Reliable Detection of Extracellular PD-L1 by DNA Computation-Mediated Microfluidics.

Extracellular vesicle PD-L1 (programmed death-1 ligand 1) is of greater value in tumor diagnosis, prognosis, and efficacy monitoring of anti-PD-1/PD-L1 immunotherapy. However, soluble PD-L1 interferes with the accurate detection of extracellular vesicle (EV) PD-L1. Here, we developed a microfluidic differentiation method for the detection of extracellular PD-L1, without the interference of soluble, by DNA computation with lipid probes and PD-L1 aptamer as inputs (DECLA). For the developed DECLA method, a cholesterol-DNA probe was designed that efficiently embeds into the EV membrane, and an aptamer-based PD-L1 probe was used for PD-L1 recognition. Due to the stable secondary structure of the designed connector, only cobinding of cholesterol-DNA and PD-L1 affinity probe induced biotin-labeled connector activation, while soluble PD-L1 cannot hybridize. As a result, PD-L1 EVs can be efficiently captured by streptavidin-functioned herringbone chip and quantified by anti-CD63-induced fluorescence signal. The high specificity of dual-input DNA computation allied to the high sensitivity of microfluidic-based detection was suitable for distinguishing lung cancer patients from healthy donors, highlighting its potential translation to clinical diagnosis and therapy monitoring.

Magnetofluid-Integrated Multicolor Immunochip for Visual Analysis of Neutralizing Antibodies to SARS-CoV-2 Variants.

The global spread of SARS-CoV-2 virus has severely affected human health, life, and work. Vaccine immunization is considered to be an effective means to protect the body from infection. Therefore, timely analysis of the antibody level is helpful to identify people with low immune response or attenuated antibodies so as to carry out targeted and precise vaccine booster immunization. Herein, we develop a magnetofluid-integrated multicolor immunochip, as a sample-to-answer system in a fully enclosed space, for visual analysis of neutralizing antibodies of SARS-CoV-2. Generally, this chip adopts an innovative three-dimensional two-phase system that utilizes mineral oil to block the connection between reagent wells in the vertical direction and provides a wide interface for rapid and nondestructive shuttle of magnetic beads during the immunoassay. In order to obtain visualized signal output, gold nanorods with a size-dependent color effect are used as the colorful chromogenic substrates for evaluation of the antibody level. Using this chip, the neutralizing antibodies were successfully detected in vaccine-immunized volunteers with 83.3% sensitivity and 100% specificity. Furthermore, changes in antibody levels of the same individual over time were also reflected by the multicolor assay. Overall, benefiting from simple operation, airtight safety, and nonrequirement of external equipment, this platform can provide a new point-of-care testing strategy for alleviating the shortage of medical resources and promoting epidemic control in underdeveloped areas.

In Situ Visualization of PD-L1-Specific Glycosylation on Tissue Sections.

Immune checkpoint therapy has provided a weapon against cancer, but its response rate has been extremely low due to the lack of effective predictors. Herein, we developed a FRET strategy based on lectin for glycan labeling and an aptamer for PD-L1 antigen recognition for visualization of PD-L1-specific glycosylation (FLAG). The FLAG strategy combines the PD-L1 aptamer, which efficiently labels the PD-L1 polyantigen with smaller steric hindrance than the PD-L1 antibody, and metabolism-free lectin labeling for glycosylation. As a result, the FLAG strategy enables in situ visualization of PD-L1-specific glycosylation on the tissue section while maintaining the spatial context and tissue architecture. Due to nonmetabolic labeling, the FLAG strategy revealed that the tissue level of PD-L1-specific glycosylation is correlated with the efficacy of PD-1/PD-L1 therapy. Overall, the FLAG strategy provides a powerful tool for revealing the significance of PD-L1 glycosylation, offering the unprecedented potential for immunophenotypic differential analysis to predict the immunotherapy response.

Quantification-Promoted Discovery of Glycosylated Exosomal PD-L1 as a Potential Tumor Biomarker.

Exosomal programmed cell death ligand 1 (exoPD-L1) has emerged as a promising biomarker for cancer diagnosis and immunotherapy outcome prediction. However, the existing quantitation methods are incapable of addressing the heterogeneity of exoPD-L1 glycosylation, which has been demonstrated to be the institutional basis for PD-L1/PD-1 interaction and the crucial participant in inhibiting the activity of CD8+ T cells. Herein, an aptamer- and lectin-induced proximity ligation assay combined with quantitative real-time polymerase chain reaction for precise quantitation of glycosylated exoPD-L1 is developed. Leveraging the metabolism-free lectin labeling of glycosylation, the glycosylation-independent aptamer tagging of PD-L1, and excellent selectivity of dual-recognition, this method enables glycosylated exoPD-L1 quantitation with high sensitivity and selectivity in a wash-free manner. As a result, this method is able to distinguish the levels of glycosylated exoPD-L1 between healthy donors and cancer patients with sensitivity and specificity of 100%. Compared with the total circulating exoPD-L1 level, glycosylated exoPD-L1 is for the first time identified to be a more reliable biomarker for tumor diagnosis. Overall, this strategy holds a great potential for revealing the significance of exoPD-L1 glycosylation and converting glycosylated exoPD-L1 into a reliable clinical indicator.