Label-free real-time imaging in microchip free-flow electrophoresis applying high speed deep UV fluorescence scanning

Köhler, Stefan; Nagl, Stefan; Fritzsche, Stefanie; Belder, Detlev

doi:10.1039/c1lc20558g

Cited by 33 publications

(32 citation statements)

References 78 publications

(76 reference statements)

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“…glass-PEG-glass FFE chips [53], or fused silica FFE microchips which allow the observation of unlabelled proteins via their UV fluorescence [54].…”

Section: Discussionmentioning

confidence: 99%

“…The pH sensor assembly integration could be adapted to work with miniaturized FFE platforms of other dimensions or materials, for example, glass-PEG-glass FFE chips [53] or fused-silica FFE microchips that allow the observation of unlabeled proteins via their UV fluorescence [54]. The developed optical setup and sensor integration technique could also be useful in other microfluidic systems for the precise and fast online observation of pH, for example in chemical reaction monitoring [55], separation or in cell culture systems [56].…”

Section: Discussionmentioning

confidence: 99%

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Development of microscopic time‐domain dual lifetime referencing luminescence detection for pH monitoring in microfluidic free‐flow isoelectric focusing

Poehler

Herzog

Suendermann

et al. 2015

Engineering in Life Sciences

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www.els-journal.com Page 3 Engineering in Life SciencesThis article is protected by copyright. All rights reserved. AbstractA lifetime-based ratiometric microscale pH sensor system and a fluorescence microscopic setup was developed for the in-line observation of pH in free-flow isoelectric focusing based on the principle of time-domain dual lifetime referencing (t-DLR). The t-DLR method has been developed for pH monitoring and various other applications in the fields of environmental monitoring and biotechnology. Here, we introduce the integration of pH sensor microstructures for t-DLR in microfluidic channels and the application of the t-DLR scheme for pH sensing in miniaturized electrophoretic procedures. The pH sensor was inkjet-printed on glass in rows with a length of 10 mm, a height of 404 ± 18 nm and a width of 371 ± 28 µm, and integrated into a microfluidic chip generated by a lasercutting and lamination technique. It had a working range from pH 4 to 8 with a pK A of 6.10 ± 0.01 and fast response times under 500 ms. The sensor was used for the in-line observation of the pH gradient during isoelectric focussing of the proteins β-lactoglobulin A, conalbumin and myoglobin and their identification by their isoelectric point (pI). The obtained pIs were in good agreement with literature data demonstrating the applicability of the pH sensor in microfluidic continuous separations.

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“…glass-PEG-glass FFE chips [53], or fused silica FFE microchips which allow the observation of unlabelled proteins via their UV fluorescence [54].…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Development of microscopic time‐domain dual lifetime referencing luminescence detection for pH monitoring in microfluidic free‐flow isoelectric focusing

Poehler

Herzog

Suendermann

et al. 2015

Engineering in Life Sciences

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“…Deep UV excited fluorescence is capable of monitoring proteins, peptides and other biomolecules via excitation of aromatic residues such as tryptophan, tyrosine and other groups. It had previously been demonstrated to be useful for monitoring of unlabeled biomolecules in several variants of electrophoresis including μFFE .…”

Section: Fluorescent Ph Sensing In Isoelectric Focusingmentioning

confidence: 99%

Micro free‐flow isoelectric focusing with integrated optical pH sensors

Nagl

2017

Engineering in Life Sciences

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Recently, a new observation method for monitoring of pH gradients in microfluidic free‐flow electrophoresis has emerged. It is based on the use of chip‐integrated fluorescent or luminescent micro sensor layers. These are able to monitor pH gradients in miniaturized separations in real time and spatially resolved; this is particularly useful in isoelectric focusing. Here these multifunctional microdevices that feature continuous separation, monitoring, and in some instances other functionalities, are reviewed. The employed microfabrication procedures to produce these devices are discussed and the different pH sensor matrices that were integrated and their applications in the separation of different types of biomolecules. The procedures for obtaining spatially resolved information about the separated molecules and the pH at the same time and different detection modalities to achieve this such as deep UV fluorescence as well as time‐resolved referenced pH sensing and the integration of a precolumn labeling step into these platforms are also highlighted.

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“…These authors also experimented quartz glass microfluidic chips decreasing the “parasite” fluorescence of the chip resulting in micromolar LOD for α‐chymotrypsinogen A, ovalbumin, and catalase . A fused‐silica chip was designed for the separation of Trp, serotonin (SER), and propranolol with a novel high‐speed deep UV fluorescence scanner, all analyte bands could be traced in one live image . A deep UV fluorescence lifetime detector based on a collinear arrangement was also built to detect small molecules and proteins.…”

Section: Applicationsmentioning

confidence: 99%

Capillary electrophoresis hyphenated with UV‐native‐laser induced fluorescence detection (CE/UV‐native‐LIF)

2016

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Native laser-induced fluorescence using UV lasers associated to CE offers now a large related literature, for now 30 years. The main works have been performed using very expensive Ar-ion lasers emitting at 257 and 275 nm. They are not affordable for routine analyses, but have numerous applications such as protein, catecholamine, and indolamine analysis. Some other lasers such as HeCd 325 nm have been used but only for few applications. Diode lasers, emitting at 266 nm, cheaper, are extensively used for the same topics, even if the obtained sensitivity is lower than the one observed using the costly UV-Ar-ion lasers. This review presents various CE or microchips applications and different UV lasers used for the excitation of native fluorescence. We showed that CE/Native UV laser induced fluorescence detection is very sensitive for detection as well as small aromatic biomolecules than proteins containing Trp and Tyr amino acids. Moreover, it is a simple way to analyze biomolecules without derivatization.

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Label-free real-time imaging in microchip free-flow electrophoresis applying high speed deep UV fluorescence scanning

Cited by 33 publications

References 78 publications

Development of microscopic time‐domain dual lifetime referencing luminescence detection for pH monitoring in microfluidic free‐flow isoelectric focusing

Development of microscopic time‐domain dual lifetime referencing luminescence detection for pH monitoring in microfluidic free‐flow isoelectric focusing

Micro free‐flow isoelectric focusing with integrated optical pH sensors

Capillary electrophoresis hyphenated with UV‐native‐laser induced fluorescence detection (CE/UV‐native‐LIF)

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