Intracellular Protein Determination Using Droplet-Based Immunoassays

Martino, Chiara; Zagnoni, Michele; Sandison, Mairi E.; Chanasakulniyom, Mayuree; Pitt, Andrew R.; Cooper, Jonathan M.

doi:10.1021/ac200876q

Cited by 50 publications

(44 citation statements)

References 41 publications

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“…Integration minimizes contamination and loss of sample, making the whole process more reliable and applicable to small samplesuseful in many clinical settings where the tissue is often limited. In addition to measuring changes in mRNA expression, the tissue-based microfluidic system can be coupled to other 'downstream' analysis modules, eg, spectrophotometric/fluorescent detection of protein release, 30 individual cells can be liberated and changed to the cell surface analyzed by on-chip flow cytometry, 24 and oligonucleotide hybridization arrays can be used to assess global changes in gene expression. 31 The combination of these technologies together with the RT-PCR described here has the potential to offer a new platform technology for studying normal or diseased tissue.…”

Section: Discussionmentioning

confidence: 99%

Integrated RNA extraction and RT-PCR for semi-quantitative gene expression studies on a microfluidic device

Shaw

Hughes

Dyer

et al. 2013

Laboratory Investigation

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This paper describes the development of a microfluidic methodology, using RNA extraction and reverse transcription PCR, for investigating expression levels of cytochrome P450 genes. Cytochrome P450 enzymes are involved in the metabolism of xenobiotics, including many commonly prescribed drugs, therefore information on their expression is useful in both pharmaceutical and clinical settings. RNA extraction, from rat liver tissue or primary rat hepatocytes, was performed using a silica-based solid-phase extraction technique. Following elution of the purified RNA, amplification of target sequences for the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the cytochrome P450 gene CYP1A2, was carried out using a one-step reverse transcription PCR. Once the microfluidic methodology had been optimized, analysis of control and 3-methylcholanthrene-induced primary rat hepatocytes were used to evaluate the system. As expected, GAPDH was consistently expressed, whereas CYP1A2 levels were found to be raised in the drug-treated samples. The proposed system offers an initial platform for development of both rapid throughput analyzers for pharmaceutical drug screening and point-of-care diagnostic tests to aid provision of drug regimens, which can be tailor-made to the individual patient.Laboratory Investigation (2013) 93, 961-966;

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Section: Discussionmentioning

confidence: 99%

Integrated RNA extraction and RT-PCR for semi-quantitative gene expression studies on a microfluidic device

Shaw

Hughes

Dyer

et al. 2013

Laboratory Investigation

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“…Control over the local chemical environment surrounding each cell is a distinguishing characteristic, because it can permit experiments that are hard to envision with flow cytometry or ELISpot. Microdroplets also permit assays on quantized cell populations and, through calibration with fluorescently labeled beads, microdroplet immunoassays can yield absolute quantitation of protein copy numbers (64). Such calibrated assays, however, have not been applied to single cells but only to very small volumes of cell lysate.…”

Section: Tools Using Cellular Staining Assaysmentioning

confidence: 99%

Microfluidics-Based Single-Cell Functional Proteomics for Fundamental and Applied Biomedical Applications

Sutherland

Wei

et al. 2014

Annual Rev. Anal. Chem.

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We review an emerging microfluidics-based toolkit for single-cell functional proteomics. Functional proteins include, but are not limited to, the secreted signaling proteins that can reflect the biological behaviors of immune cells or the intracellular phosphoproteins associated with growth factor-stimulated signaling networks. Advantages of the microfluidics platforms are multiple. First, 20 or more functional proteins may be assayed simultaneously from statistical numbers of single cells. Second, cell behaviors (e.g., motility) may be correlated with protein assays. Third, extensions to quantized cell populations can permit measurements of cell-cell interactions. Fourth, rare cells can be functionally identified and then separated for further analysis or culturing. Finally, certain assay types can provide a conduit between biology and the physicochemical laws. We discuss the history and challenges of the field then review design concepts and uses of the microchip platforms that have been reported, with an eye toward biomedical applications. We then look to the future of the field. 275

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“…In addition to cell lysis, a droplet-extraction system can be used to extract acidic analytes from urine; this is followed by capillary electrophoresis and laser-induced fluorescence detection [217]. The passive isolation of T Fig.…”

Section: Sample Preparationmentioning

confidence: 99%