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 −1 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.
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Pd@Pt Nanodendrites as Peroxidase Nanomimics for Enhanced Colorimetric ELISA of Cytokines with Femtomolar Sensitivity
Colorimetric enzyme-linked immunosorbent assay (ELISA) has been widely applied as the gold-standard method for cytokine detection for decades. However, it has become a critical challenge to further improve the detection sensitivity of ELISA, as it is limited by the catalytic activity of enzymes. Herein, we report an enhanced colorimetric ELISA for ultrasensitive detection of interleukin-6 (IL-6, as a model cytokine for demonstration) using Pd@Pt core@shell nanodendrites (Pd@Pt NDs) as peroxidase nanomimics (named “Pd@Pt ND ELISA”), pushing the sensitivity up to femtomolar level. Specifically, the Pd@Pt NDs are rationally engineered by depositing Pt atoms on Pd nanocubes (NCs) to generate rough dendrite-like Pt skins on the Pd surfaces via Volmer–Weber growth mode. They can be produced on a large scale with highly uniform size, shape, composition, and structure. They exhibit significantly enhanced peroxidase-like catalytic activity with catalytic constants (Kcat) more than 2000-fold higher than those of horseradish peroxidase (HRP, an enzyme commonly used in ELISA). Using Pd@Pt NDs as the signal labels, the Pd@Pt ND ELISA presents strong colorimetric signals for the quantitative determination of IL-6 with a wide dynamic range of 0.05–100 pg mL−1 and an ultralow detection limit of 0.044 pg mL−1 (1.7 fM). This detection limit is 21-fold lower than that of conventional HRP-based ELISA. The reproducibility and specificity of the Pd@Pt ND ELISA are excellent. More significantly, the Pd@Pt ND ELISA was validated for analyzing IL-6 in human serum samples with high accuracy and reliability through recovery tests. Our results demonstrate that the colorimetric Pd@Pt ND ELISA is a promising biosensing tool for ultrasensitive determination of cytokines and thus is expected to be applied in a variety of clinical diagnoses and fundamental biomedical studies.
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- Award ID(s):
- 1943302
- NSF-PAR ID:
- 10414294
- Date Published:
- Journal Name:
- Chemosensors
- Volume:
- 10
- Issue:
- 9
- ISSN:
- 2227-9040
- Page Range / eLocation ID:
- 359
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/chemosensors10090359
