Improved native UV laser induced fluorescence detection for single cell analysis in poly(dimethylsiloxane) microfluidic devices

Hellmich, Wibke; Greif, Dominik; Pelargus, Christoph; Anselmetti, Dario; Ros, Alexandra

doi:10.1016/j.chroma.2006.06.008

Cited by 51 publications

(40 citation statements)

References 30 publications

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“…Fluorescence is the most sensitive and popular technique used for the detection of biomolecules, cancer progression in cells and biochemical activities in microfluidic systems. Laser induced fluorescence detection in single cell microbioreactors has been reported in a number of publications [137][138][139][140].…”

Section: Optical Methodsmentioning

confidence: 99%

Microfluidic Bioreactors for Cell Culturing: A Review

Pasirayi

Auger

Scott

et al. 2011

MNS

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Section: Optical Methodsmentioning

confidence: 99%

Microfluidic Bioreactors for Cell Culturing: A Review

Pasirayi

Auger

Scott

et al. 2011

MNS

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“…Epifluorescence quickly became very popular because of the simplicity of the epifluorescence design and the availability of commercial microscopes designed with epifluorescence capability. Currently, it is quite common to find microfluidic epifluorescence with LODs of zmol or better [21][22][23][24]. However, these detectors may not be fully adequate in mitochondrial analyses in which there are a significant number of events occurring very close to the LOD [25,26].…”

Section: Introductionmentioning

confidence: 99%

Determining under‐ and oversampling of individual particle distributions in microfluidic electrophoresis with orthogonal laser‐induced fluorescence detection

et al. 2008

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This report investigates the effects of sample size on the separation and analysis of individual biological particles using microfluidic devices equipped with an orthogonal LIF detector. A detection limit of 17 6 1 molecules of fluorophore is obtained using this orthogonal LIF detector under a constant flow of fluorescein, which is a significant improvement over epifluorescence, the most common LIF detection scheme used with microfluidic devices. Mitochondria from rat liver tissue and cultured 143B osteosarcoma cells are used as model biological particles. Quantile-quantile (q-q) plots were used to investigate changes in the distributions. When the number of detected mitochondrial events became too large (.72 for rat liver and .98 for 143B mitochondria), oversampling occurs. Statistical overlap theory is used to suggest that the cause of oversampling is that separation power of the microfluidic device presented is not enough to adequately separate large numbers of individual mitochondrial events. Fortunately, q-q plots make it possible to identify and exclude these distributions from data analysis. Additionally, when the number of detected events became too small (,55 for rat liver and ,81 for 143B mitochondria) there were not enough events to obtain a statistically relevant mobility distribution, but these distributions can be combined to obtain a statistically relevant electrophoretic mobility distribution.

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“…Compared to earlier attempts with full carbon-blackened PDMS microfluidic devices on fused silica cover slides, 26 we improved the fabrication procedure in order to maintain optical transparency for chip handling in combination with strong reduction of the UV background fluorescence (k em ¼ 335 nm-385 nm). We investigated the background signals of a microfluidic channel filled with Tris buffer at pH 9.5 over a time period of 60 s. Except for reference measurements of clear PDMS, the focus is in the center of the bPDMS droplet and in the middle of the microchannel (see Fig.…”

Section: A Background Fluorescence Of Different Pdms To Cbp Ratiosmentioning

confidence: 99%

“…Consequently improving our work with label-free protein fingerprinting of single cells in microfluidic devices already published, 10,26,32 we aspire for an extension of our approach of label-free UV-LIF detection. As an example, Sf9-insect cells distinctively expressing a single GFP-labeled protein were used (c-PKC fused with GFP).…”

Section: Single Cell Experimentsmentioning

confidence: 99%

“…Based on our previous studies of UV-LIF detection and with respect to our prior experimental enhancements by adjusting laser power, pinhole size, and separation buffer properties (especially concerning dynamic surface coatings), 10,26 we report here the application of a droplet of PDMS with carbon black pigments (CBP) as additive placed only at the point of detection keeping the rest of the chip transparent. Compared to previous attempts with fully carbon-blackened PDMS chips, 26 optical control in chip handling and therefore the possibility to conduct SCA can be maintained. In order to quantify the effect of the CBP-additive, the fluorescence background intensities were measured for different PDMS/CBP ratios.…”

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confidence: 99%

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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

et al. 2012

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In single cell analysis (SCA), individual cell-specific properties and inhomogeneous cellular responses are being investigated that is not subjected to ensembleaveraging 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 ¼ 3.5) was possible, providing a theoretical limit of detection of 86 nM (with S/N ¼ 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 GFPlabeled 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 c-PKC and envisions a further feasible identification of more than one single protein in the future.

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Improved native UV laser induced fluorescence detection for single cell analysis in poly(dimethylsiloxane) microfluidic devices

Cited by 51 publications

References 30 publications

Microfluidic Bioreactors for Cell Culturing: A Review

Microfluidic Bioreactors for Cell Culturing: A Review

Determining under‐ and oversampling of individual particle distributions in microfluidic electrophoresis with orthogonal laser‐induced fluorescence detection

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

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