RESUMO
The underlying physical properties of microfluidic tools have led to new biological insights through the development of microsystems that can manipulate, mimic and measure biology at a resolution that has not been possible with macroscale tools. Microsystems readily handle sub-microlitre volumes, precisely route predictable laminar fluid flows and match both perturbations and measurements to the length scales and timescales of biological systems. The advent of fabrication techniques that do not require highly specialized engineering facilities is fuelling the broad dissemination of microfluidic systems and their adaptation to specific biological questions. We describe how our understanding of molecular and cell biology is being and will continue to be advanced by precision microfluidic approaches and posit that microfluidic tools - in conjunction with advanced imaging, bioinformatics and molecular biology approaches - will transform biology into a precision science.
Assuntos
Técnicas Analíticas Microfluídicas , Microfluídica/instrumentação , Animais , Bioensaio , Perfilação da Expressão Gênica , Genômica , Humanos , Modelos Biológicos , Análise de Célula ÚnicaRESUMO
MicroRNAs (miRNA) are short, noncoding RNAs that have been implicated in many diseases, including cancers. Because miRNAs are dysregulated in disease, miRNAs show promise as highly stable biomarkers. Formalin-fixed, paraffin-embedded (FFPE) tissue is a valuable sample type to assay for biomolecules because it is a convenient storage method and is often used by pathologists for histological staining. However, extracting biomolecules from FFPE tissue is challenging because of the presence of cellular and extracellular proteins, formaldehyde cross-links, and paraffin. Moreover, most protocols to measure miRNA in FFPE tissue are time-consuming and laborious. Here, we report a simple protocol to directly measure miRNA from formalin-fixed cells, FFPE tissue sections after paraffin is removed, and FFPE tissue sections using encoded hydrogel microparticles fabricated using stop flow lithography. Measurements by these particles show agreement between formalin-fixed cells and fresh cells, and measurement of FFPE tissue with paraffin is 10% less than FFPE tissue when paraffin is removed before the assay. When normal and tumor FFPE tissue are compared using this microparticle assay, we observe differential miRNA signal for oncogenic miRNAs and tumor suppressing miRNAs. This approach reduces assay times, reduces the use of hazardous chemicals to remove paraffin, and provides a sensitive, quantitative, and multiplexed measurement of miRNA in FFPE tissue.
Assuntos
Formaldeído/química , Hidrogéis , MicroRNAs/química , Inclusão em Parafina , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Feminino , Humanos , Pulmão/metabolismo , Neoplasias Pulmonares/genética , Camundongos , Camundongos Nus , Tamanho da PartículaRESUMO
Isoelectric focusing (IEF) is a powerful separation method, useful for resolving subtle changes in the isoelectric point of unlabeled proteins. While microfluidic IEF has reduced the separation times from hours in traditional benchtop IEF to minutes, the enclosed devices hinder post-separation access to the sample for downstream analysis. The two-layer open IEF device presented here comprises a photopatterned hydrogel lid layer containing the chemistries required for IEF and a thin polyacrylamide bottom layer in which the analytes are separated. The open IEF device produces comparable minimum resolvable difference in isoelectric point and gradient stability to enclosed microfluidic devices while providing post-separation sample access by simple removal of the lid layer. Further, using simulations, we determine that the material properties and the length of the separation lanes are the primary factors that affect the electric field magnitude in the separation region. Finally, we demonstrate self-indexed photomasks for alignment-free fabrication of multi-domain hydrogels. We leverage this approach to generate arrayed pH gradients with a total of 80 concurrent separation lanes, which to our knowledge is the first demonstration of multiple IEF separations in series addressed by a single pair of electrodes.
Assuntos
Hidrogéis/química , Focalização Isoelétrica/métodos , Microfluídica/métodos , Proteínas/análiseRESUMO
To measure protein isoforms in individual mammalian cells, we report single-cell resolution isoelectric focusing (scIEF) and high-selectivity immunoprobing. Microfluidic design and photoactivatable materials establish the tunable pH gradients required by IEF and precisely control the transport and handling of each 17-pL cell lysate during analysis. The scIEF assay resolves protein isoforms with resolution down to a single-charge unit, including both endogenous cytoplasmic and nuclear proteins from individual mammalian cells.
Assuntos
Focalização Isoelétrica/métodos , Isoformas de Proteínas/análise , Linhagem Celular Tumoral , Corantes Fluorescentes/química , Humanos , Concentração de Íons de Hidrogênio , Imunoensaio , Dispositivos Lab-On-A-Chip , Análise de Célula ÚnicaRESUMO
Chemical mobilization following IEF enables single-point detection of an ideally stationary equilibrium electrophoresis mode. Despite prior studies exploring optimization of chemical mobilization conditions and recent insight from numerical simulations, understanding of both chemical mobilization mechanisms and the implications of mobilization on IEF analytical performance remains limited. In this study, we utilize full-field imaging of microchannel IEF to assess the performance of a range of canonical chemical mobilization schemes. We empirically demonstrate and characterize key areas where limited understanding of performance implications exists, including: the effects of mobilization solution pH and ion concentration, differences between ionic and zwitterionic mobilization, and diffusion as a source of zone broadening. We utilize Simul5 simulations to gain insight into the sources of the measured performance differences. Measurements of the location, linearity, and slope of the IEF pH gradient (via fluorescent pH markers imaged before and during mobilization) as well as mobilization-associated broadening of focused analytes were performed to quantify performance and determine the dominant sources of variability. Our results suggest that nonuniform broadening of the pH gradient and changes in the pH gradient linearity stem from conductivity nonuniformities in the separation channel and not diffusion-associated band broadening during mobilization.
Assuntos
Focalização Isoelétrica/métodos , Concentração de Íons de Hidrogênio , Focalização Isoelétrica/instrumentação , Concentração Osmolar , Reprodutibilidade dos TestesRESUMO
By combining isoelectric focusing (IEF) with subsequent gel electrophoresis, two-dimensional electrophoresis (2DE) affords more specific characterization of proteins than each constituent unit separation. In a new approach to integrating the two assay dimensions in a microscope slide-sized glass device, we introduce microfluidic 2DE using photopatterned polyacrylamide (PA) gel elements housed in a millimeter-scale, 20-µm-deep chamber. The microchamber minimizes information loss inherent to channel network architectures commonly used for microfluidic 2DE. To define the IEF axis along a "lane" at the top of the chamber, we used free solution carrier ampholytes and immobilized acrylamido buffers in the PA gels. This approach yielded high-resolution (0.1 pH unit) and rapid (<20 min) IEF. Next, protein transfer to the second dimension was accomplished by chemical mobilization perpendicular to the IEF axis. Mobilization drove focused proteins off the IEF lane and into a region for protein gel electrophoresis. Using fluorescently labeled proteins, we observed transfer-induced band broadening factors ~7.5-fold lower than those observed in microchannel networks. Both native polyacrylamide gel electrophoresis (PAGE) and pore-limit electrophoresis (PLE) were studied as the second assay dimension and completed in <15 min. PLE yields protein molecular mass information without the need for ionic surfactant or reducing agents, simplifying device design and operation. Microchamber-based 2DE unifies two independent separation dimensions in a single device with minimal transfer-associated information losses. Peak capacities for the total assay ranged from 256 to 35 with <1 h assay duration. The rapid microchamber 2DE assay has the potential to bridge an existing gap in targeted proteomics for protein biomarker validation and systems biology that may complement recent innovation in mass spectrometry.
Assuntos
Eletroforese em Gel Bidimensional , Técnicas Analíticas Microfluídicas , Proteínas/análise , Resinas Acrílicas/química , Desenho de EquipamentoRESUMO
We describe a novel isoelectric point photoswitching phenomenon in both wild-type Aequorea victoria (av) GFP and the amino acid 222 E-to-G mutant Aequorea coerulescens (ac) GFP. A combination of time-resolved microfluidic isoelectric focusing (IEF) and in situ antibody blotting IEF was employed to monitor dark (nonfluorescent) and bright (fluorescent) GFP populations. Through IEF, each population was observed to exhibit distinct isoelectric points (pI) and, thus, distinct formal electrostatic charges. Experimentally observed interconversion between the dark, higher pI and bright, lower pI GFP populations is tightly controlled by differential UV and blue light exposure. The stoichiometry and kinetics of charge transfer tied to this reversible photobleaching process are deduced. In concert with a reaction-transport model of bistable reversible charge and fluorescence photoswitching, the on-chip measurements of population interconversion rates suggest the potential for both rheostatic and discrete switch-like modulation of the electrostatic charge of GFPs depending on the illumination profile. We estimate that 3-4 formal charges distinguish the bright and dark populations of avGFP, as compared to one charge for those of acGFP. Given the proposed role of E222 as a bridge between internal and exit hydrogen-bond clusters within the GFP ß-barrel, the difference in charge switching magnitude between the two mutants provides intriguing evidence for the proton wire hypothesis of proton transport within the GFP structure, and of proton exchange with the bulk solvent. Our facile dynamic and probed IEF assays should find widespread use in analytical screening and quantitative kinetic analysis of photoswitching and other charge switching processes in response to stimuli including light, temperature, or binding/cleavage events.
Assuntos
Proteínas de Fluorescência Verde/química , Hibridização in Situ Fluorescente , Focalização Isoelétrica , Técnicas Analíticas Microfluídicas , Animais , Antozoários/química , Proteínas de Fluorescência Verde/genética , Ponto Isoelétrico , Processos FotoquímicosRESUMO
Microfluidic technologies are playing an increasingly important role in biological inquiry. Sophisticated approaches to the microanalysis of biological specimens rely, in part, on the fine fluid and material control offered by microtechnology, as well as a sufficient capacity for systems integration. A suite of techniques that utilize photopatterning of polymers on fluidic surfaces, within fluidic volumes, and as primary device structures underpins recent technological innovation in bioanalysis. Well-characterized photopatterning approaches enable previously fabricated or commercially fabricated devices to be customized by the user in a straight-forward manner, making the tools accessible to laboratories that do not focus on microfabrication technology innovation. In this review of recent advances, we summarize reported microfluidic devices with photopatterned structures and regions as platforms for a diverse set of biological measurements and assays.
RESUMO
Spatially resolved gene expression patterns are emerging as a key component of medical studies, including companion diagnostics, but technologies for quantification and multiplexing are limited. We present a method to perform spatially resolved and multiplexed microRNA (miRNA) measurements from formalin-fixed, paraffin-embedded (FFPE) tissue. Using nanoliter well arrays to pixelate the tissue section and photopatterned hydrogels to quantify miRNA, we identified differentially expressed miRNAs in tumors from a genetically engineered mouse model for non-small cell lung cancer (K-rasLSL-G12D/+; p53fl/fl). This technology could be used to quantify heterogeneities in tissue samples and lead to informed, biomarker-based diagnostics.
RESUMO
MicroRNAs (miRNAs) have recently emerged as promising biomarkers for the profiling of diseases. Translation of miRNA biomarkers to clinical practice, however, remains a challenge due to the lack of analysis platforms for sensitive, quantitative, and multiplex miRNA assays that have simple and robust workflows suitable for translation. The platform we present here utilizes functionalized hydrogel posts contained within isolated nanoliter well reactors for quantitative and multiplex assays directly from unprocessed cell samples without the need of prior nucleic acid extraction. Simultaneous reactor isolation and delivery of miRNA extraction reagents is achieved by sealing an array of wells containing the functionalized hydrogel posts and cells against another array of wells containing lysis and extraction reagents. The nanoliter well array platform features >100× better sensitivity compared to previous technology utilizing hydrogel particles without relying on signal amplification and enables >100 parallel assays in a single device. These advances provided by this platform lay the groundwork for translatable and robust analysis technologies for miRNA expression profiling in samples with small populations of cells and in precious, material-limited samples.
Assuntos
Separação Celular/instrumentação , MicroRNAs/metabolismo , Nanotecnologia/instrumentação , Linhagem Celular Tumoral , Humanos , MicroRNAs/genética , Análise de Sequência com Séries de OligonucleotídeosRESUMO
The importance of biological assays spans from clinical diagnostics to environmental monitoring. Simultaneous detection of multiple analytes enhances the efficacy of bioassays by providing more data per assay under standardized conditions. Nevertheless, simultaneous handling and assaying of multiple samples, targets, and experimental conditions can be laborious, reagent consuming, and time intensive. Given these demands, microfluidic platforms have emerged over the past two decades as well-suited approaches for multiplexed assays. Microfluidic design supports integration of assay steps and reproducible sample manipulation across large sets of conditions--all relevant to multiplexed assays. Taken together, reduced reagent consumption, faster assay times, and potential for automation stemming from microfluidic assay design are attractive and needed multiplexed assay performance attributes. This review highlights recent advances in multiplexed bioanalyses benefitting from microfluidic integration.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Microfluídica/métodos , Automação Laboratorial/métodos , Ensaios de Triagem em Larga Escala/economia , Microfluídica/economia , Fatores de TempoRESUMO
Degas-driven flow is a novel phenomenon used to propel fluids in poly(dimethylsiloxane) (PDMS)-based microfluidic devices without requiring any external power. This method takes advantage of the inherently high porosity and air solubility of PDMS by removing air molecules from the bulk PDMS before initiating the flow. The dynamics of degas-driven flow are dependent on the channel and device geometries and are highly sensitive to temporal parameters. These dependencies have not been fully characterized, hindering broad use of degas-driven flow as a microfluidic pumping mechanism. Here, we characterize, for the first time, the effect of various parameters on the dynamics of degas-driven flow, including channel geometry, PDMS thickness, PDMS exposure area, vacuum degassing time, and idle time at atmospheric pressure before loading. We investigate the effect of these parameters on flow velocity as well as channel fill time for the degas-driven flow process. Using our devices, we achieved reproducible flow with a standard deviation of less than 8% for flow velocity, as well as maximum flow rates of up to 3 nL∕s and mean flow rates of approximately 1-1.5 nL∕s. Parameters such as channel surface area and PDMS chip exposure area were found to have negligible impact on degas-driven flow dynamics, whereas channel cross-sectional area, degas time, PDMS thickness, and idle time were found to have a larger impact. In addition, we develop a physical model that can predict mean flow velocities within 6% of experimental values and can be used as a tool for future design of PDMS-based microfluidic devices that utilize degas-driven flow.