Confocal Microscopy Studies of Trypsin Immobilization on Porous Glycidyl Methacrylate Beads

Malmsten, Martin; Xing, Kezhao; Ljunglöf, Anders

doi:10.1006/jcis.1999.6540

Cited by 44 publications

(42 citation statements)

References 52 publications

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“…In our case, because we attempted a quantitative single-bead analysis RI matching between the solid skeleton of a porous particle and its saturating liquid appears most important [48,[56][57][58]. Any RI mismatch inside particles will lead to inevitable intensity loss in BODIPY  fluorescence depending on the actual penetration depth, thus, on radial position.…”

Section: Refractive Index Matchingmentioning

confidence: 99%

Perfusive flow and intraparticle distribution of a neutral analyte in capillary electrochromatography

Tallarek¹,

Pačes²,

Rapp³

2003

Electrophoresis

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The relevance and magnitude of an electroosmotic perfusion mechanism in electrochromatography is analyzed. To systemize our studies we first considered the transport of an electroneutral and nonadsorbing tracer. Based on the refractive index matching in a microfluidic setup containing fixed spherical porous particles, we conducted a quantitative analysis in real time of the spatio-temporal distribution of fluorescent tracer molecules during their uptake by (and a release from) single particles using confocal laser scanning microscopy. Even under conditions of a significant electrical double layer overlap the intraparticle electroosmotic flow produces due to its unidirectional nature and in striking contrast to the symmetric (spherical) distributions typical for purely diffusive transport strongly asymmetric concentration profiles inside spherical particles as the locally charged pore liquid begins to respond to the externally applied electrical field. The profiles retain an axisymmetric nature, i.e., rotational symmetry with respect to the field direction. Results of our measurements could be successfully interpreted and further analyzed by a compact mathematical model. Intraparticle Peclet numbers of up to 150 have been realized and found to significantly enhance the mass transport on particle scale towards the convection-dominated regime when compared to a conventional (diffusion-limited) kinetics.

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Section: Refractive Index Matchingmentioning

confidence: 99%

Perfusive flow and intraparticle distribution of a neutral analyte in capillary electrochromatography

Tallarek¹,

Pačes²,

Rapp³

2003

Electrophoresis

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“…The immobilized-enzyme reactors are used to digest proteins through a heterogeneous liquid-solid interface, and this concept of enzyme immobilization on a solid support for protein digestion has gained much attention because it offers advantages such as good reproducibility, attributed to the reduced enzyme denaturation rate, and reusability, attributed to the reduced or almost avoidable enzyme autoproteolysis during or after the digestion [38][39][40][41][42][43][44][45][46][47][48][49][50][51].…”

Section: Immobilized-enzyme Reactor For On-line Protein Digestionmentioning

confidence: 99%

“…Years later, supports based on microchips [39][40][41][42], porous beads [43][44][45][46], micro membranes [47][48], micro channels [49], silica-based monoliths [50][51] have been developed. These supports have a similarity, i.e.…”

Section: Immobilized-enzyme Reactor For On-line Protein Digestionmentioning

confidence: 99%

Development of Micro-Flow-Controlled Techniques and Novel Stationary Phases in Capillary Liquid Chromatography

Lim

2015

Chromatography

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This focusing review summarizes our recent works on the dissemination of capillary liquid chromatography through various approaches, i.e. simple stepwise-gradient elution system, on-line sample enrichment system, alternate-pumping recycle chromatography system, on-line immobilized-enzyme reactor, development of novel stationary phases, etc. Capillary liquid chromatography saves energy and waste, and it possesses several advantages over the conventional liquid chromatography; however, it has not been much popularized since its appearance and most users still prefer to use conventional liquid chromatography if there is a choice between them. Our research focuses on, but not limited to, developing versatile capillary liquid chromatography systems that are not only environmental-friendly but also user-friendly, and novel stationary phases that could be operated under mixed modes conditions by manipulating the eluent, which are applicable to real samples analyses and will therefore eventually switch the users from "conventional" to "capillary".

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“…This holds true for both scanning modes commonly applied, namely horizontal and vertical scanning ( Table 3, H/V). A common method to evaluate and correct for light attenuation effects is the so-called 'refractive index matching' technique which is based on the addition of additives, e.g., aqueous sugar solution [30], benzyl alcohol [66], dimethyl sulfoxide [29,33], in order to adjust the refractive index of the fluid phase. When using this approach, however, CLSM analysis only reflects a significantly altered chromatographic system caused by the changes in fluid phase conditions.…”

Section: Light Attenuation Effectsmentioning

confidence: 99%

Confocal laser scanning microscopy as an analytical tool in chromatographic research

Hubbuch

Kula

2008

Bioprocess Biosyst Eng

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In recent years, confocal laser scanning microscopy has been developed into a non-invasive tool to probe intra-particle profiles of protein in chromatographic adsorbents. A necessary prerequisite when using this technique lies in the labeling of proteins with fluorescent probes. The quality of the obtained results is thus strongly dependent on the probes used, its sensitivity on experimental parameters and the change of protein characteristics upon binding. In this review, the fundamental issues when using fluorescent probes are described, before giving a critical evaluation on published literature in the field of confocal laser scanning microscopy for the analysis of chromatographic principles.

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Confocal Microscopy Studies of Trypsin Immobilization on Porous Glycidyl Methacrylate Beads

Cited by 44 publications

References 52 publications

Perfusive flow and intraparticle distribution of a neutral analyte in capillary electrochromatography

Perfusive flow and intraparticle distribution of a neutral analyte in capillary electrochromatography

Development of Micro-Flow-Controlled Techniques and Novel Stationary Phases in Capillary Liquid Chromatography

Confocal laser scanning microscopy as an analytical tool in chromatographic research

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