研究目的
To develop an ultrasensitive and simultaneous detection method for two cytokines (VEGF and IL-8) secreted by single cells using a SERS-microfluidic droplet platform with magnetic field amplification.
研究成果
The SERS-microfluidic droplet platform enables ultrasensitive and simultaneous detection of VEGF and IL-8 at the single-cell level, with a detection limit of 1.0 fg/mL. It reveals cell-to-cell heterogeneity in cytokine secretion and suggests that cell-cell interactions up-regulate VEGF and IL-8, potentially promoting angiogenesis in cancer cells. This method is promising for large-scale studies of cytokine roles in tumor biology.
研究不足
The method may have limitations in handling very high cell densities or complex biological samples, and the microfluidic setup requires precise control of flow rates and conditions. Potential optimizations include improving droplet stability and expanding to more cytokines or cell types.
1:Experimental Design and Method Selection:
The study uses a SERS-microfluidic droplet platform combined with magnetic field amplification for high-sensitivity detection. It involves immune-sandwich configurations with antibody-conjugated nanoparticles and magnetic beads, and microfluidic droplet generation for single-cell encapsulation.
2:Sample Selection and Data Sources:
Cancer cell lines (MDA-MB-231, A549, SGC) were used, stimulated with PMA to secrete cytokines. Samples included droplets containing single cells and immune-particles.
3:List of Experimental Equipment and Materials:
Microfluidic chip, Horiba J-Y Aramis spectrometer, TEM (JEOL JEM-2100F), UV-Vis spectrometer (Ocean Optics USB4000), DLS (Malvern Zetasizer Nano ZS), Fluorinert HFE-7500 oil, PFPE-PEG surfactant, antibodies, silver nanoparticles, magnetic beads, Raman reporters (4-ABP, AAD).
4:Experimental Procedures and Operational Workflow:
Droplets were formed using a cross-typed microfluidic chip with specific flow rates. Cells and immune-particles were encapsulated, and SERS measurements were performed after magnetic field-induced aggregation. Data were collected using a Raman spectrometer.
5:Data Analysis Methods:
SERS spectra were analyzed, with intensity ratios used for quantification. Linear models were fitted for calibration curves, and detection limits were calculated based on signal-to-noise ratios.
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Transmission Electron Microscope
JEM-2100F
JEOL
Used to measure the size and morphology of immune-particles.
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UV-Vis Spectrometer
USB4000
Ocean Optics
Used to measure the plasmonic properties of nanoparticles.
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Dynamic Light Scattering Spectrometer
Zetasizer Nano ZS
Malvern
Used to measure the hydrodynamic size and zeta potential of particles.
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Raman Spectrometer
Aramis
Horiba J-Y
Used for SERS measurements with a 632.8 nm laser.
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Fluorocarbon Oil
HFE-7500
3M
Used as the oil phase for suspending microfluidic droplets.
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Surfactant
PFPE-PEG block-copolymer
Ranbiotech
Added to oil to stabilize droplets against coalescence.
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Microfluidic Chip
Cross-typed
Used to generate water-in-oil droplets for single-cell encapsulation.
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