RESUMEN
Isogenic cell populations possess heterogeneous gene expression patterns. Most methods for mRNA expression analysis start with the reverse transcription of mRNA into cDNA, a process that can introduce strong signal variations not related to the actual mRNA levels. Miniaturized lab-on-a-chip systems offer properties - e.g. low sample dilution, low contamination - that enable new reaction schemes for molecular analyses. To enable transcription-free mRNA expression analysis of few single cells, a one-step cell lysis, target labelling and hybridisation approach as well as a corresponding passive multiwell chip with a volume of 25.5 nL/well were developed. The method enabled the parallel analysis of up to 96 samples and 6 target genes per sample. Preceding light microscopy of the living cells allowed correlating mRNA levels and cell number. As a proof-of-principle, the pancreatic cancer cell line Panc-1 was investigated for expression heterogeneity of a reference gene plus 5 genes reported to be overexpressed in cancer stem cells (CSCs). A good correlation (r(51)=0.739, p<0.001; rs(51)=0.744, p<0.001) between the cell number per well and the number of detected reference gene mRNA confirmed the proper function of the device. Moreover, a heterogeneous expression of the CSC-associated target genes was found which matched well with reports on the presence of CSCs in the Panc-1 cell line.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Dispositivos Laboratorio en un Chip , Células Madre Neoplásicas/metabolismo , Neoplasias Pancreáticas/metabolismo , ARN Mensajero/metabolismo , Análisis de Matrices Tisulares/instrumentación , Línea Celular Tumoral , Enzimas , Diseño de Equipo , Análisis de Falla de Equipo , Humanos , Hibridación Fluorescente in Situ/instrumentación , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , ARN Mensajero/genética , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Peptide ligands have great potential as selective agents for diagnostic imaging and therapeutic targeting of human cancers. A number of high-throughput assays for screening potential candidate peptides have been developed. Although these screening assays are indispensable for the identification of peptide ligands at a large scale, it is crucial to validate peptide binding and selectivity for targeted receptors in a live-cell context. For testing high-affinity peptide-receptor interactions in the plasma membrane of living cells, we developed cell-resistant, micro-structured glass surfaces with high-density and high-contrast peptide features. Cell adhesion and recruitment of fluorescent receptors to micro-patterned peptides in the live-cell membrane were evaluated by reflection interference contrast (RIC) and total internal reflection (TIRF) microscopy, respectively. To demonstrate both the specificity and modularity of the assay, co-patterning of fluorescent receptors with three different immobilized micro-structured ligands was shown: first, interaction of green fluorescent protein (GFP)-tagged epidermal growth factor (EGF) receptor expressed in Jurkat cells with immobilized EGF was detected and quantified. Second, using Jurkat cells, we demonstrated specific interaction of yellow fluorescent protein (YFP)-tagged ß3 integrin with c(RGDfK) peptide. Third, we identified indirect recruitment of GFP-tagged α5 integrin to an 11-mer peptide. In summary, our results show that the developed micro-structured surfaces are a useful tool for the validation and quantification of peptide-receptor interactions in their natural cellular environment.
Asunto(s)
Técnicas Biosensibles , Péptidos/química , Receptores de Péptidos/aislamiento & purificación , Secuencia de Aminoácidos/genética , Adhesión Celular/genética , Membrana Celular/química , Membrana Celular/metabolismo , Humanos , Células Jurkat , Ligandos , Microscopía Fluorescente , Receptores de Péptidos/genéticaRESUMEN
Heterogeneity of cell populations in various biological systems has been widely recognized, and the highly heterogeneous nature of cancer cells has been emerging with clinical relevance. Single-cell analysis using a combination of high-throughput and multiparameter approaches is capable of reflecting cell-to-cell variability, and at the same time of unraveling the complexity and interdependence of cellular processes in the individual cells of a heterogeneous population. In this review, analytical methods and microfluidic tools commonly used for high-throughput, multiparameter single-cell analysis of DNA, RNA, and proteins are discussed. Applications and limitations of currently available technologies for cancer research and diagnostics are reviewed in the light of the ultimate goal to establish clinically applicable assays.
Asunto(s)
Ácidos Nucleicos/análisis , Análisis de la Célula Individual/métodos , Animales , Citometría de Flujo , Genoma , Genómica , Humanos , Ligandos , Espectrometría de Masas , Ratones , Técnicas Analíticas Microfluídicas/métodos , Microfluídica , Microscopía , Metástasis de la Neoplasia , Neoplasias/diagnóstico , Proteínas , Proteómica , Análisis de Secuencia de ARN , TranscriptomaRESUMEN
Biominerals produced by biological systems in physiologically relevant environments possess extraordinary properties that are often difficult to replicate under laboratory conditions. Understanding the mechanism that underlies the process of biomineralisation can lead to novel strategies in the development of advanced materials. Using microfluidics, we have demonstrated for the first time, that an extrapallial (EP) 28 kDa protein, located in the extrapallial compartment between mantle and shell of Mytilus edulis, can influence, at both micro- and nanoscopic levels, the morphology, structure and polymorph that is laid down in the shell ultrastructure. Crucially, this influence is predominantly dependent on the existence of an EP protein concentration gradient and its consecutive interaction with Ca²(+) ions. Novel lemon-shaped hollow vaterite structures with a clearly defined nanogranular assembly occur only where particular EP protein and Ca²(+) gradients co-exist. Computational fluid dynamics enabled the progress of the reaction to be mapped and the influence of concentration gradients across the device to be calculated. Importantly, these findings could not have been observed using conventional bulk mixing methods. Our findings not only provide direct experimental evidence of the potential influence of EP proteins in crystal formation, but also offer a new biomimetic strategy to develop functional biomaterials for applications such as encapsulation and drug delivery.
Asunto(s)
Calcificación Fisiológica , Proteínas de Unión al Calcio/química , Microfluídica/métodos , Animales , Tampones (Química) , Carbonato de Calcio/síntesis química , Carbonato de Calcio/química , Cloruro de Calcio/química , Carbonatos/química , Cristalización/métodos , Técnicas Analíticas Microfluídicas/métodos , Microscopía Electrónica de Rastreo , Mytilus edulis/química , Proteína G de Unión al Calcio S100/química , Albúmina Sérica Bovina/química , Espectrometría RamanRESUMEN
Biomineralization is the process where biological systems produce well-defined composite structures such as shell, teeth, and bones. Currently, there is substantial momentum to investigate the processes implicated in biomineralization and to unravel the complex roles of proteins in the control of polymorph switching. An understanding of these processes may have wide-ranging significance in health care applications and in the development of advanced materials. We have demonstrated a microfluidic approach toward these challenges. A reversibly sealed T-junction microfluidic device was fabricated to investigate the influence of extrapallial (EP) fluid proteins in polymorph control of crystal formation in mollusk shells. A range of conditions were investigated on chip, allowing fast screening of various combinations of ion, pH, and protein concentrations. The dynamic formation of crystals was monitored on chip and combined with in situ Raman to reveal the polymorph in real time. To this end, we have demonstrated the unique advantages of this integrated approach in understanding the processes involved in biomineralization and revealing information that is impossible to obtain using traditional methods.