Single cell analysis in full body quartz glass chips with native UV laser-induced fluorescence detection

Greif, Dominik; Galla, Lukas; Ros, Alexandra; Anselmetti, Dario

doi:10.1016/j.chroma.2008.07.013

Cited by 27 publications

(29 citation statements)

References 34 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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“…In order to separate and analyze label-free biological compounds like proteins from single cells, microfluidics provides an effective and sensitive investigation tool in combination with UV laser induced fluorescence (UV-LIF) detection. 10,11 Promising alternative detection systems are integrated optical waveguides and electrochemical detectors for direct sensing in microchannels. 12 Therefore, Shadpour et al reported a limit of detection (LOD) of 3.3 lM for proteins evaluated with the first multichannel microchip electrophoresis (MCE) device with integrated contact conductivity measurements.…”

Section: B)mentioning

confidence: 99%

“…For analyte injection and separation, the pinched injection method was used. Single cell experiments were performed as previously published 10,20 and can be briefly divided into four steps. After isolating and guiding a single cell of interest to the intersection position of the microdevice via hydraulic pressure (comparable to Ref.…”

Section: E Chip Operationsmentioning

confidence: 99%

“…By using full body quartz glass chips, Greif et al were able to reduce the theoretical LOD for tryptophan down to 2.5 nM. 10 However, due to its simple and low-cost processing for disposable devices, 11 polydimethylsiloxane (PDMS) as an alternative material for the fabrication of microfluidic devices yields a number of advantages. Unfortunately, conventional PDMS formulations often suffer from relatively high autofluorescence intensities around 360 nm, making useful and sensitive applications in UV-LIF detection a challenging task.…”

mentioning

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.…”

mentioning

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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Electrophoresis microchip with integrated waveguides for simultaneous native UV fluorescence and absorbance detection

et al. 2009

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Simultaneous label-free detection of UV absorbance and native UV-excited fluorescence in an electrophoresis microchip is presented. UV transparent integrated waveguides launch light at a wavelength of 254 nm from a mercury lamp along the length of a 1-mm long detection cell. Transmitted UV light is collected by another waveguide in the opposite end of the detection cell, while visible fluorescence is collected vertically through the lid of the chip. The background of scattered excitation light is suppressed by detection perpendicular to the excitation, the limited UV transparency of the borosilicate lid and by choosing a PMT insensitive to the excitation light. This way, the need for a fluorescence filter is eliminated. Calibration curves were measured for serotonin, tryptophan, propranolol and acetaminophen, and separations of the four compounds were demonstrated by electrophoresis and MEKC. All compounds could be detected in the micromolar range by absorbance detection, but fluorescence detection improved detection limits for compounds displaying native UV fluorescence up to ten times. The simultaneous detection also proved useful for the identification of compounds with similar retention times and even enables accurate quantification of co-eluting compounds.

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Single cell analysis in full body quartz glass chips with native UV laser-induced fluorescence detection

Cited by 27 publications

References 34 publications

Microfluidic Bioreactors for Cell Culturing: A Review

Microfluidic Bioreactors for Cell Culturing: A Review

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

Electrophoresis microchip with integrated waveguides for simultaneous native UV fluorescence and absorbance detection

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