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.

The growth of large-area graphene on catalytic metal substrates is a topic of both fundamental and technological interest. We have developed an atmospheric pressure chemical vapor deposition (CVD) method that is potentially more cost-effective and compatible with industrial production than approaches based on synthesis under high vacuum. Surface morphology of the catalytic Cu substrate and the concentration of carbon feedstock gas were found to be crucial factors in determining the homogeneity and electronic transport properties of the final graphene film. The use of an electropolished metal surface and low methane concentration enabled the growth of graphene samples with single layer content exceeding 95%. Field effect transistors fabricated from CVD graphene made with the optimized process had room temperature hole mobilities that are a factor of 2−5 larger than those measured for samples grown on as-purchased Cu foil with larger methane concentration. A kinetic model is proposed to explain the observed dependence of graphene growth on catalyst surface roughness and carbon source concentration.

We used covalent attachments to single-walled carbon nanotubes (SWNTs) to fabricate single-molecule electronic devices. The technique does not rely on submicrometer lithography or precision mechanical manipulation, but instead uses circuit conductance to monitor and control covalent attachment to an electrically connected SWNT. Discrete changes in the circuit conductance revealed chemical processes happening in real time and allowed the SWNT sidewalls to be deterministically broken, reformed, and conjugated to target species. By controlling the chemistry through electronically controlled electrochemical potentials, we were able to achieve single chemical attachments. We routinely functionalized pristine, defect-free SWNTs at one, two, or more sites and demonstrated three-terminal devices in which a single attachment controls the electronic response.

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We have developed a cost-effective and portable graphene-enabled biosensor to detect Zika virus with a highly specific immobilized monoclonal antibody. Field Effect Biosensing (FEB) with monoclonal antibodies covalently linked to graphene enables real-time, quantitative detection of native Zika viral (ZIKV) antigens. The percent change in capacitance in response to doses of antigen (ZIKV NS1) coincides with levels of clinical significance with detection of antigen in buffer at concentrations as low as 450pM. Potential diagnostic applications were demonstrated by measuring Zika antigen in a simulated human serum. Selectivity was validated using Japanese Encephalitis NS1, a homologous and potentially cross-reactive viral antigen. Further, the graphene platform can simultaneously provide the advanced quantitative data of nonclinical biophysical kinetics tools, making it adaptable to both clinical research and possible diagnostic applications. The speed, sensitivity, and selectivity of this first-of-its-kind graphene-enabled Zika biosensor make it an ideal candidate for development as a medical diagnostic test.