Protein Labeling Enhances Aptamer Selection by Methods of Kinetic Capillary Electrophoresis

Jong, Stephanie de; Krylov, Sergey N.

doi:10.1021/ac201242r

Cited by 18 publications

(13 citation statements)

References 31 publications

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“…To increase the sensitivity of this technique, all proteins were fluorescently labeled with Chromeo P503. This enabled the use of much lower sample concentrations than otherwise necessary for UV detection .…”

Section: Resultsmentioning

confidence: 99%

A semipermanent coating for preventing protein adsorption at physiological pH in kinetic capillary electrophoresis

Jong¹,

Epelbaum²,

Liyanage³

et al. 2012

Electrophoresis

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Protein adsorption to the inner capillary wall hinders the use of kinetic capillary electrophoresis (KCE) when studying noncovalent protein-ligand interactions. Permanent and dynamic capillary coatings have been previously reported to alleviate much of the problems associated with protein adsorption. The characteristic limitations associated with permanent and dynamic coatings motivated us to look at a third type of coating - semipermanent. Here, we demonstrate that a semipermanent capillary coating, designed by Lucy and co-workers, comprised of dioctadecyldimethylammonium bromide (DODAB) and polyoxyethylene (POE) stearate, greatly reduces protein adsorption at physiological pH - a necessary requirement for KCE. The coating (i) does not inhibit protein-DNA complex formation, (ii) prevents the adsorption of the analytes, and (iii) supports an electoosmotic flow required for many applications of KCE. The coating was tested in three physiological buffers using a well-known DNA aptamer and four proteins that severely bind to bare silica capillaries as standards. For every protein, a condition was found under which the semipermanent coating effectively suppresses protein adhesion. While no coating can completely prevent the adsorption of all proteins, our findings suggest that the DODAB/POE stearate coating can have a broad impact on CE at large, as it prevents the absorption of several well studied, highly adhesive proteins at physiological pH.

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Section: Resultsmentioning

confidence: 99%

A semipermanent coating for preventing protein adsorption at physiological pH in kinetic capillary electrophoresis

Jong¹,

Epelbaum²,

Liyanage³

et al. 2012

Electrophoresis

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“…As a new fluorescence dye, Chromeo P503 was tested for use for the fluorescent labeling of bovine serum albumin, human serum albumin, R1-acid glycoprotein, myoglobin and R-lactalbumin. It has been proved that the dye does not affect the electrophoretic properties of these proteins or the binding of the target protein and library, and does not react with the electrolytes in running buffer such as tris and glycine containing an amino group (De Jong & Krylov, 2011).…”

Section: Kinetic Cementioning

confidence: 99%

Capillary electrophoresis‐based methodology for screening of oligonucleotide aptamers

Guo

Chen

2021

Biomedical Chromatography

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As a new molecular recognition element, oligonucleotide aptamer not only has higher affinity and specificity to target molecules, but also has the advantages of wide recognition range, in vitro synthesis and chemical stability compared with conventional antibodies. Since a kind of screening method termed systematic evolution of ligands by exponential enrichment (SELEX) was reported, scientists have extensively researched the methodology of how to highly and efficiently screen out aptamers from a library consisting of a large number of random oligonucleotides. Certainly capillary electrophoresis-based screening methodologies, including nonequilibrium capillary electrophoresis of equilibrium mixtures, equilibrium capillary electrophoresis of equilibrium mixtures, non-SELEX, ideal-filter capillary electrophoresis, capillary transient isotachophoresis, etc., are revolutionary. Compared with conventional SELEX, these capillary electrophoresis-based methodologies show incomparable advantages such as the single-round screening of aptamers and increased successful screening rate. Methodology studies on the screening process of aptamers are comprehensively reviewed.

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“…In addition, Chromeo forms a relatively stable molecule and its stock solution does not deteriorate, so it can be used for several months [33]. Moreover, the dye can be conjugated with a protein without changing the charge of the protein during the process in order to preserve its native form [34]. Our wave-mixing setup is not a commercial closed system, thus subtle capillary movement, inconsistent temperature, and other outside elements including dust and noise can affect the laser wave mixing CE signal, especially at zepto-mole levels.…”

Section: Uv-visible Analysis Of α-Synuclein and Amine-reactive Labelsmentioning

confidence: 99%

Sensitive analysis of α-synuclein by nonlinear laser wave mixing coupled with capillary electrophoresis

Iwabuchi

Hetu

Tong

2016

Analytical Biochemistry

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Multi-photon nonlinear laser wave-mixing spectroscopy is a novel absorption-based technique that offers excellent detection sensitivity for biomedical applications, including early diagnosis and investigation of neurodegenerative diseases. α-Synuclein is linked to Parkinson's disease (PD), and characterization of its oligomers and quantification of the protein may contribute to understanding PD. The laser wave-mixing signal has a quadratic dependence on analyte concentration, and hence the technique is effective in monitoring small changes in concentration within biofluids. A wide variety of labels can be employed for laser wave-mixing detection due to its ability to detect both chromophores and fluorophores. In this investigation, two fluorophores and a chromophore are studied and used as labels for the detection of α-synuclein. Wave-mixing detection limits of PD-related protein conjugated with fluorescein isothiocyanate, QSY 35 acetic acid, succinimidyl ester, and Chromeo P503 were determined to be 1.4 × 10(-13) M, 1.4 × 10(-10) M, and 1.9 × 10(-13) M, respectively. Based on the laser probe volume used, the corresponding mass detection limits were determined to be 1.1 × 10(-23) mol, 1.1 × 10(-20) mol, and 1.5 × 10(-23) mol. This study also presents molecular-based separation and quantification of α-synuclein by laser wave mixing coupled with capillary electrophoresis.

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Protein Labeling Enhances Aptamer Selection by Methods of Kinetic Capillary Electrophoresis

Cited by 18 publications

References 31 publications

A semipermanent coating for preventing protein adsorption at physiological pH in kinetic capillary electrophoresis

A semipermanent coating for preventing protein adsorption at physiological pH in kinetic capillary electrophoresis

Capillary electrophoresis‐based methodology for screening of oligonucleotide aptamers

Sensitive analysis of α-synuclein by nonlinear laser wave mixing coupled with capillary electrophoresis

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