Spectroscopic Characteristics of the Cationic Dye Basic Orange 21 in

Leukocytes

Z. Eizig

, D. T. Major

, H. L. Kasdan

, E. Afrimzon

, N. Zurgil

and M. Deutsch

The Biophysical Interdisciplinary Jerome Schottenstein Center for the Research and the Technology of the Cellome,

Physics Department, Bar Ilan University, Ramat Gan, Israel

Department of Chemistry, Bar-Ilan University, Ramat-Gan, Israel

IRIS Diagnostics Division, IRIS International Inc., Chatsworth, CA, U.S.A.

1 STAGE OF THE RESEARCH

A comprehensive literature review regarding BO21

and other metachromatic dyes has been completed

and yields many open questions regarding the cause

for BO21 metachromasia. Recognizing the need for

a more satisfactory basis for machine interpretation

of leukocytes, Kass (Kass, 1986) found useful,

reaction properties of Basic Orange #21 (BO21),

which acts supravitally to induce metachromasia in

leukocytes.

However, surprisingly, and most probably due to

the low quantum yield of BO21, reference to its

florescence characteristics and polarization is

missing.

Therefore, the spectroscopic aspect of BO21 is

extensively investigated in our work. First, the

dependency of BO21 metachromasia upon a variety

of factors (pH, viscosity, salts, proteins temperature,

etc.) have been investigated. In all experiments,

phosphate buffered saline buffer (PBS) was used as

the hosting medium in order to retain a constant pH

level.

At first, the influence of these factors upon BO21

was assessed by changes (red or blue shift) in the

absorption spectrum of BO21. Practically, the ratio

between the absorption (A) measured within the

wavelength windows 481 – 490nm and within 461 –

470nm was calculated for suspending media without

(S) and with reagent (R), after which the Absorption

Ratio (AR) was calculated (Equation 1).

(481 490) (461 470)







(1)

Though some influence of the acidity level and the

viscosity could be observed via the ARs ratio, they

were negligible in respect to that obtained with

heparin, an anion organic molecule which exists in

some types of leukocytes. It has been found that

heparin induces a blue shift in the absorption

spectrum (AR ~ 0.84), yielding a red hue BO21

solution. Results are summarized in Table 1.

The absorption (solid line) and emission dashed

line) spectra of BO21 in the absence (blue curves),

and in the presence (red curves) of heparin, are

shown in Figure 1. The measured absorption

wavelength windows are indicated by the blue

blocks.

Table 1: Influence of factors upon BO21 absorption

spectra.

Variable Reference Variable density Abs. Ratio

Concentration 10uM BO21 50uM 1.00

pH pH 7.4 pH 5 0.99

pH pH 7.4 pH 9 1.00

Viscosity 1cP 219cP 1.07

CaCl

BO21 in H2O 0.09 uM 1.00

CuSO

BO21 in H2O 1.35 nM 1.00

KCl BO21 in H2O 600 mM 1.00

MgCl

BO21 in H2O 70 mM 1.00

NaCl BO21 in H2O 300 mM 1.00

BSA/PBS BO21 in PBS 100 uM 1.04

BSA/H2O BO21 in H2O 100 uM 1.01

heparin BO21 in H2O 0.8 uM 0.84

Figure 1: absorption (solid line) and emission (dashed

line) spectra of BO21 (10uM) in the absence (blue) and in

the presence (red) of 0.8uM heparin. The two orthogonal

blocks represent the said wavelength windows.

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

Spectroscopic Characteristics of the Cationic Dye Basic Orange 21 in Leukocytes.

 2014 SCITEPRESS (Science and Technology Publications, Lda.)

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.

1.1 Mechanism

Changes in absorption spectrum might evolve from

electrostatic bonds between BO21 molecules

(formation of dimers, trimers, etc.), as well as the

presence of heparin, on which the dye cation

aggregates, occupying adjacent sites on the poly-

anion. In order to distinguish between the two

possible mechanisms, two types of experiments were

performed. First, the electrostatic bonds between

BO21 and heparin were neutralized by replacing the

heparin with small anion molecules. Results are

listed in Table 2. As can be seen, no influence of the

salts upon BO21 absorbance could be observed; the

control (BO21 in water or in PBS) values (~ 0.59)

were, for all practical purposes, the same as with

salts (3

column from left). Moreover, the presence

of heparin caused no noticeable change as well (4th

column from left).

Table 2: Influence of anion and cation salts upon BO21

absorption spectra with and without heparin.

Absorbance at 484nm

Sub. Concentration BO21+ Sub.

BO21+ Sub.+

heparin

H2O control

0.59 0.49

PBS control 0.57 0.57

(NH

)SO

0.2M 0.58 0.59

NaAC 0.2M

0.58 0.59

NaCl 0.2M

0.58 0.58

Sub.- Substance

Next, the role of BO21 aggregation in

metachromasia was examined independently by

tracing the dependency of BO21 absorption upon its

concentration. In order to avoid measurement errors

due to inner filter effects, a special cuvette was

designed with an optical path of 0.15mm. In Figure

2, two cuvettes are shown, each containing the same

BO21 concentration. Nevertheless, with the regular

1cm cuvette (item a in the figure) a red shift is

evident due to enhanced inner filter effect, while

with the 0.15mm cuvette (item b in the figure),

either no shift at all is evident, or a significantly

smaller shift can be seen. Results of absorption

measurements with the latter are given in the lower

panel of Figure 2. As can be seen, the higher the

BO21 concentration, the more significant the blue

shift is – a finding which supports BO21 aggregation

(which generates dimers, trimers and more complex

formations of BO21) as the main cause of

metachromasia.

Figure 2: BO21 absorbance at high concentration. Upper

panel: 8mM dye concentration in 10mm (a) and in

0.15mm (b)

cuvettes. Lower panel: absorption spectra of

0.05mM BO21 (blue curve) and 8mM (red curve), as

measured in the 0.15mm cuvette.

The level of blue shift was also found to be

heparin concentration dependent, i.e. the higher the

heparin concentration the greater the blue shift is

(see Figure 3).

Figure 3: Absorption spectra of 10uM BO21 at various

concentrations of heparin.

0,9

0,92

0,94

0,96

0,98

1,02

450 470 490

NormalizedAbs.

Wavelength(nm)

0.05mM 8mM

350 400 450 500 550 600

0.1

0.2

0.3

0.4

0.5

Wavelenght

Abs.

0.00 uM

0.10 uM

0.20 uM

0.41 uM

0.81 uM

1.63 uM

3.26 uM

6.51 uM

13.03 uM

26.05 uM

39.08 uM

45.59 uM

SpectroscopicCharacteristicsoftheCationicDyeBasicOrange21inLeukocytes

However, a close look into Figure 3 shows that

the shift has a maximum at 0.8uM heparin, after

which it decreases.

Our interpretation of these results follows

Bradley’s and Wolf‘s (1959). The idea is illustrated

in Figure 4. Denoting BO21 as D and heparin

binding site as P, we propose that at low heparin

concentrations, namely P/D<1,

the heparin binding

sites are completely occupied with BO21 and the

surplus BO21 remains unbound in the solution

(Figure 4a). In the case where P/D~1 (Figure 4b), all

BO21 molecules are expected to occupy all heparin

binding sites and consequently, to yield maximum

metachromasia. With further increase of the heparin

concentration, P/D>1 (Figure 4c), the competition

between heparin molecules on BO21 molecules will

finally lead to an equilibrium state where the BO21

molecules only partially occupy heparin binding

sites, hence lessening the proximity between BO21

molecules attached to each of the heparin molecules

and consequently lowering the chance for BO21

aggregation to occur on a single heparin.

Figure 4: Aggregation schematic of the dye (D) on the

anionic binding site of the polymer (P). a) Low polymer

concentration leaves free dye molecules in the hosting

solution. b) At an equal number of polymer binding sites

and dyes, the dye molecules occupy all polymer binding

sites. c) The number of polymer anionic binding sites is

larger than that of dye molecules, hence yielding partial

occupancy of the binding sites by the dye.

The experimental results and proposed

mechanism was further strengthened by the use of

computational chemistry. BO21 molecules is a

multi-bodied electronic structure containing 47

nuclei and 169 electrons (despite the chlorine that

dissolves in water), and therefore, the Schrödinger

equation is impractical as a modelling method,

hence, the Density Functional Theory (DFT)

approach was applied (Hohenberg and Kohn 1964).

DFT is a quantum mechanical modelling method

which investigates the electronic structure

(principally the ground state) of many-body systems.

With this theory, instead of using N wave function

that depend on 3N coordination for x,y,z, the

properties of a many-electron system can be

determined by using the charge density ρ. This

method is based on two Hohenberg-Kohn theorems

(H-K). The first H-K theorem demonstrates that the

ground state properties of a many-electron system

are uniquely determined by an electron density that

depends on only 3 spatial coordinates, while the

second H-K theorem defines energy functional for

the system, and proves that the correct ground state

electron density minimizes this energy functional.

We calculated the electric dipole of the BO21

molecule. This molecule is cation due to a positive

charge surrounding the nitrogen atom connected to

the methyl group. Based on density function, a 3-d

MEP (Molecular Electrostatic Potential) was

produced in Figure 5a.

The molecule electric dipole moment µ was

estimated by the formula:



nucl





 



 





rr r

(2)

Where ࣋

ሺ

࢘

ሻ

is the density function, Z is atomic

number and α is the nuclei index. The integral is

above the electron coordinates and the summation

yields the nucleus contribution to the dipole

moment. The dipole moment vector of BO21 was

found to be

1.43 10







, which is twice

the water molecule (

6.2 10







21BO





is indicated in Figure 5a by a light blue

arrow in respect to the molecule axis. In order to

support the aggregation theorem that causes the blue

shift, we test the feasibility of dimer or trimer

formation of BO21 molecules. In the tested model,

the molecular electrical dipoles of dimers and

trimers of BO21 are oriented in a parallel fashion as

shown in Figure 5b. Surprisingly, the related binding

energies were found to be even lower than those

calculated for anti-parallel arrangements, a fact

which explains the blue shift in the absorption

spectrum.

2 OUTLINE OF OBJECTIVES

 To study the spectroscopic characteristics of

BO21 in bulk solution in the presence and in the

absence of biomolecules in general, as well as

those which exist in leukocytes, in particular.

 To explore unique spectroscopic features of

BO21 stained leukocytes, if they exist

BIOSTEC2014-DoctoralConsortium

 To correlate via the MCR, on same cell basis,

between the explored spectroscopic features of

BO21 stained sub types of leukocytes, and

between traditional methods for differential

leukocyte counts.

Figure 5: Molecular Electrostatic Potential from most

negative (blue) to positive (red). a) BO21 monomer,

electric dipole moment direction marked in light blue

arrow. b) Parallel (dipoles) BO21 dimer.

3 RESEARCH PROBLEM

 Overcoming inner filter problem by the

construction of unique cuvettes which will allow

high concentration fluorescence and absorption

measurements

 Development of theoretical tools for the

examination of correctness level of the above

unique cuvette performance

 Preparation of upright epifluorescence

microscope for spectroscopic measurement of

BO21-stained cells in a single cell resolution,

while preserved within the MCR.

 Development of DFT-based algorithm for the

investigation of dye-dye and dye-heparin

interactions (primary results discussed above).

 Development of protocols for treatment of

leukocytes within the MCR, e.g. cell loading,

cell staining with BO21, fixation, Giemsa/Wright

staining, etc.

 Evaluation of the diagnostic potential of BO21.

4 STATE OF THE ART

Presently, the leukocyte differential count test is

mainly

based on measuring individual cell electrical

impedance, fluorescence and light scattering, which

are all methods based on signals from an entire cell

and not from the detailed image of a cell. Signals are

acquired using flow cytometer and hematology

analyzers that require large amounts of reagent and

blood samples. Cell analysis on flow cytometers

typically involves two steps: first, labeling target

cells with detection assays, e.g. fluorophore-

conjugated antibodies, and second, detecting target

cells by corresponding optical signals, e.g.,

fluorescence assays (Yun et al., 2010) of

fluorophore-conjugated antibodies for leukocyte

analysis on the microflow cytometers. However, the

low temperature needed for reagent storage makes

this assay less than ideal for point-of-care

applications. In comparison, assays of fluorescent

dyes, have been proven as useful alternatives in cell

analysis (Shi et al., 2013). Shi used the combination

of FITC, PI and BO21 for classifying four types of

leukocytes, though the spectroscopic aspect of BO21

was barely studied in respect to both FITC and PI.

Furthermore, to the best of our knowledge, studies

of BO21 fluorescence and its polarization do not

exist.

5 RESEARCH METHODOLOGY

5.1 Bulk Spectroscopy of BO21

In this chapter we intend to further investigate

spectroscopic features (Absorbance, FI and FP,

fluorescence lifetime-FLT and polarization decay-

PD) of BO21 in general, and at high concentrations

in particular, in the absence and in the presence of

heparin, in a variety of concentrations. This will be

realized via our Cary UV spectrophotometer and

Cary eclipse spectrofluorometer (Agilent, USA). In

addition, FLT and PD and the evaluation of the

rotational relaxation time of BO21 in solution will

be realized via the DCS-120 confocal FLIM system

(Becker and Hickl GmbH Berlin, Germany).

Especially with the last type of measurements,

which are time dependent, the extremely low

quantum yield of fluorescence of BO21 (about 1000

times less than that of fluorescein), should be

SpectroscopicCharacteristicsoftheCationicDyeBasicOrange21inLeukocytes

carefully considered in order to improve the

expected low S/N ratio.

Further investigation of the complexes: BO21

dimers (trimers) and BO21-heparin will be realized

via Density Functional Theory (DFT). Initial

computational quantum chemistry based calculations

teache that the molecular electrical dipoles of BO21

in dimers and trimers tends to be parallel oriented.

Moreover, and quite surprisingly, the related binding

energies are even lower than those calculated for

anti-parallel arrangements. The BO21-heparin

complexes formed, seem to be governed by

electrostatic interactions, wherein the positive

charge of BO21 interacts with the negative charge

located in the heparin sites. Additionally, the π-

cation interaction between stacked BO21 molecules

stabilizes the complexes. Quantum calculations were

performed with the DMol3 module in Material

Studio (Accelery, USA).

5.2 Bulk Spectroscopy of BO21-stained

Leukocytes Suspension

In this chapter we intend to repeat the bulk

measurement discussed above, but in suspension of

BO21 stained leukocyte, utilizing the Cary UV

spectrophotometer and the Cary eclipse

spectrofluorometer (Agilent, USA).

5.3 Single Cell Resolution Spectroscopy

of BO21-stained Leukocytes

In this chapter we intend to measure the

spectroscopic characteristics of intra leukocyte

BO21 at a single-cell resolution. This will be carried

out by loading the leukocytes in a Microtiter plate

Cell Retainer (MCR). MCR is a high throughput

Microtiter plate that has been developed in our

Center (Deutsch et al., 2006) to enable high-content,

time-dependent analysis of the same single non-

adherent and non-anchored cells in a large cell

population while bio-manipulating the cells. The

identity of each cell in the investigated population is

secured, even during bio-manipulation, by cell

retention in a specially designed concave microlens

(Figure 6), acting as a picoliter well. The MCR

technique combines micro-optical features and

microtiter plate methodology.

While solutions for fluorescence measurement at

a single cell resolution is quite common, solutions

for single cell absorption (1nm spectral resolution) is

slow to appear. Hence, for the realization of the

latter in a single cell resolution, we intend to

extensively upgrade our Olympus upright BX61

microscope (Tokyo, Japan) to enable medium

throughput absorption measurements of BO21

stained cell.

Figure 6: An SEM image of Jurkat T cells in MCR. Scale

bar: 20.

In short, to the existing Olympus upright BX61

microscope, which is equipped with a sub-micron

Marzhauser–Wetzlar motorized stage (types SCAN,

with an Lstep controller, Wetzlar–Steindorf,

Germany), a xenon lamp, 1nm spectral resolution

monochromator, and a CCD camera will be added,

adjusted and calibrated. The entire system will be

controlled by designated / software. The captured

images will be processed using Matlab (MathWorks,

USA) to produce spectral quantile (SQ) maps, which

present a surface plot showing absorption quantile

amplitudes as a function of wavelength (see Figure

7).

Neutrophil Lymphocyte

Monocyte Eosinophil

Figure 7: Spectral-Quantile (SQ) plots for four of the five

normal leukocyte subtypes showing how BO21

metachromasia allows easy identification of the subtypes.

Axis from left to right is the Quantile axis (50 quantiles).

Axis from right to left is the wavelength axis (51

wavelengths from 400nm through 650nm at 5nm

increments).

BIOSTEC2014-DoctoralConsortium

Finally, in order to explore possible identifying

features which might be used for differentiating

between types of leukocytes and between leukocytes

and other type of cells, pre- versus post-fixation

(with Wright Giemsa staining) correlation of BO21

stained cells will be performed.

6 EXPECTED OUTCOME

 Evaluating the ability of a single dye BO21 to

classify types of leukocytes via maps of

absorption, FI and FP spectra.

 Exploring the mechanism of BO21-Heparin

interactions/structures in general, and that which

stands behind the measured high FP of BO21 in

water in particular.

REFERENCES

Deutsch, M., Deutsch, A., Shirihai, O., Hurevich, I.,

Afrimzon, E., Shafran, Y., and Zurgil, N. (2006) ‘A

Novel Miniature Cell Retainer for Correlative High-

Content Analysis of Individual Untethered Non-

Adherent Cells’. Lab on a Chip 6 (8), 995–1000

Hohenberg, P. and Kohn, W. (1964) ‘Inhomogeneous

Electron Gas’. Physical Review 136 (3B), B864–B871

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]

Shi, W., Guo, L., Kasdan, H., and Tai, Y.-C. (2013) ‘Four-

Part 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]

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.

SpectroscopicCharacteristicsoftheCationicDyeBasicOrange21inLeukocytes