RESUMO
Synthetic biology is bringing together engineers and biologists to design and build novel biomolecular components, networks and pathways, and to use these constructs to rewire and reprogram organisms. These re-engineered organisms will change our lives over the coming years, leading to cheaper drugs, 'green' means to fuel our cars and targeted therapies for attacking 'superbugs' and diseases, such as cancer. The de novo engineering of genetic circuits, biological modules and synthetic pathways is beginning to address these crucial problems and is being used in related practical applications.
Assuntos
Biotecnologia/tendências , Engenharia Genética , Animais , Materiais Biocompatíveis , Biocombustíveis , Técnicas Biossensoriais , Tratamento Farmacológico , Redes Reguladoras de Genes , Humanos , Tecnologia FarmacêuticaRESUMO
Rapid, accurate and frequent detection of the RNA of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and of serological host antibodies to the virus would facilitate the determination of the immune status of individuals who have Coronavirus disease 2019 (COVID-19), were previously infected by the virus, or were vaccinated against the disease. Here we describe the development and application of a 3D-printed lab-on-a-chip that concurrently detects, via multiplexed electrochemical outputs and within 2 h, SARS-CoV-2 RNA in saliva as well as anti-SARS-CoV-2 immunoglobulins in saliva spiked with blood plasma. The device automatedly extracts, concentrates and amplifies SARS-CoV-2 RNA from unprocessed saliva, and integrates the Cas12a-based enzymatic detection of SARS-CoV-2 RNA via isothermal nucleic acid amplification with a sandwich-based enzyme-linked immunosorbent assay on electrodes functionalized with the Spike S1, nucleocapsid and receptor-binding-domain antigens of SARS-CoV-2. Inexpensive microfluidic electrochemical sensors for performing multiplexed diagnostics at the point of care may facilitate the widespread monitoring of COVID-19 infection and immunity.
Assuntos
COVID-19 , SARS-CoV-2 , Anticorpos Antivirais , COVID-19/diagnóstico , Humanos , Dispositivos Lab-On-A-Chip , Plasma , RNA Viral , Saliva , Glicoproteína da Espícula de CoronavírusRESUMO
Materials that sense and respond to biological signals in their environment have a broad range of potential applications in drug delivery, medical devices and diagnostics. Nucleic acids are important biological cues that encode information about organismal identity and clinically relevant phenotypes such as drug resistance. We recently developed a strategy to design nucleic acid-responsive materials using the CRISPR-associated nuclease Cas12a as a user-programmable sensor and material actuator. This approach improves on the sensitivity of current DNA-responsive materials while enabling their rapid repurposing toward new sequence targets. Here, we provide a comprehensive resource for the design, synthesis and actuation of CRISPR-responsive hydrogels. First, we provide guidelines for the synthesis of Cas12a guide RNAs (gRNAs) for in vitro applications. We then outline methods for the synthesis of both polyethylene glycol-DNA (PEG-DNA) and polyacrylamide-DNA (PA-DNA) hydrogels, as well as their controlled degradation using Cas12a for the release of cargos, including small molecules, enzymes, nanoparticles and living cells within hours. Finally, we detail the design and assembly of microfluidic paper-based devices that use Cas12a-sensitive hydrogels to convert DNA inputs into a variety of visual and electronic readouts for use in diagnostics. Following the initial validation of the gRNA and Cas12a components (1 d), the synthesis and testing of either PEG-DNA or PA-DNA hydrogels require 3-4 d of laboratory time. Optional extensions, including the release of primary human cells or the design of the paper-based diagnostic, require an additional 2-3 d each.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Técnicas e Procedimentos Diagnósticos , Sistemas de Liberação de Medicamentos/métodos , Liberação Controlada de Fármacos , Materiais Inteligentes/química , Resinas Acrílicas/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas Associadas a CRISPR/metabolismo , DNA/química , DNA/genética , Endodesoxirribonucleases/metabolismo , Humanos , Células K562 , Polietilenoglicóis/química , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Stimuli-responsive materials activated by biological signals play an increasingly important role in biotechnology applications. We exploit the programmability of CRISPR-associated nucleases to actuate hydrogels containing DNA as a structural element or as an anchor for pendant groups. After activation by guide RNA-defined inputs, Cas12a cleaves DNA in the gels, thereby converting biological information into changes in material properties. We report four applications: (i) branched poly(ethylene glycol) hydrogels releasing DNA-anchored compounds, (ii) degradable polyacrylamide-DNA hydrogels encapsulating nanoparticles and live cells, (iii) conductive carbon-black-DNA hydrogels acting as degradable electrical fuses, and (iv) a polyacrylamide-DNA hydrogel operating as a fluidic valve with an electrical readout for remote signaling. These materials allow for a range of in vitro applications in tissue engineering, bioelectronics, and diagnostics.
Assuntos
Proteínas de Bactérias/química , Materiais Biocompatíveis/química , Técnicas Biossensoriais , Proteínas Associadas a CRISPR/química , DNA/química , Endodesoxirribonucleases/química , Hidrogéis/química , Patologia Molecular , Engenharia Tecidual , Resinas Acrílicas/química , Células/química , Reagentes de Ligações Cruzadas/química , Clivagem do DNA , DNA de Cadeia Simples/química , Dispositivos Lab-On-A-Chip , Nanopartículas/química , Permeabilidade , Polietilenoglicóis/químicaRESUMO
Hands-on demonstrations greatly enhance the teaching of science, technology, engineering, and mathematics (STEM) concepts and foster engagement and exploration in the sciences. While numerous chemistry and physics classroom demonstrations exist, few biology demonstrations are practical and accessible due to the challenges and concerns of growing living cells in classrooms. We introduce BioBits™ Explorer, a synthetic biology educational kit based on shelf-stable, freeze-dried, cell-free (FD-CF) reactions, which are activated by simply adding water. The FD-CF reactions engage the senses of sight, smell, and touch with outputs that produce fluorescence, fragrances, and hydrogels, respectively. We introduce components that can teach tunable protein expression, enzymatic reactions, biomaterial formation, and biosensors using RNA switches, some of which represent original FD-CF outputs that expand the toolbox of cell-free synthetic biology. The BioBits™ Explorer kit enables hands-on demonstrations of cutting-edge science that are inexpensive and easy to use, circumventing many current barriers for implementing exploratory biology experiments in classrooms.