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Profiling protein-surface interactions of multicomponent suspensions via flow cytometry.

Langmuir : the ACS journal of surfaces and colloids (2007-12-11)
Darby Kozak, Annie Chen, Matt Trau
ABSTRACT

This study presents the use of flow cytometry as a high-throughput quantifiable technique to study multicomponent adsorption interactions between proteins and surfaces. Flow cytometry offers the advantage of high-throughput analysis of multiple parameters on a very small sampling scale. This enables flow cytometry to distinguish between individual adsorbent particles and adsorbate components within a suspension. As a proof of concept study, the adsorption of three proteins--bovine serum albumin (BSA), bovine immunoglobulin gamma (IgG) and fibrinogen--onto five surface-modified organosilica microsphere surfaces was used as a model multicomponent system for analysis. By uniquely labeling each protein and solid support type with spectrally distinguishable fluorescent dyes, the adsorption process could be "multiplexed" allowing for simultaneous screening of multiple adsorbate (protein) and adsorbent (particle surface) interactions. Protein adsorption experiments quantified by flow cytometry were found to be comparable to single-component adsorption studies by solution depletion. Quantitative distribution of the simultaneous competitive adsorption of BSA and IgG indicated that, at concentrations below surface saturation, both proteins adsorbed onto the surface. However, at concentrations greater than surface saturation, BSA preferentially adsorbed. Multiplexed particle suspensions of optically encoded particles were modified to produce a positively and negatively charged surface, a grafted 3400 MW poly(ethylene glycol) layer, or a physisorbed BSA or IgG layer. It was observed that adsorption was rapid and irreversible on all of the surfaces, and preadsorbed protein layers were the most effective in preventing further protein adsorption.

MATERIALS
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Sigma-Aldrich
Atto 488 NHS ester, BioReagent, suitable for fluorescence, ≥80% (coupling to amines)
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Atto 488 maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling to thiols)
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Atto 488 amine
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Atto 550 NHS ester, BioReagent, suitable for fluorescence, ≥80% (coupling to amines)
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Atto 550-Biotin, BioReagent, suitable for fluorescence, ≥90% (HPCE)
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Atto 550 azide, BioReagent, suitable for fluorescence, ≥80.0% (HPCE)
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Atto 488 iodoacetamide
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Atto 390 azide, suitable for fluorescence
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Atto 390 NHS ester, BioReagent, suitable for fluorescence, ≥80% (coupling to amines)
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Atto 620, BioReagent, suitable for fluorescence, ≥90% (HPCE)
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Atto 620 NHS ester, BioReagent, suitable for fluorescence, ≥80% (coupling to amines)

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