A fluorescent microbead-based microfluidic immunoassay chip for immune cell cytokine secretion quantification

Abstract

Quantitative and dynamic analyses of immune cell secretory cytokines are essential for precise determination and characterization of the "immune phenotype" of patients for clinical diagnosis and treatment of immune-related diseases. Although multiple methods including the enzyme-linked immunosorbent assay (ELISA) have been applied for cytokine detection, such measurements remain very challenging in real-time, high-throughput, and high-sensitivity immune cell analysis. In this paper, we report a highly integrated microfluidic device that allows for on-chip isolation, culture, and stimulation, as well as sensitive and dynamic cytokine profiling of immune cells. Such a microfluidic sensing chip is integrated with cytometric fluorescent microbeads for real-time and multiplexed monitoring of immune cell cytokine secretion dynamics, consuming a relatively small extracted sample volume (160 nl) without interrupting the immune cell culture. Furthermore, it is integrated with a Taylor dispersion-based mixing unit in each detection chamber that shortens the immunoassay period down to less than 30 minutes. We demonstrate the profiling of multiple pro-inflammatory cytokine secretions (e.g. interleukin-6, interleukin-8, and tumor necrosis factors) of human peripheral blood mononuclear cells (PBMCs) with a sensitivity of 20 pg ml and a sample volume of 160 nl per detection. Further applications of this automated, rapid, and high-throughput microfluidic immunophenotyping platform can help unleash the mechanisms of systemic immune responses, and enable efficient assessments of the pathologic immune status for clinical diagnosis and immune therapy.

Microfluidic implementation of functional cytometric microbeads for improved multiplexed cytokine quantification

“…This testing device is based on our recently reported work. 40 Each detection region contains a microchamber (diameter: 1 mm; height: 1 mm; and volume: 0.79 μ l) and a bypass channel. The device contains three layers: control/mixing layer ( red/blue ), flow layer ( black ), and chamber layer ( green ).…”

“…For example, the dynamic variations of cytokine levels in the body reflect more representatively the corresponding immune status and immune diseases. 39 Recently, we have reported a microfluidic mixing-assisted multiplexed dynamic cytokine profiling strategy compatible with the commercially available cytometric microbeads, 40 yet the configuration parameters for the liquid handling, reagent mixing, and measurement performance have not been optimized. This work describes the required procedures and expected results of configuring the measurement parameters (e.g., reagent mixing time and image analysis strategy) for the multiplex molecular quantification with the reduced detection limit and process time.…”

Analysis of Leukocyte Behaviors on Microfluidic Chips

“…The gates are opened after cell stimulation for cytokine–bead interaction and detection. Reproduced with permission . Copyright 2018, The Royal Society of Chemistry.…”

“…Besides, detection of leukocyte secreted cytokines is also informative to determine the “immune phenotype” and even the disease‐related pathways of patients. Cui et al developed a comprehensive microfluidic device to dynamically quantify cytokine secretion with the use of fluorescent microbeads. Their device has separate regions for leukocyte culture and stimulation, and the cytokine detection was performed after leukocyte stimulation following the steps shown in Figure B.…”

Biofunctionalization of Glass‐ and Paper‐Based Microfluidic Devices: A Review

“…Microfluidic devices and lab‐on‐a‐chip technology have been distinguished as a promising platform for ultrasensitive bioanalytical devices, point‐of‐care (POC) diagnostics and health monitoring systems, investigation of cell biology and tissue engineering, mixing and separation of biospecies, drug discovery, biohazard detection, as well as automating synthetic biology to create micro‐organism and DNA foundries . Incorporating microfluidics in different bioanalytical systems such as those based on fluorescence, colorimetric, electrochemical, and chemiluminescence readout, as well as mass spectrometry has significantly enhanced the limit of detection (LOD) of these assays . In cell biology, microfluidics enables the integration of multiple cell lines to mimic “organs‐on‐chips” with the desired physiological conditions and hemodynamic forces.…”