Am. in minute quantity 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. A.?Introduction Advances in modern medicine have increased the average lifespan worldwide which has highlighted the need to address the diseases of old age. These geriatric conditions have become endemic, posing unsustainable economic and social burdens1. Heterochronic parabiosis studies have provided proof-of-principle results that aging is both reversible and inducible through the specific composition of the circulatory milieu2,3. Specifically, a number of old tissues including muscle, heart, liver, brain, bones, etc. were rejuvenated by heterochronic parabiosis, and at the same time, young mice displayed a broad decline in tissue health and maintenance2C4. Recent studies utilizing heterochronic blood exchange in mice further confirmed that age-related changes in the blood play a major role in attenuating tissue health and function, and have uncoupled the rejuvenating properties of the endocrineCsystemic milieu from the changes or adaptations caused by the sharing of organ systems and/or being joined for several weeks5. Combination of blood heterochronicity and BONCAT has the promise to directly and comprehensively identify the regulators of tissue aging and rejuvenation, aiding in the development of better diagnostics and treatments for a broad class of degenerative and metabolic diseases. However, BONCAT demands downstream analysis such as Mass Spectrometry for specific protein identification. Mass Spectrometry requires milligrams of Gadoxetate Disodium starting material, which Gadoxetate Disodium is challenging to obtain in studies of small animals and stem cell research. Additionally, with Mass Spectrometry based profiling, the proteins6, which contain ANL, are conjugated to biotin through click-chemistry and can be enriched on affinity columns before their identification7. However, because ANL-labeling is not to saturation, the unlabeled proteins, which can bind non-specifically to the enrichment column, can introduce false positives. Furthermore, false positives can occur when a mixture of ANL-labeled and unlabeled proteins are processed for Mass Spectrometry directly, due to salt-adducts; the additional mass of ANL (172.19 g/mol) over methionine (149.21 g/mol) is ~22.98 g/mol, which is very close to the molecular weight of a sodium ion (22.99 g/mol) (See Table S1 in the Supporting Information). An approach that utilizes ANL-adapted Antibody Array proteomics, takes advantage of biotin clicked to ANL-labeled proteins, which are detected by streptavidin-Cy3 fluorescence8. This method minimized false positives, however, it is limited by cost, the bias of antibody choice, and by the relatively low sensitivity i.e., intensity of fluorescence signal. Furthermore, in GATA6 Mass Spectrometry and modified Antibody Arrays, some ANL-labeled proteins might be missed through their dilution by the unlabeled protein pool, conceivably generating false negative results. Gadoxetate Disodium Finally, all of the above assays involve many steps and take many days to complete. Therefore, there is a need.