RESUMEN
The primary goal of bioprocess cell line development is to obtain high product yields from robustly growing and well-defined clonal cell lines in timelines measured in weeks rather than months. Likewise, high-throughput screening of B cells and hybridomas is required for most cell line engineering workflows. A substantial bottleneck in these processes is detecting and isolating rare clonal cells with the required characteristics. Traditionally, this was achieved by the resource-intensive method of limiting dilution cloning, and more recently aided by semiautomated technologies such as cell sorting (e.g., fluorescence-activated cell sorting) and colony picking. In this paper we report on our novel Cyto-Mine Single Cell Analysis and Monoclonality Assurance System, which overcomes the limitations of current technologies by screening hundreds of thousands of individual cells for secreted target proteins, and then isolating and dispensing the highest producers into microtiter plate wells (MTP). The Cyto-Mine system performs this workflow using a fully integrated, microfluidic Cyto-Cartridge. Critically, all reagents and Cyto-Cartridges used are animal component-free (ACF) and sterile, thus allowing fast, robust, and safe isolation of desired cells.
Asunto(s)
Células Clonales/citología , Ensayos Analíticos de Alto Rendimiento/métodos , Análisis de la Célula Individual/métodos , Programas Informáticos , Animales , Antígenos/metabolismo , Células CHO , Células Inmovilizadas/citología , Cricetulus , Citometría de Flujo , Transferencia Resonante de Energía de Fluorescencia , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunoglobulina G/metabolismo , RatonesRESUMEN
Acoustic sensors that exploit resonating quartz crystals to directly detect the binding of an analyte to a receptor are finding increasing utility in the quantification of clinically relevant analytes. We have developed a novel acoustic detection technology, which we term resonant acoustic profiling (RAP). This technology builds on the fundamental basics of the "quartz crystal microbalance" or "QCM" with several key additional features including two- or four-channel automated sample delivery, in-line referencing and microfluidic sensor 'cassettes' that are pre-coated with easy-to-use surface chemistries. Example applications are described for the quantification of myoglobin concentration and its interaction kinetics, and for the ranking of enzyme-cofactor specificities.
Asunto(s)
Técnicas Biosensibles , Microfluídica , Cinética , Unión Proteica , Proteínas/metabolismoRESUMEN
BACKGROUND: Acoustic sensors that exploit resonating quartz crystals directly detect the binding of an analyte to a receptor. Applications include detection of bacteria, viruses, and oligonucleotides and measurement of myoglobin, interleukin 1beta (IL-1beta), and enzyme cofactors. METHODS: Resonant Acoustic Profiling was combined with a microfluidic lateral flow device incorporating an internal reference control, stable linker chemistry, and immobilized receptors on a disposable sensor "chip". Analyte concentrations were determined by analyzing the rate of binding of the analyte to an appropriate receptor. RESULTS: The specificity and affinity of antibody-antigen and enzyme-cofactor interactions were determined without labeling of the receptor or the analyte. We measured protein concentrations (recombinant human IL-1beta and recombinant human myoglobin) and quantified binding of cofactors (NADP+ and NAD+) to the enzyme glucose dehydrogenase. Lower limits of detection were approximately 1 nmol/L (17 ng/mL) for both IL-1beta and human myoglobin. The equilibrium binding constant for NADP+ binding to glucose dehydrogenase was 2.8 mmol/L. CONCLUSIONS: Resonant Acoustic Profiling detects analytes in a relatively simple receptor-binding assay in <10 min. Potential applications include real-time immunoassays and biomarker detection. Combination of this technology platform with existing technologies for concentration and presentation of analytes may lead to simple, label-free, high-sensitivity methodologies for reagent and assay validation in clinical chemistry and, ultimately, for real-time in vitro diagnostics.
Asunto(s)
Acústica , Técnicas Biosensibles/métodos , Glucosa 1-Deshidrogenasa/análisis , Interleucina-1/análisis , Mioglobina/análisis , Animales , Especificidad de Anticuerpos , Técnicas Biosensibles/instrumentación , Humanos , Ratones , Técnicas Analíticas Microfluídicas/métodos , NAD/análisis , NADP/análisis , Proteínas Recombinantes/análisis , Sensibilidad y Especificidad , Especificidad por Sustrato , Propiedades de Superficie , Factores de TiempoRESUMEN
BLM prepared on electrode substrates by supporting or tethering were tested for 'pin-hole' character, comparing data from cyclic voltammetry (CV), surface plasmon resonance (SPR) and rotating disc electrodes (RDE). 1-hexadecylamine tethered BLMs on SAM modified gold electrodes were compared with BLMs assembled on modified polyHEMA or sol-gel layers. BLM formation followed by SPR showed that the initial phase of the assembly was complete in 5-20 minutes and produced layers of thickness >5 nm, compared with the expected final BLM thickness of approximately 3 nm. The CVs of the K(3)[Fe(CN)(6)] couple were significantly suppressed irrespective of the method of BLM assembly, without major differences emerging for the different methods. However, data from the RDE distinguished the 'pin-hole' character of the different preparations. The data were consistent with incomplete initial (<1 h, SPR estimated BLM thickness >5 nm) vesicle fusion leaving 'pin-holes' of approximately 2 microm (HDA-11-mercaptoundecanoic acid (MUA) tethered BLM) to approximately 3 microm (tetraethylorthosilicate sol-gel supported BLM) followed by a slow maturation (>15 h; impedance spectroscopy estimated thickness approximately 3 nm) and lateral spreading and fusion, resulting in loss of 'pin-hole' character (<1 microm). The BLM could be used in conjunction with potentiometric measurement to observe the incorporation of nystatin into the BLM and the rate of incorporation adjusted according to original permeability of the BLM. The 'pin-hole-free' BLM construction with lowest permeability (TEOS supported, 4 x 10(-10) cm s(-1) compared with HDA-MUA, 3 x 10(-9) cm s(-1)) gave a potentiometric signal independent of bulk ion-concentration across 5 decades change in concentration. Formed on an ion-selective electrode, nystatin incorporation could be followed as a change in potential, over >2 h, whereas the TEOS supported BLM with permeability 1 x 10(-9) cm s(-1) shows nystatin incorporation within 1 h. In this instance, addition of ConA reduced the potential to the same value as prior to nystatin incorporation, consistent with nystatin channel closure.