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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.

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.

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.

Most methods for the detection of nucleic acids require many reagents and expensive and bulky instrumentation. Here, we report the development and testing of a graphene-based field-effect transistor that uses clustered regularly interspaced short palindromic repeats (CRISPR) technology to enable the digital detection of a target sequence within intact genomic material. Termed CRISPR-Chip, the biosensor uses the gene-targeting capacity of catalytically deactivated CRISPR-associated protein 9 (Cas9) complexed with a specific single-guide RNA and immobilized on the transistor to yield a label-free nucleic-acid-testing device whose output signal can be measured with a simple handheld reader. We used CRISPR-Chip to analyse DNA samples collected from HEK293T cell lines expressing blue fluorescent protein, and clinical samples of DNA with two distinct mutations at exons commonly deleted in individuals with Duchenne muscular dystrophy. In the presence of genomic DNA containing the target gene, CRISPR-Chip generates, within 15 min, with a sensitivity of 1.7 fM and without the need for amplification, a significant enhancement in output signal relative to samples lacking the target sequence. CRISPR-Chip expands the applications of CRISPR-Cas9 technology to the on-chip electrical detection of nucleic acids.

Studies of heterochronic parabiosis, where two animals of different ages are joined surgically, provided proof-of-principle results that systemic proteins have broad age-specific effects on tissue health and repair. In an effort to identify these systemic proteins, we previously developed a method to selectively label the proteome of only one animal joined in parabiosis utilizing bio-orthogonal non-canonical amino acid tagging (BONCAT), which can metabolically label proteins during their de novo synthesis by incorporating a methionine substitute, azido-nor-leucine (ANL), in cells expressing a mutant methionyl-tRNA synthetase (MetRSL274G). Once labeled, we can selectively identify the proteins produced by the MetRSL274G transgenic mouse in the setting of heterochronic parabiosis. This approach enabled the detection of several rejuvenating protein candidates from the young parabiont, which were transferred to the old mammalian tissue through their shared circulation. Although BONCAT is a very powerful technology, the challenges associated with its complexity including large starting material requirements and cost of ANL-labeled protein detection, such as modified antibody arrays and mass spectrometry, limit its application. Herein, we propose a lab-on-a-chip technology, termed Click-A+Chip for facile and rapid digital detection of ANL-labeled proteomes present in minute amount of sample, to replace conventional assays. Click-A+Chip is a graphene-based field effect biosensor (gFEB) which utilizes novel on-chip click-chemistry to specifically bind to ANL-labeled biomolecules. In this study, Click-A+Chip is utilized for the capture of ANL-labeled proteins transferred from young to old parabiotic mouse partners. Moreover, we were able to identify the young-derived ANL-labeled Lif-1 and leptin in parabiotic systemic milieu, confirming previous data as well as providing novel findings on the relative levels of these factors in young versus old parabionts. Summarily, our results demonstrate that Click-A+Chip can be used for rapid detection and identification of ANL-labeled proteins, significantly reducing the sample size, complexity, cost and time associated with BONCAT analysis.

Therapeutics based on transcription factors have the potential to revolutionize medicine but have had limited clinical success as a consequence of delivery problems. The delivery of transcription factors is challenging because it requires the development of a delivery vehicle that can complex transcription factors, target cells and stimulate endosomal disruption, with minimal toxicity. Here, we present a multifunctional oligonucleotide, termed DARTs (DNA assembled recombinant transcription factors), which can deliver transcription factors with high efficiency in vivo. DARTs are composed of an oligonucleotide that contains a transcription-factor-binding sequence and hydrophobic membrane-disruptive chains that are masked by acid-cleavable galactose residues. DARTs have a unique molecular architecture, which allows them to bind transcription factors, trigger endocytosis in hepatocytes, and stimulate endosomal disruption. The DARTs have enhanced uptake in hepatocytes as a result of their galactose residues and can disrupt endosomes efficiently with minimal toxicity, because unmasking of their hydrophobic domains selectively occurs in the acidic environment of the endosome. We show that DARTs can deliver the transcription factor nuclear erythroid 2-related factor 2 (Nrf2) to the liver, catalyse the transcription of Nrf2 downstream genes, and rescue mice from acetaminophen-induced liver injury.

A digital point-of-care biosensor for measuring reactive oxygen species is presented based on novel reactive oxygen species responsive polymer-based electrodes. The biosensor is able to detect a drug-induced liver injury by monitoring the oxidative stress in the blood.

A new turn-off fluorescent substrate for horseradish peroxidase is introduced, termed clics, which enhances the sensitivity of commercial enzyme-linked immunosorbant assays (ELISA) by 32-fold and detects chlamydial infections in patients. Clics are cost-effective, stable, and can be easily incorporated in commercial ELISA kits, and therefore should have a great impact in the fields of medicine and biology.