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Biomicrofluidics / Volume 6 / Issue 1 / REGULAR ARTICLES

Biomicrofluidics 6, 014104 (2012); http://dx.doi.org/10.1063/1.3675608 (10 pages)

Microfluidic carbon-blackened polydimethylsiloxane device with reduced ultra violet background fluorescence for simultaneous two-color ultra violet/visible-laser induced fluorescence detection in single cell analysis

Lukas Galla, Dominik Greif, Jan Regtmeier, and Dario Anselmetti

Experimental Biophysics and Applied Nanoscience, Faculty of Physics, Bielefeld University, 33615 Bielefeld, Germany

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(Received 4 August 2011; accepted 16 December 2011; published online 12 January 2012)

In single cell analysis (SCA), individual cell-specific properties and inhomogeneous cellular responses are being investigated that is not subjected to ensemble-averaging or heterogeneous cell population effects. For proteomic single cell analysis, ultra-sensitive and reproducible separation and detection techniques are essential. Microfluidic devices combined with UV laser induced fluorescence (UV-LIF) detection have been proposed to fulfill these requirements. Here, we report on a novel microfluidic chip fabrication procedure that combines straightforward production of polydimethylsiloxane (PDMS) chips with a reduced UV fluorescence background (83%-reduction) by using PDMS droplets with carbon black pigments (CBP) as additives. The CBP-droplet is placed at the point of detection, whereas the rest of the chip remains transparent, ensuring full optical control of the chip. We systematically studied the relation of the UV background fluorescence at CBP to PDMS ratios (varying from 1:10 to 1:1000) for different UV laser powers. Using a CBP/PDMS ratio of 1:20, detection of a 100 nM tryptophan solution (S/N = 3.5) was possible, providing a theoretical limit of detection of 86 nM (with S/N = 3). Via simultaneous two color UV/VIS-LIF detection, we were able to demonstrate the electrophoretic separation of an analyte mixture of 500 nM tryptophan (UV) and 5 nM fluorescein (VIS) within 30 s. As an application, two color LIF detection was also used for the electrophoretic separation of the protein content from a GFP-labeled single Spodoptera frugiperda (Sf9) insect cell. Thereby just one single peak could be measured in the visible spectral range that could be correlated with one single peak among others in the ultraviolet spectra. This indicates an identification of the labeled protein γ-PKC and envisions a further feasible identification of more than one single protein in the future.

© 2012 American Institute of Physics

Article Outline

  1. INTRODUCTION
  2. EXPERIMENTAL
    1. Chemicals and reagents
    2. Chip fabrication
    3. Fluorescence detection
    4. Cell culture
    5. Chip operations
  3. RESULTS AND DISCUSSION
    1. Background fluorescence of different PDMS to CBP ratios
    2. Analyte injection and separation
    3. Single cell experiments
  4. CONCLUSION

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KEYWORDS, PACS, and IPC

Keywords

biochemistry, biological techniques, bioMEMS, carbon, cellular biophysics, electrophoresis, fluorescence, lab-on-a-chip, microfluidics, proteins, separation

PACS

  • 87.80.Ek

    Mechanical and micromechanical techniques

  • 87.80.Qk

    Biochemical separation processes

  • 47.85.-g

    Applied fluid mechanics

  • 82.45.-h

    Electrochemistry and electrophoresis

  • 85.85.+j

    Micro- and nano-electromechanical systems (MEMS/NEMS) and devices

  • 87.17.-d

    Cell processes

  • 87.15.R-

    Reactions and kinetics

International Patent Classification (IPC)

  • B01D

    Separation

  • B01D57/02

    By electrophoresis

  • B81B

    Micro-structural devices or systems, e.g. micro-mechanical devices

  • C12

    Biochemistry; Beer; Spirits; Wine; Vinegar; Microbiology; Enzymology; Mutation or genetic engineering

  • C25

    Electrolytic or electrophoretic processes; Apparatus therefor

  • F15D

    Fluid dynamics, i.e. methods or means for influencing the flow of gases or liquids

ARTICLE DATA

Digital Object Identifier
http://dx.doi.org/10.1063/1.3675608

PUBLICATION DATA

ISSN

1932-1058 (online)

Publisher

$abstract.society.value is a Member of CrossRef American Institute of Physics

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