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A microfluidic platform for high-throughput multiplexed protein quantitation.

ABSTRACT: 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-1?, TNF-?, 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.

SUBMITTER: Volpetti F

PROVIDER: S-EPMC4334502 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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A microfluidic platform for high-throughput multiplexed protein quantitation.

Volpetti Francesca F Garcia-Cordero Jose J Maerkl Sebastian J SJ

PloS one 20150213 2

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 ...[more]

PMID: 25680117

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Project description:Adoptive cell transfer against solid tumors faces challenges to overcome tumor microenvironment (TME), which plays as a physical barrier and provides immuno-suppressive conditions. Classical cytotoxicity assays are widely used to measure killing ability of the engineered cytotoxic lymphocytes as therapeutics, but the results cannot represent the performance in clinical application due to the absence of the TME. This paper describes a 3D cytotoxicity assay using an injection molded plastic array culture (CACI-IMPACT) device for 3D cytotoxicity assay to assess killing abilities of cytotoxic lymphocytes in 3D microenvironment through a spatiotemporal analysis of the lymphocytes and cancer cells embedded in 3D extra cellular matrix (ECM). Rail-based microfluidic design was integrated within a single 96-well and the wells were rectangularly arrayed in 2 × 6 to enhance the experimental throughput. The rail-based microstructures facilitate hydrogel patterning with simple pipetting so that hydrogel pre-solution aspirated with 10 μl pipette can be patterned in 10 wells within 30 s. To demonstrate 3D cytotoxicity assay, we patterned HeLa cells encapsulated by collagen gel and observed infiltration, migration and cytotoxic activity of NK-92 cells against HeLa cells in the collagen matrix. We found that 3D ECM significantly reduced migration of cytotoxic lymphocytes and access to cancer cells, resulting in lower cytotoxicity compared with 2D assays. In dense ECM, the physical barrier function of the 3D matrix was enhanced, but the cytotoxic lymphocytes effectively killed cancer cells once they contacted with cancer cells. The results implied ECM significantly influences migration and cytotoxicity of cytotoxic lymphocytes. Hence, the CACI-IMPACT platform, enabling high-throughput 3D co-culture of cytotoxic lymphocyte with cancer cells, has the potential to be used for pre-clinical evaluation of cytotoxic lymphocytes engineered for immunotherapy against solid tumors.

Project description:BackgroundBiomarkers play a key role in risk assessment, assessing treatment response, and detecting recurrence and the investigation of multiple biomarkers may also prove useful in accurate prediction and prognosis of cancers. Immunohistochemistry (IHC) has been a major diagnostic tool to identify therapeutic biomarkers and to subclassify breast cancer patients. However, there is no suitable IHC platform for multiplex assay toward personalized cancer therapy. Here, we report a microfluidics-based multiplexed IHC (MMIHC) platform that significantly improves IHC performance in reduction of time and tissue consumption, quantification, consistency, sensitivity, specificity and cost-effectiveness.Methodology/principal findingsBy creating a simple and robust interface between the device and human breast tissue samples, we not only applied conventional thin-section tissues into on-chip without any additional modification process, but also attained perfect fluid control for various solutions, without any leakage, bubble formation, or cross-contamination. Four biomarkers, estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR) and Ki-67, were examined simultaneously on breast cancer cells and human breast cancer tissues. The MMIHC method improved immunoreaction, reducing time and reagent consumption. Moreover, it showed the availability of semi-quantitative analysis by comparing Western blot. Concordance study proved strong consensus between conventional whole-section analysis and MMIHC (n = 105, lowest Kendall's coefficient of concordance, 0.90). To demonstrate the suitability of MMIHC for scarce samples, it was also applied successfully to tissues from needle biopsies.Conclusions/significanceThe microfluidic system, for the first time, was successfully applied to human clinical tissue samples and histopathological diagnosis was realized for breast cancers. Our results showing substantial agreement indicate that several cancer-related proteins can be simultaneously investigated on a single tumor section, giving clear advantages and technical advances over standard immunohistochemical method. This novel concept will enable histopathological diagnosis using numerous specific biomarkers at a time even for small-sized specimens, thus facilitating the individualization of cancer therapy.

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A microfluidic platform for high-throughput multiplexed protein quantitation.

Publications

A microfluidic platform for high-throughput multiplexed protein quantitation.

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