alexa Asymmetric Mono- and Bis-Squarylium Dyes as Pre-Column and On-Column Labels for Protein Analysis by Capillary Electrophoresis with Laser-Induced Fluorescence Detection | Abstract
ISSN: 2155-9872

Journal of Analytical & Bioanalytical Techniques
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Research Article

Asymmetric Mono- and Bis-Squarylium Dyes as Pre-Column and On-Column Labels for Protein Analysis by Capillary Electrophoresis with Laser-Induced Fluorescence Detection

Xiuli Lin1, Stephanie Rockett1, Tara L. Massie1,2, Geoffrey B. Turner1,3, Takeshi Maeda4, Hiroyuki Nakazumi4, and Christa L. Colyer1*

1Department of Chemistry, Wake Forest University, Winston-Salem, NC 27109, United States

2NC BioNetwork Pharmaceutical Center, 391 Technology Way, Suite 162, Winston-Salem, NC 27101, United States

3National Renewable Energy Lab, Colorado, USA

4Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka

*Corresponding Author:
C. L. Colyer
Department of Chemistry
Wake Forest University
1834 Wake Forest Road
Winston-Salem, NC 27109, United States
Fax: 336-758-4656;
E-mail: [email protected]

Received date: April 07, 2012; Accepted May 26, 2012; Published date: June 06, 2012

Citation: Lin X, Rockett S, Massie TL, Turner GB, Maeda T, et al. (2012) Asymmetric Mono- and Bis-Squarylium Dyes as Pre-Column and On-Column Labels for Protein Analysis by Capillary Electrophoresis with Laser-Induced Fluorescence Detection. J Anal Bioanal Tech S9:001. doi: 10.4172/2155-9872.S9-001

Copyright: © 2012 Lin X, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Labeling proteins with fluorescent dyes offers a powerful tool for monitoring protein interactions in vitro and in vivo. In order for this tool to be effective, the nature of the dyes - absorbance and emission properties, solution stability, pH range, and mechanisms for protein interaction - must first be considered. Two new asymmetric, squarylium dyes, bis-SQHN-4d and SQHN-3c, were shown to be only weakly fluorescent in aqueous buffers in the absence of proteins. However, their spectra showed a dramatic increase in fluorescence intensity upon the addition of Human Serum Albumin (HSA) or Bovine Serum Albumin (BSA) as model proteins. The enhanced fluorescence properties, attributed to noncovalent binding, allowed the use of the new squarylium dyes as probes for the low-level detection of proteins in a mixture (including myoglobin (pI=7.16), transferrin (pI=5.9), and HSA (pI=4.8)), separated by Capillary Electrophoresis with Laser-Induced Fluorescence detection (CE-LIF). Because of the low background fluorescence of these probes, on-column labeling was feasible and led to simple and rapid protein detection. This labeling protocol offered greater sensitivity than the more conventional pre-column labeling protocol (with a 10-fold lower limit of detection for HSA with bis-SQHN-4d). A limit of detection for HSA (by CE-LIF with on-column labeling with bis-SQHN-4d) of 3.42 x 10-8 M indicates that this dye is well suited to the development of other protein assays.


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