RESUMO
Cell-based biosensors have great potential to detect various toxic and pathogenic contaminants in aqueous environments. However, frequently they cannot meet practical requirements due to insufficient sensing performance. To address this issue, we investigated a modular, cascaded signal amplifying methodology. We first tuned intracellular sensory receptor densities to increase sensitivity, and then engineered multi-layered transcriptional amplifiers to sequentially boost output expression level. We demonstrated these strategies by engineering ultrasensitive bacterial sensors for arsenic and mercury, and improved detection limit and output up to 5,000-fold and 750-fold, respectively. Coupled by leakage regulation approaches, we developed an encapsulated microbial sensor cell array for low-cost, portable and precise field monitoring, where the analyte can be readily quantified via displaying an easy-to-interpret volume bar-like pattern. The ultrasensitive signal amplifying methodology along with the background regulation and the sensing platform will be widely applicable to many other cell-based sensors, paving the way for their real-world applications.
Assuntos
Arsênio/análise , Técnicas Biossensoriais , Telefone Celular , Metais Pesados/análise , Técnicas Analíticas Microfluídicas , Arsênio/efeitos adversos , Técnicas Biossensoriais/instrumentação , Telefone Celular/instrumentação , Humanos , Metais Pesados/efeitos adversos , Técnicas Analíticas Microfluídicas/instrumentaçãoRESUMO
Synthetically engineered cells are powerful and potentially useful biosensors, but it remains problematic to deploy such systems due to practical difficulties and biosafety concerns. To overcome these hurdles, we developed a microfluidic device that serves as an interface between an engineered cellular system, environment, and user. We created a biodisplay consisting of 768 individually programmable biopixels and demonstrated that it can perform multiplexed, continuous sampling. The biodisplay detected 10 µg/L sodium-arsenite in tap water using a research grade fluorescent microscope, and reported arsenic contamination down to 20 µg/L with an easy to interpret "skull and crossbones" symbol detectable with a low-cost USB microscope or by eye. The biodisplay was designed to prevent release of chemical or biological material to avoid environmental contamination. The microfluidic biodisplay thus provides a practical solution for the deployment and application of engineered cellular systems.
Assuntos
Bacillus subtilis , Engenharia Celular , Escherichia coli , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas , Bacillus subtilis/citologia , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Engenharia Celular/instrumentação , Engenharia Celular/métodos , Escherichia coli/citologia , Escherichia coli/genética , Escherichia coli/metabolismo , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodosRESUMO
Microfluidic diagnostic devices have the potential to transform the practice of medicine. We engineered a multiplexed digital-analog microfluidic platform for the rapid and highly sensitive detection of 3-4 biomarkers in quadruplicate in 16 independent and isolated microfluidic unit cells requiring only a single 5 µL sample. We comprehensively characterized the platform by performing single enzyme and digital immunoassays, achieving single molecule detection and measured as low as â¼10 fM (330 fg/mL) GFP in buffer and â¼12 fM GFP in human serum. We applied our integrated digital detection mechanism to multiplexed detection of 1pM anti-Ebola IgG in human serum and were able to differentiate three common Ebola strains. To ascertain that the device can be applied in environments beyond clinical point-of-care settings, we developed a low-cost, portable hardware system to control and read out the microfluidic device and detected anti-Ebola IgG in ultralow volume whole blood samples to levels of 100 pM in a multiplexed assay format.
RESUMO
We present a high-throughput microfluidic platform capable of quantitating up to 384 biomarkers in 4 distinct samples by immunoassay. The microfluidic device contains 384 unit cells, which can be individually programmed with pairs of capture and detection antibody. Samples are quantitated in each unit cell by four independent MITOMI detection areas, allowing four samples to be analyzed in parallel for a total of 1,536 assays per device. We show that the device can be pre-assembled and stored for weeks at elevated temperature and we performed proof-of-concept experiments simultaneously quantitating IL-6, IL-1ß, TNF-α, PSA, and GFP. Finally, we show that the platform can be used to identify functional antibody combinations by screening 64 antibody combinations requiring up to 384 unique assays per device.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Microfluídica/métodos , Proteínas/química , Proteínas/metabolismo , Biomarcadores , Ensaios de Triagem em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/normas , Imunoensaio/instrumentação , Imunoensaio/métodos , Imunoensaio/normas , Microfluídica/instrumentação , Microfluídica/normas , Reprodutibilidade dos Testes , Fluxo de TrabalhoRESUMO
Microfluidics and miniaturization of biosensors are fundamental for the development of point-of-care (PoC) diagnostic and analytical tools with the potential of decreasing reagent consumption and time of analysis while increasing portability. However, interfacing microfluidics with fluid control systems is still a limiting factor in practical implementation. We demonstrate an innovative capillary microfluidic design that allows sequential insertion of controlled volumes of liquids into a microfluidic channel with general applicability. The system requires only the placing of liquids at the corresponding inlets. Subsequently, the different solutions flow inside the microfluidic device sequentially and autonomously without the use of valves using integrated capillary pumps. The capillary microfluidic system is demonstrated with a model immunoassay.