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 Reprints and permissions information is available at www.nature.com/reprints. * kiana_aran@kgi.edu. Correspondence and requests for materials should be addressed to K.A. Author contributions R.H. optimized the CRISPR-Chip design, performed the CRISPR-Chip DMD experiments, data collection and analysis, LOD optimization, HEK-BFP calibration methodologies in the presence and absence of contamination, and kinetic analysis, and prepared the manuscript. S.B. assisted in optimization of the CRISPR-Chip assay protocols, performed the MB-dRNP studies, DMD patient sample analysis, HEK-BFP PCR experiments and analysis, and prepared the manuscript. T.T. assisted with the initial CRISPR-Chip design, performed initial CRISPR-Chip protocols for HEK-BFP studies, and prepared the manuscript. T.d. performed the synthesis of sgRNA for the bfp and Scram studies, genomic purification and initial system design, and helped with manuscript preparation. J.E. contributed to the design of the DMD-based validation of CRISPR-Chip and provided the PCR and sequencing data for the DMD studies. M.S. contributed to the design of the DMD-based validation of CRISPR-Chip and assisted in manuscript preparation. N.A.W. and J.-Y.C. assisted T.D. with the synthesis of sgRNAs for bfp studies and assisted with sample preparation. J.N. and B.G. assisted with CRISPR-Chip data analysis and manuscript preparation. M.A. and J.P. assisted with manuscript preparation and data analysis. R.P. assisted with the design of threshold experiments, data analysis and CRISPR-Chip validation. N.M. supervised the synthesis of sgRNAs for the bfp and Scram studies. I.M.C. assisted with technology design, DMD validation and manuscript preparation. K.A. designed and developed the technology, planned and supervised the project, analysed, interpreted and integrated the data, and prepared the manuscript.

Perfluorinated acids have received increasing scientific attention due to their widespread global distribution, environmental persistence and bioaccumulation in wildlife and humans. For perfluorohexane sulfonate (PFHxS, C(6)F(13)SO(3) (-), m/z 399), all existing human data have been generated using high-performance liquid chromatography (HPLC) and its most sensitive tandem mass spectrometric (MS/MS) transitions (m/z 399/80 [SO(3)](-) or m/z 399/99 [SO(3)F](-)), but this may be problematic because of co-eluting endogenous steroid sulfates that share common fragmentation pathways. We examined the magnitude of over-reporting for PFHxS in pregnant women (n = 29), and in pooled serum of males, non-pregnant and pregnant females (n = 3, 100 samples per pool), by comparing m/z 399/80 and 399/99 data with an interference-free transition, m/z 399/119. PFHxS concentrations in pregnant women determined using m/z 399/80 and 399/99 (p < 0.05), but not m/z 399/119, were positively correlated to the response of the steroid sulfates. This led to an average overestimation of PFHxS by 1.5- and 4.7-fold, using m/z 399/80 and 399/99, respectively, and validated the use of m/z 399/119 for the first time. The interferences were a problem in all human serum samples, and analysis of pooled serum revealed statistically significant over-reporting by m/z 399/80 and 399/99 for pregnant women > non-pregnant women > men. The magnitude of over-reporting here represents a worst-case scenario, but the extent to which the published literature values are biased is unknown due to limited details of methods in existing reports. Instead of using the less sensitive m/z 399/119 transition, we showed that an alternative selection of column and mobile phase can allow for sufficient chromatographic separation of the interferences. In conclusion, it was shown that routine analytical methods are prone to systematically overestimating PFHxS concentrations in serum of men or women, but that this can be avoided by alternative chromatographic steps or MS/MS transitions.

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.