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 prevailing philosophy in biological testing has been to focus on simple tests with easy to interpret information such as ELISA or lateral flow assays. At the same time, there has been a decades long understanding in device physics and nanotechnology that electrical approaches have the potential to drastically improve the quality, speed, and cost of biological testing provided that computational resources are available to analyze the resulting complex data. This concept can be conceived of as “the internet of biology” in the same way miniaturized electronic sensors have enabled “the internet of things.” It is well established in the nanotechnology literature that techniques such as field effect biosensing are capable of rapid and flexible biological testing. Until now, access to this new technology has been limited to academic researchers focused on bioelectronic devices and their collaborators. Here we show that this capability is retained in an industrially manufactured device, opening access to this technology generally. Access to this type of production opens the door for rapid deployment of nanoelectronic sensors outside the research space. The low power and resource usage of these biosensors enables biotech engineers to gain immediate control over precise biological and environmental data.
The unique antiwetting properties of superhydrophobic (SH) surfaces prevent the adhesion of water and bodily fluids, including blood, urine, and saliva. While typical manufacturable approaches to create SH surfaces rely on chemical and structural modifications, such approaches are expensive, require postprocessing, and are often not biocompatible. By contrast, it is demonstrated that purely structural SH features are easily formed using high throughput roll-to-roll (R2R) manufacturing by shrinking a prestressed thermoplastic with a thin, stiff layer of silver and calcium. These features are subsequently embossed into any commercially available and Food and Drug Administration (FDA)-approved plastic. The R2R SH surfaces have contact angles >150° and contact angle hysteresis <10°. Importantly, the surfaces minimize blood adhesion, leading to reduced blood coagulation without the need for anticoagulants. SH surfaces have >4200× reduction of blood residue area compared to the nonstructured controls of the same material. In addition, blood clotting is reduced >5× using whole blood directly from the patient. Furthermore, these surfaces can be easily configured into 3D shapes, as demonstrated with SH tubes. With the simple scale-up production and the eliminated need for anticoagulants to prevent clotting, the proposed conformable SH surfaces can be impactful for a wide range of medical tools, including catheters and microfluidic channels.
We present a rapid, simple, and scalable approach to achieve superhydrophobic (SH) substrates directly in commodity shrink wrap film utilizing Argon (Ar) plasma. Ar plasma treatment creates a stiff skin layer on the surface of the shrink film. When the film shrinks, the mismatch in stiffness between the stiff skin layer and bulk shrink film causes the formation of multiscale hierarchical wrinkles with nano-textured features. Scanning electron microscopy (SEM) images confirm the presence of these biomimetic structures. Contact angle (CA) and contact angle hysteresis (CAH) measurements, respectively, defined as values greater than 150° and less than 10°, verified the SH nature of the substrates. Furthermore, we demonstrate the ability to reliably pattern hydrophilic regions onto the SH substrates, allowing precise capture and detection of proteins in urine. Finally, we achieved self-driven microfluidics via patterning contrasting superhydrophilic microchannels on the SH Ar substrates to induce flow for biosensing.
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