The Microfluidic Toolbox for Analyzing Exosome Biomarkers of Aging.

As the fields of aging and neurological disease expand to liquid biopsies, there is a need to identify informative biomarkers for the diagnosis of neurodegeneration and other age-related disorders such as cancers. A means of high-throughput screening of biomolecules relevant to aging can facilitate this discovery in complex biofluids, such as blood. Exosomes, the smallest of extracellular vesicles, are found in many biofluids and, in recent years, have been found to be excellent candidates as liquid biopsy biomarkers due to their participation in intercellular communication and various pathologies such as cancer metastasis. Recently, exosomes have emerged as novel biomarkers for age-related diseases. Hence, the study of exosomes, their protein and genetic cargo can serve as early biomarkers for age-associated pathologies, especially neurodegenerative diseases. However, a disadvantage of exosome studies includes a lack in standardization of isolating, detecting, and profiling exosomes for downstream analysis. In this review, we will address current techniques for high-throughput isolation and detection of exosomes through various microfluidic and biosensing strategies and how they may be adapted for the detection of biomarkers of age-associated disorders

Small-animal blood exchange is an emerging approach for systemic aging research

We describe a small-animal blood exchange approach developed for aging research as an alternative to heterochronic parabiosis or plasma injections. In parabiosis, animals are surgically coupled, which has several disadvantages, including difficulty controlling experimental procedure, the effects of shared organs, environmental enrichment from jointly exploring the housing enclosure, involuntary exercise and an imprecise onset of blood sharing. Likewise, in plasma injections, the added volumes need to be small, and there is little flexibility in changing the relative contributions of ectopic to endogenous blood components. These factors complicate the conclusions and interpretations, including the identification of key mechanisms and molecular or cellular determinants. Our approach, where blood is exchanged between animals without them being surgically coupled, is less invasive than parabiosis. The percentage of exchanged blood or other exchanged fluids is known and precise. The age of plasma and cells can be mixed and matched at all desired relative contributions to the endogenous systemic milieu, and the onset of the effects can be accurately delineated. In this protocol, we describe the preparatory and animal surgery steps required for small-animal blood exchange in mice and compare this process with parabiosis and plasma injections. We also provide the design, hardware and software for the blood exchange device and compare automated and manual exchange methods. Lastly, we report mathematical modeling of the dilution of blood factors. The fluid exchange takes ~30 min when performed by a well-trained biomedical scientist; the entire process takes ~2 h

Emerging Biosensing Technologies for the Diagnostics of Viral Infectious Diseases

Several viral infectious diseases appear limitless since the beginning of the 21st century, expanding into pandemic lengths. Thus, there are extensive efforts to provide more efficient means of diagnosis, a better understanding of acquired immunity, and improved monitoring of inflammatory biomarkers, as these are all crucial for controlling the spread of infection while aiding in vaccine development and improving patient outcomes. In this regard, various biosensors have been developed recently to streamline pathogen and immune response detection by addressing the limitations of traditional methods, including isothermal amplification-based systems and lateral flow assays. This review explores state-of-the-art biosensors for detecting viral pathogens, serological assays, and inflammatory biomarkers from the material perspective, by discussing their advantages, limitations, and further potential regarding their analytical performance, clinical utility, and point-of-care adaptability. Additionally, next-generation biosensing technologies that offer better sensitivity and selectivity, and easy handling for end-users are highlighted. An emerging example of these next-generation biosensors are those powered by novel synthetic biology tools, such as clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated proteins (Cas), in combination with integrated point-of-care devices. Lastly, the current challenges are discussed and a roadmap for furthering these advanced biosensing technologies to manage future pandemics is provided

The Microfluidic Toolbox for Analyzing Exosome Biomarkers of Aging

As the fields of aging and neurological disease expand to liquid biopsies, there is a need to identify informative biomarkers for the diagnosis of neurodegeneration and other age-related disorders such as cancers. A means of high-throughput screening of biomolecules relevant to aging can facilitate this discovery in complex biofluids, such as blood. Exosomes, the smallest of extracellular vesicles, are found in many biofluids and, in recent years, have been found to be excellent candidates as liquid biopsy biomarkers due to their participation in intercellular communication and various pathologies such as cancer metastasis. Recently, exosomes have emerged as novel biomarkers for age-related diseases. Hence, the study of exosomes, their protein and genetic cargo can serve as early biomarkers for age-associated pathologies, especially neurodegenerative diseases. However, a disadvantage of exosome studies includes a lack in standardization of isolating, detecting, and profiling exosomes for downstream analysis. In this review, we will address current techniques for high-throughput isolation and detection of exosomes through various microfluidic and biosensing strategies and how they may be adapted for the detection of biomarkers of age-associated disorders

Mechanisms and Minimization of False Discovery of Metabolic Bioorthogonal Noncanonical Amino Acid Proteomics

Metabolic proteomics has been widely used to characterize dynamic protein networks in many areas of biomedicine, including in the arena of tissue aging and rejuvenation. Bioorthogonal noncanonical amino acid tagging (BONCAT) is based on mutant methionine-tRNA synthases (MetRS) that incorporates metabolic tags, for example, azidonorleucine [ANL], into newly synthesized proteins. BONCAT revolutionizes metabolic proteomics, because mutant MetRS transgene allows one to identify cell type-specific proteomes in mixed biological environments. This is not possible with other methods, such as stable isotope labeling with amino acids in cell culture, isobaric tags for relative and absolute quantitation and tandem mass tags. At the same time, an inherent weakness of BONCAT is that after click chemistry-based enrichment, all identified proteins are assumed to have been metabolically tagged, but there is no confirmation in mass spectrometry data that only tagged proteins are detected. As we show here, such assumption is incorrect and accurate negative controls uncover a surprisingly high degree of false positives in BONCAT proteomics. We show not only how to reveal the false discovery and thus improve the accuracy of the analyses and conclusions but also approaches for avoiding it through minimizing nonspecific detection of biotin, biotin-independent direct detection of metabolic tags, and improvement of signal to noise ratio through machine learning algorithms