficiency improves 100‐fold and the capture efficiency is enhanced more than 300 % compared with a monovalent aptamer‐modified chip. Moreover, the captured cancer cells can be released through a thiol exchange reaction with up to 80 % efficiency and 96 % viability, which is fully compatible with downstream mutation detection and CTC culture. Using the chip, we were able to find CTCs in all cancer samples analyzed.
Octopus chip cell capture: Inspired by the high efficiency of the prey mechanism of the octopus, an aptamer‐tailed octopus chip (AP‐Octopus‐Chip) for CTC enrichment was developed. The design of the chip and the high binding affinity of the multivalent structures against the target cells significantly improved the CTC capture efficiency and enrichment. The enriched cancer cells can be released through a thiol exchange reaction, which is fully compatible with downstream mutation detection and CTC culture.
Immunotherapy has revolutionized cancer treatment, but its efficacy is severely hindered by the lack of effective predictors. Herein, we developed a homogeneous, low‐volume, efficient, and sensitive ...exosomal programmed death‐ligand 1 (PD‐L1, a type of transmembrane protein) quantitation method for cancer diagnosis and immunotherapy response prediction (HOLMES‐ExoPD‐L1). The method combines a newly evolved aptamer that efficiently binds to PD‐L1 with less hindrance by antigen glycosylation than antibody, and homogeneous thermophoresis with a rapid binding kinetic. As a result, HOLMES‐ExoPD‐L1 is higher in sensitivity, more rapid in reaction time, and easier to operate than existing enzyme‐linked immunosorbent assay (ELISA)‐based methods. As a consequence of an outstanding improvement of sensitivity, the level of circulating exosomal PD‐L1 detected by HOLMES‐ExoPD‐L1 can effectively distinguish cancer patients from healthy volunteers, and for the first time was found to correlate positively with the metastasis of adenocarcinoma. Overall, HOLMES‐ExoPD‐L1 brings a fresh approach to exosomal PD‐L1 quantitation, offering unprecedented potential for early cancer diagnosis and immunotherapy response prediction.
An aptamer‐induced thermophoresis quantitation of exosomal programmed death‐ligand 1 (PD‐L1, a transmembrane protein) was developed, which integrates effective recognition of aptamer and homogeneous thermophoresis. The facile technique is more sensitive and efficient than the current enzyme‐linked immunosorbent assay (ELISA)‐based methods. Translation of the method into standard clinical practice for immunotherapy prediction and monitoring is anticipated.
The COVID‐19 pandemic caused by SARS‐CoV‐2 is threating global health. Inhibiting interaction of the receptor‐binding domain of SARS‐CoV‐2 S protein (SRBD) and human ACE2 receptor is a promising ...treatment strategy. However, SARS‐CoV‐2 neutralizing antibodies are compromised by their risk of antibody‐dependent enhancement (ADE) and unfavorably large size for intranasal delivery. To avoid these limitations, we demonstrated an aptamer blocking strategy by engineering aptamers’ binding to the region on SRBD that directly mediates ACE2 receptor engagement, leading to block SARS‐CoV‐2 infection. With aptamer selection against SRBD and molecular docking, aptamer CoV2‐6 was identified and applied to prevent, compete with, and substitute ACE2 from binding to SRBD. CoV2‐6 was further shortened and engineered as a circular bivalent aptamer CoV2‐6C3 (cb‐CoV2‐6C3) to improve the stability, affinity, and inhibition efficacy. cb‐CoV2‐6C3 is stable in serum for more than 12 h and can be stored at room temperature for more than 14 days. Furthermore, cb‐CoV2‐6C3 binds to SRBD with high affinity (Kd=0.13 nM) and blocks authentic SARS‐CoV‐2 virus with an IC50 of 0.42 nM.
We propose an aptamer blocking strategy to inhibit SARS‐CoV‐2 infection. With the advantages of small size, rapid kinetics, high stability, sophisticated programmability and high security, our aptamers have great potential as prophylactic and therapeutic agents, which could greatly assist in the intervention of prevailing and emerging infectious diseases other than COVID‐19.
Equipment-free devices with quantitative readout are of great significance to point-of-care testing (POCT), which provides real-time readout to users and is especially important in low-resource ...settings. Among various equipment-free approaches, distance-based visual quantitative detection methods rely on reading the visual signal length for corresponding target concentrations, thus eliminating the need for sophisticated instruments. The distance-based methods are low-cost, user-friendly and can be integrated into portable analytical devices. Moreover, such methods enable quantitative detection of various targets by the naked eye. In this review, we first introduce the concept and history of distance-based visual quantitative detection methods. Then, we summarize the main methods for translation of molecular signals to distance-based readout and discuss different microfluidic platforms (glass, PDMS, paper and thread) in terms of applications in biomedical diagnostics, food safety monitoring, and environmental analysis. Finally, the potential and future perspectives are discussed.
Equipment-free devices with quantitative readout are of great significance to point-of-care testing (POCT), which provides real-time readout to users and is especially important in low-resource settings.
Herein, we propose a metabolic d‐amino acid‐based labeling and in situ hybridization‐facilitated (MeDabLISH) strategy for the quantitative analysis of the indigenous metabolic status of gut bacteria. ...The fluorescent d‐amino acid (FDAA)‐based labeling intensities of bacteria were found to highly correlate with their temporal and steady‐state metabolic status. Then, after taxonomic identification of bacterial genera in the in vivo FDAA‐labeled mouse gut microbiota, by corresponding fluorescence in situ hybridization (FISH) probes, the metabolic activities of different gut bacterial genera are quantified by flow cytometry, using FISH signals to differentiate genera and FDAA signals to indicate their basal metabolic levels. It was found that Gram‐negative genera in the mouse microbiota have stronger metabolic activities during the daytime, and Gram‐positive genera have higher activities at the night. Our strategy will be instrumental in deepening our understanding of the highly complex microbiota.
Gut sensing: The indigenous metabolic status of mouse gut microbiota is quantified by flow cytometry using the signal from fluorescent d‐amino acid‐based in vivo labeling as the indicator of bacterial metabolic activities, and signals from fluorescence in situ hybridization to indicate their taxonomic identification.
Microfluidic Single‐Cell Omics Analysis Xu, Xing; Wang, Junxia; Wu, Lingling ...
Small (Weinheim an der Bergstrasse, Germany),
03/2020, Letnik:
16, Številka:
9
Journal Article
Recenzirano
The commonly existing cellular heterogeneity plays a critical role in biological processes such as embryonic development, cell differentiation, and disease progress. Single‐cell omics‐based ...heterogeneous studies have great significance for identifying different cell populations, discovering new cell types, revealing informative cell features, and uncovering significant interrelationships between cells. Recently, microfluidics has evolved to be a powerful technology for single‐cell omics analysis due to its merits of throughput, sensitivity, and accuracy. Herein, the recent advances of microfluidic single‐cell omics analysis, including different microfluidic platform designs, lysis strategies, and omics analysis techniques, are reviewed. Representative applications of microfluidic single‐cell omics analysis in complex biological studies are then summarized. Finally, a few perspectives on the future challenges and development trends of microfluidic‐assisted single‐cell omics analysis are discussed.
Microfluidics is considered as a powerful methodology for single‐cell omics analysis with merits in throughput, sensitivity, and accuracy, which can be applied in the study of complex biological processes to identify different cell populations, discover new cell types, reveal informative cell features, and uncover significant interrelationships between cells.
Retinal endothelial cells (RECs) are the primary target cells for diabetes-induced vascular damage. The P2X7/NLRP3 pathway plays an essential role in amplifying inflammation via an ATP feedback loop, ...promoting the inflammatory response, pyroptosis, and apoptosis of RECs in the early stages of diabetic retinopathy induced by hyperglycemia and inflammation. 3TC, a type of nucleoside reverse transcriptase inhibitor, is effective against inflammation, as it can targeting formation of the P2X7 large pore formation. Hence, our aim was to evaluated the anti-inflammatory effects and potential mechanisms of action of 3TC in vitro in retinal microvascular endothelial cells treated with high-glucose (HG) and lipopolysaccharide (LPS), as well as in vivo in the retinas of C57BL/6J male mice with streptozotocin-induced diabetes. The expression of inflammasome-related proteins P2X7 and NLRP3, and apoptosis in the retinas of 3TC-treated diabetic mice were compared to those of untreated diabetic mice. Furthermore, the anti-inflammatory, anti-apoptotic, and anti-pyroptotic effects of 3TC were evaluated in vitro in cultured mice retinal endothelial cells. Co-application of HG and LPS significantly increased the secretion of IL-6, IL-1β, and TNF-α, and ATP levels, whereas 3TC decreased cell inflammation, apoptosis, and pyroptosis. Inhibition of P2X7R and NLRP3 inflammasome activation decreased NLRP3 inflammasome-mediated injury. 3TC prevented cytokine and ATP release following co-application of HG and LPS/BzATP. Our findings provide new insights regarding the mechanisms of action of 3TC in diabetic environment-induced retinal injury, including apoptosis and pyroptosis.
Even though the diagnostic and prognostic value of circulating tumor cells (CTCs) has been demonstrated, their clinical utility and widespread adoption have been limited. Herein, we describe a new ...device, size‐dictated immunocapture chip (SDI‐Chip), for efficient, sensitive, and spatially resolved capture and detection of CTCs. SDI‐Chip enables selective, frequent, and extended interaction of CTCs with hydrodynamically optimized immunocoated micropillar surfaces. CTCs with different antigen expression levels can be efficiently captured and spatially resolved around the micropillars. Capture efficiency greater than 92 % with a purity of 82 % was achieved with blood samples. CTCs were detected in non‐metastasis colorectal (CRC) patients, while none was detected from healthy volunteers. We believe that SDI‐Chip will facilitate the transition of tumor diagnosis from anatomical pathology to molecular pathology in localized CRC patients.
A size‐dictated immunocapture chip (SDI‐Chip) was designed through the rigorous computational analysis of various parameters. Frequent contact with and prolonged retention time on the immunocoated surface, as well as optimum hydrodynamic forces provided by the device, enables the efficient capture of CTCs from cancer patient samples as well as spatial profiling based on antigen expression levels.