Spectroscopic Characteristics of the Cationic Dye Basic Orange 21 in Leukocytes

Z. Eizig, D. T. Major, H. L. Kasdan, E. Afrimzon, N. Zurgil, M. Deutsch

2014

Abstract

Leukocyte staining with the cationic dye, Basic Orange #21 (BO21) can improve leukocyte differential counting, routinely used to assess general health, help diagnose the cause of an abnormal white blood cell count, and/or monitor other diseases and conditions that affect specific leukocytes . The spectroscopic aspects of BO21 and their changes upon interaction with biological molecules within the live cell in general, and various leukocytes (white blood cells) in particular, were examined. Spectroscopic characteristics were studied in bulk solutions under several conditions (pH, viscosity) and various solvents (salts, proteins). Results show negligible changes in absorption spectra under most conditions and solvents. However, Heparin, an anion organic molecule common to a specific type of leukocyte, causes a blue shift of the spectra from 484 nm in its absence to 465 nm when Heparin is present. Next, it was found that in the presence of heparin the emission peak of BO21 was extremely red shifted; from 519 to 550nm for excitation of BO21 at 490nm. Finally, Fluorescence polarization (FP) measurements of BO21 in water yielded, contrary to all expectations, FP ~ 0.450. However, the presence of heparin produced a dramatically lower FP; about 0.200. may be caused either by an electrostatic bond between the cationic-dye and the polyanions (Heparin) or by effective aggregation of the dye cations due to their occupying adjacent sites on the polyanion. Utilizing different small anion molecules and high dye concentration, we concluded that it is not the electrostatic bond which is responsible for the blue spectral shift of the dye, but rather the aggregation of dye ions. We proposed a mechanism using computational chemistry which indicates that parallel setting of the molecular electrical dipoles of BO21, of which the dimers and trimers are composed, is indeed feasible. Surprisingly, the related binding energies are even lower than those calculated for anti-parallel arrangement.

References

  1. Deutsch, M., Deutsch, A., Shirihai, O., Hurevich, I., Afrimzon, E., Shafran, Y., and Zurgil, N. (2006) 'A Novel Miniature Cell Retainer for Correlative HighContent Analysis of Individual Untethered NonAdherent Cells'. Lab on a Chip 6 (8), 995-1000
  2. Hohenberg, P. and Kohn, W. (1964) 'Inhomogeneous Electron Gas'. Physical Review 136 (3B), B864-B871
  3. Kass, L. (1986) Individual Leukocyte Determination by Means of Differential Metachromatic ... 4581223. available from <http://www.google.co.il/patents?id= RHk8AAAAEBAJ> [3 February 2013]
  4. Shi, W., Guo, L., Kasdan, H., and Tai, Y.-C. (2013) 'FourPart Leukocyte Differential Count Based on Sheathless Microflow Cytometer and Fluorescent Dye Assay'. Lab on a Chip [online] available from <http://pubs.rsc.org/en/content/articlelanding/2013/lc/ c3lc41059e> [25 February 2013]
  5. Yun, H., Bang, H., Min, J., Chung, C., Chang, J. K., and Han, D.-C. (2010) 'Simultaneous Counting of Two Subsets of Leukocytes Using Fluorescent Silica Nanoparticles in a Sheathless Microchip Flow Cytometer'. Lab on a Chip 10 (23), 3243.
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Paper Citation


in Harvard Style

Eizig Z., T. Major D., L. Kasdan H., Afrimzon E., Zurgil N. and Deutsch M. (2014). Spectroscopic Characteristics of the Cationic Dye Basic Orange 21 in Leukocytes . In Doctoral Consortium - DCBIOSTEC, (BIOSTEC 2014) ISBN Not Available, pages 82-87


in Bibtex Style

@conference{dcbiostec14,
author={Z. Eizig and D. T. Major and H. L. Kasdan and E. Afrimzon and N. Zurgil and M. Deutsch},
title={Spectroscopic Characteristics of the Cationic Dye Basic Orange 21 in Leukocytes},
booktitle={Doctoral Consortium - DCBIOSTEC, (BIOSTEC 2014)},
year={2014},
pages={82-87},
publisher={SciTePress},
organization={INSTICC},
doi={},
isbn={Not Available},
}


in EndNote Style

TY - CONF
JO - Doctoral Consortium - DCBIOSTEC, (BIOSTEC 2014)
TI - Spectroscopic Characteristics of the Cationic Dye Basic Orange 21 in Leukocytes
SN - Not Available
AU - Eizig Z.
AU - T. Major D.
AU - L. Kasdan H.
AU - Afrimzon E.
AU - Zurgil N.
AU - Deutsch M.
PY - 2014
SP - 82
EP - 87
DO -