Investigation of benzophenoxazine derivatives for the detection of latent fingerprints on porous surfaces

Article abstract of DOI:10.1016/j.jphotochem.2020.112416

In this report, two classes of benzophenoxazine dyes, Nile red and Nile blue derivatives, were evaluated for the detection of latent fingerprints on porous surfaces. The efficiency to develop fingerprints is influenced by the physical properties of the dye molecules including hydrophobicity as characterized by distribution coefficient value (logD), and other factors such as chemical structure of the dye and hydrogen bonds. Both Nile red and the basic form of Nile blue showed excellent to acceptable ability to detect fingerprints due to their great hydrophobicity at 515 and Crime Scene Search (CSS) settings of the forensic light source. Higher hydrophobicity derivatives of Nile red and Nile blue (in the basic form) improved both quality and sensitivity of fingerprint detection in comparison with their corresponding parent dyes. They developed strong luminescent and visible fingerprints, and a better contrast was achieved between impressions and the background surface suggesting the potential use of these compounds in forensic investigation. Therefore, the hydrophobic derivatives are considered dual-fingerprint reagents because the developed prints can be seen by the naked eye and under illumination process. However, lower hydrophobicity derivatives of Nile red and Nile blue and derivatives with different substituents developed weak or non-luminescent fingerprints with poor contrast. A complete analysis of what dye properties are the most important in fingerprint detection is discussed along with dye optimization for improved performance.

Full text of DOI:10.1016/j.jphotochem.2020.112416

Journal Pre-proof
Investigation of Benzophenoxazine Derivatives for the Detection of Latent
Fingerprints on Porous Surfaces
Eman Alsolmy, Walid M. Abdelwahab, Vincent Martinez, Maged
Henary, Gabor Patonay
PII:
S1010-6030(19)30500-3
DOI:
https://doi.org/10.1016/j.jphotochem.2020.112416
JPC 112416
Reference:
To appear in:
Journal of Photochemistry & Photobiology, A: Chemistry
Received Date:
Revised Date:
Accepted Date:
5 April 2019
16 January 2020
27 January 2020
Please cite this article as: Alsolmy E, Abdelwahab WM, Martinez V, Henary M, Patonay G,
Investigation of Benzophenoxazine Derivatives for the Detection of Latent Fingerprints on
Porous Surfaces, Journal of Photochemistry and amp; Photobiology, A: Chemistry (2020),
doi: https://doi.org/10.1016/j.jphotochem.2020.112416
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Investigation of Benzophenoxazine Derivatives for the Detection of Latent Fingerprints on
Porous Surfaces
Eman Alsolmya,b,*, Walid M. Abdelwahaba,c, Vincent Martineza, Maged Henarya, Gabor Patonaya
a
Department of Chemistry, Georgia State University, P.O. Box 3965, Atlanta, Georgia 30302-
3965, USA.
b
Chemistry Department, Faculty of Science, University of Jeddah, P.O. Box 80327, Jeddah,
21589, Saudi Arabia.
c Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini St.,
Cairo 11562, Egypt.
* Corresponding author
Tel: (+1) 404-413-5557; fax: (+1) 404-413-5505
E-mail address: ealsolmy1@student.gsu.edu
1

Graphical Abstract
Fingerprints Development
Forensic
Light
Source
Benzophenoxazine Corresponding
Latent fingerprints deposition
Derivative
parent dye
Camera
Highlights
 The hydrophobicity of the dye plays a significant role for developing fingerprints.
 The fingerprints detection is also influenced by the chemical structure of the dye, hydrogen
bonds, and interactions with solvents.
 Nile red and Nile blue detect fingerprints with excellent to acceptable definition due to the
great hydrophobicity.
 Highly hydrophobic benzophenoxazine derivatives could be considered dual-fingerprint
reagents because they develop luminescent and visible fingerprints.
 Less hydrophobic benzophenoxazine derivatives or hydrophobic derivatives containing
interference substituents develop weak luminescent fingerprints.
2

Abstract
In this report, two classes of benzophenoxazine dyes, Nile red and Nile blue derivatives,
were evaluated for the detection of latent fingerprints on porous surfaces. The efficiency to develop
fingerprints is influenced by the physical properties of the dye molecules including hydrophobicity
as characterized by distribution coefficient value (logD), and other factors such as chemical
structure of the dye and hydrogen bonds. Both Nile red and the basic form of Nile blue showed
excellent to acceptable ability to detect fingerprints due to their great hydrophobicity at 515 and
Crime Scene Search (CSS) settings of the forensic light source. Higher hydrophobicity derivatives
of Nile red and Nile blue (in the basic form) improved both quality and sensitivity of fingerprint
detection in comparison with their corresponding parent dyes. They developed strong luminescent
and visible fingerprints, and a better contrast was achieved between impressions and the
background surface suggesting the potential use of these compounds in forensic investigation.
Therefore, the hydrophobic derivatives are considered dual-fingerprint reagents because the
developed prints can be seen by the naked eye and under illumination process. However, lower
hydrophobicity derivatives of Nile red and Nile blue and derivatives with different substituents
developed weak or non-luminescent fingerprints with poor contrast. A complete analysis of what
dye properties are the most important in fingerprint detection is discussed along with dye
optimization for improved performance.
Keywords: Benzophenoxazine derivatives; Nile red; Nile blue; Fingerprint detection;
Hydrophobicity; Luminescent
1. Introduction
Fingerprint identification is considered one of the most crucial clues of physical evidence
in forensic investigation. The common form of fingerprints found at a crime scene is latent
3

fingerprints, the invisible residue deposited by a person’s hand when touching a surface. Due to
the complexity of fingerprint residue, revealing the prints requires physical or chemical treatment
to enable their detection. Several developing reagents have been used for fingerprint detection that
target various components such as amino acids or lipids in fingerprint residue. Amino acid-
sensitive reagents such as ninhydrin, 1,8-diazafluoren-9-one (DFO), and 1,2-indanedione are
insufficient for revealing fingerprints on porous surfaces that have been wetted or found in humid
conditions [1-3]. In this case, amino acids are likely to be dissolved or washed away due to their
solubility in water. Therefore, lipid-sensitive reagent is the reagent of choice to detect fingerprints
on wet porous substance. Physical developer (PD) is the standard technique for developing
fingerprints in such applications. However, this technique has been recognized as a complex and
time-consuming process due to high sensitivity to contaminations and the need for freshly prepared
working solutions [4]. Many studies have been conducted on alternative lipid sensitive reagents
such as dyes [5-7]. Oil Red O is a diazo dye used in histological staining for lipidic compounds
and belongs to the family of Sudan dyes. It has been studied extensively for recovering fingerprints
as red visible prints on wet porous surfaces due to the high affinity of the dye to lipids [8, 9].
Solvent black 3 is another diazo dye that comes from the same family of Sudan dyes. Similarly,
the dye has been investigated in revealing fingerprints but to a lesser extent than Oil Red O. Solvent
black 3 is more effective in developing fingerprints on greasy contaminated substances [10].
Fingerprints developed by the previously mentioned reagents are visible to the naked eye
which limit their applications to light-colored surfaces. However, the use of organic dyes that
fluoresce upon exposure to the light source has the advantage of revealing fingerprints with
sufficient contrast on dark-colored surfaces. The fluorescent dye eosin-blue with a phase transfer
catalyst was used to detect fingerprints on many surfaces [11]. Moreover, a formulation based on
4

Coomassie brilliant blue G-250 dye has been proposed as a developing reagent to differentiate
between male and female fingerprints [12]. Furthermore, several fluorescent dyes have been
reported as dusting agents for detecting fingerprints including acridine orange, acridine yellow,
crystal violet, coumarin 6, and rhodamine B [13]. In addition, micro-structured fluorescent
powders based on benzazole dyes have been applied for the development of latent fingerprints on
many surfaces [14]. Moreover, the fluorescence properties of such compounds enhance the
sensitivity of fingerprints developed with regular reagents. Rhodamine 6G, for example, was used
as post-treatment of fingerprints developed with Oil Red O on dark porous surfaces [15].
Recently, benzophenoxazine dyes such as Nile red and Nile blue have been applied in the
field of fingerprint detection. Nile red is a neutral fluorescent dye used in histology for staining of
some lipid compounds such as triacylglycerols, phospholipids, cholesteryl esters, and lipoproteins
[16]. Nile blue is a cationic dye used in the histological staining of neutral fats, phospholipids, and
fatty acids [17]. These compounds are present in fingerprint residue as a result of sebaceous gland
secretions [18]. The optical properties of Nile red and Nile blue are highly influenced by the
solvent polarity and dielectric constant. In non-polar media, the fluorescence spectrum of both
dyes is in the visible to the near-infrared region, and high quantum yield values are recorded. In
polar media, however, the fluorescence spectrum of Nile Red and Nile blue in its basic form shows
a red shift with low quantum yield values due to hydrogen bonding [19-21]. The early use of Nile
red in fingerprint detection was a post-treatment for cyanoacrylate fuming. It was reported that the
performance of Nile red for the enhancement of cyanoacrylate-developed latent fingerprint was
considerably superior to that for rhodamine 6G [22]. Similarly, Nile blue was initially used to
improve the contrast of latent fingerprints developed by cyanoacrylate [23]. Both dyes have been
used for the detection of latent lip prints on multicolored surfaces, and Nile red performed better
5

than Nile blue especially with old prints [24]. Nile red has been used effectively to develop
fingerprints on different porous surfaces in a methanolic-aqueous formulation. In addition, Nile
red performance was compared to that of PD with fresh and aged fingerprints [25]. On the other
hand, an aqueous solution of Nile blue has been proposed as an alternative fingerprint reagent to
the previous formulation of Nile red. The procedure was described as simple, less toxic, and lower
cost and can be applied on different surfaces [26]. The efficiency of this solution is due to the small
presence of Nile red resulting from the spontaneous hydrolysis of Nile blue [27]. The presence of
the two dyes in one formulation stains both neutral lipids and acidic moieties [17]. Recently, an
aqueous microemulsion of Nile red has been developed for visualizing both natural and charged
fingerprints. The performance of the solution was comparable to the methanolic-aqueous
formulation of Nile red and superior to the aqueous solution of Nile blue [28]. More recently, Nile
red has been encapsulated into mesoporous silica nanoparticles for developing fingerprints on
thermal paper without the need of organic solvents [29].
Most fluorescent dyes used in forensic detection as developing reagents are hydrophobic.
The hydrophobicity of a compound is measured by the partition coefficient (logP) or the
distribution coefficient (logD). Both terms describe the ratio of the concentration of a compound
in organic and aqueous phases (octanol and water) at equilibrium. However, logD is more
extensively used than logP to express such property because logP deals with unionized form only
while logD takes into consideration both ionized and unionized forms. Moreover, logD is pH-
dependent whereas logP is pH-independent [30, 31]. LogP value was first mentioned in forensic
science as an indication of the great hydrophobicity of Oil Red O explaining the extensive use of
the dye in histological staining [9]. The hydrophobicity characterized by logD for common
developing reagents is summarized in Table 1.
6

Fingerprint reagents can be classified based on the appearance of fingerprints after
treatment process into three types: visible-fingerprint reagent, luminescent-fingerprint reagent, and
dual-fingerprint reagent. Visible-fingerprint reagents such as ninhydrin, and Oil Red O bind to
fingerprint components and the resulting impression can be seen only by the naked eye.
Luminescent-fingerprint reagents such as Nile red and Nile blue interact with fingerprint residue
to produce fluorescent impressions which can be seen only upon exposure to a light source. Dual-
fingerprint reagents produce fingerprint impressions that are both visible and luminescent. Few
reagents have been known as dual-fingerprint reagents, one of which is a natural pigment called
Genipin. It is extracted from gardenia fruit and produces blue color with various amino acids [32].
This reagent detects amino acids in fingerprint residue to produce visible blue impressions and red
impressions under illumination [33]. Our preliminary results indicated that some
benzophenoxazine derivatives synthesized by our group were dual reagents as they developed
colored and luminescent fingerprints.
To the best of our knowledge, most of the fingerprint detection reports have studied the
common developing reagents and compared their performance with new analogous compounds
for enhanced detection [34-37]. However, very few reports have studied the relationship of the
chemical structure of a reagent and its efficiency for fingerprint development. The purpose of our
work was to compare the performance of two fingerprint reagents Nile red and Nile blue (in the
basic form) in terms of their chemical and physical properties such as hydrophobicity. The study
aimed also to evaluate several modified structures of benzophenoxazine derivatives as reagents for
fingerprint detection with respect to the two parent reagents. The use of benzophenoxazine
derivatives in forensic investigation has the advantage of revealing luminescent fingerprints on
7

porous surfaces. In addition, some derivatives of higher hydrophobicity developed visible and
luminescent prints and thus are considered as dual-fingerprint reagents.
2. Materials and methods
2.1 Chemicals and reagents
Nile red (NR) and Nile blue (NB) were obtained from Sigma-Aldrich (St. Louis, MO). The
synthesis of all Nile red and Nile blue derivatives except NR1 and NR2 will be illustrated in
Martinez et al. (in preparation). The chemical synthesis of NR1 and NR2 is described in the
following section. The chemical structures of all dyes are shown in Fig. 1. Ethanol (200 proof
ethanol) was obtained from Pharmco-AAPER (Brookfield, CT). Sodium hydroxide (99%) was
obtained from Fisher Scientific (Fair Lawn, NJ). Absorbance spectra were acquired using a Cary
3G UV-visible spectrophotometer (Varian Inc., Palo Alto, CA) interfaced to a PC. All
measurements were performed in disposable plastic cuvette with a path length of 1.0 cm. Nanopure
water was obtained using an ELGA Purelab Classic water purification system. The nuclear
magnetic resonance spectra were obtained by high quality Kontes NMR tubes (Kimble Chase,
Vineland, NJ) rated to 500 MHz and were recorded on a Bruker Avance 400 MHz spectrometer
interfaced to a PC using Topspin 3.1. High-resolution accurate mass spectra were obtained at the
Georgia State University Mass Spectrometry Facility using a Waters Q-TOF micro (ESI-Q-TOF)
mass spectrometer.
2.2. Synthesis of the dialkylated Nile red derivatives (NR1 and NR2)
All chemical reactions were maintained under a positive pressure of nitrogen unless
otherwise stated. The reaction progress was monitored using silica gel 60 F254 thin layer
chromatography plates (Merck EMD Millipore, Darmstadt, Germany). Open column
8

chromatography was utilized for the purification of all final compounds using 60−200 μm, 60A
classic column silica gel (Dynamic Adsorbents, Norcross, GA).
According to Scheme 1, compounds (2a-b) were prepared by dissolving 3-aminophenol,
compound 1, (2.00 g) with two molar equivalents of potassium carbonate in isopropanol/water
(1:1, v/v). Two molar equivalents of iodobutane were added to the mixture of 2a and two molar
equivalents of 1-bromo-3-phenylpropane were added to the mixture of 2b, both mixtures were
heated to 70°C for 3 h. The 2b solution was evaporated under reduced pressure then extracted with
ethyl acetate. For both compounds, the organic layer was separated and concentrated under
reduced pressure to yield brown oil. This was purified by regular phase column chromatography
using hexane /ethyl acetate (95:5, v/v). 0.6 g of Compounds (2a-b) were dissolved in isopropanol
(2.0 mL) to facilitate its dissolution. Then 4.0 mL (32% HCl) and 8.0 g of crushed ice were added.
The mixture was chilled in an ice bath and a solution of NaNO2 (0.4 g in 6.0 mL water) was added
dropwise over one hour. This was continuously stirred for 3 additional hours at 0º C. For compound
2a, the liquid medium had changed color to dark orange which was then concentrated under
reduced pressure. The nitroso product, compound 3a, was dried under reduced pressure and used
in the next step without further purification due its instability. For compound 2b, a yellow-orange
precipitate had formed by this time. This precipitate was filtered and dried under reduced pressure.
Due to instability of nitroso compounds, the product was used in the next step without purification.
Compounds (4a-b) were prepared by dissolving compounds (3a-b) and one molar equivalent of
1-hydroxynaphthalene in 15.0 mL ethanol. The mixture was heated to 70°C for 3 h to afford a
highly impure dye. The solution was neutralized with ammonium hydroxide and then extracted
with CH2Cl2. The organic layer was separated and then removed under reduced pressure. This was
9

purified by regular phase column chromatography using CH2Cl2/methanol (99:1, v/v) and (90:10,
v/v) for compounds 4a and 4b, respectively.
1
9-(dibutylamino)-5H-benzo[a]phenoxazin-5-one (NR1): 41% yield. H NMR (400 MHz,
CDCl3) δ 8.68 (d, 1H, J = 8 Hz), 8.33 (d, 1H, J = 8 Hz), 7.75 (t, 1H, J = 7.6 Hz ), 7.70 (t, 1H, J =
7.6 Hz), 7.63 (d, 1H, J = 8.8 Hz), 6.65 (d, 1H, J = 8.8 Hz), 6.48 (s, 1H), 6.42 (s, 1H), 3.43 (t, 4H,
J = 8 Hz), 1.69 (m, 4H, J = 8 Hz), 1.44 (m, 4H, J = 8 Hz), 1.03 (t, 6H, J = 8 Hz); MS (ESI): 375
(M+); m.p. >210 °C.
9-(bis(3-phenylpropyl)amino)-5H-benzo[a]phenoxazin-5-one (NR2): 23% yield. 1H NMR
(400 MHz, CDCl3) δ 8.68 (d, 1H), 8.33 (d, 1H), 7.74 (t, 1H), 7.68 (t, 1H), 7.58 (t, 2H), 7.31-7.39
(m, 4H), 7.21-7.30 (m, 5H), 6.48 (d, 1H), 6.41 (s, 1H), 6.26 (s, 1H), 3.40 (t, 4H), 2.71 (t, 4H), 2.00
(m, 4H); MS (ESI): 499 (M+); m.p. >220 °C.
2.3. Classification of benzophenoxazine derivatives
Nile red derivatives can be classified into three types according to the substituents attached
to benzophenoxazine backbone in two positions: the 2-position and the 9-position. The first type
is dialkylated derivatives containing a tertiary amine at the 9-position including NR1 and NR2,
the second type is 2-hydroxy-dialkylated derivatives containing a hydroxyl group at the 2-position
and a tertiary amine at the 9-position including NR3, NR4 and NR5, and the third type is 2-
hydroxy-monoalkylated derivatives containing a hydroxyl group at the 2-position and a secondary
amine at the 9-position including NR6 and NR7 as shown in Fig. 1a. Nile blue derivatives can be
classified into two types according to the type of amine attached to benzophenoxazine backbone
at the 9-position. The first type is dialkylated derivatives containing a tertiary amine at the 9-
position including NB1 and NB2, and the second type is monoalkylated derivatives containing a
secondary amine at the 9-position including NB3 and NB4 as shown in Fig. 1b.
10

2.4. Preparation of working solutions
Stock solutions of Nile red and Nile blue and their derivatives (4.0×10-4 M) were prepared
in ethanol and sonicated for 15 minutes to ensure complete dissolution of the dye. Sodium
hydroxide solution (0.01 M, pH 12.0) was prepared in deionized water. The working solution was
prepared by slowly adding 15.0 mL of sodium hydroxide solution to 15.0 mL of the dye with
constant stirring and used immediately after preparation. Final concentration of the dye in the
working solution was 2.0 ×10-4 M in ethanol: aqueous sodium hydroxide (1:1, v/v).
2.5. Fingerprint detection procedure
This work aims to provide a better understanding of the effect of chemical structure of the
dye on fingerprint development. Therefore, one donor and one porous substrate (paper) were
selected for the study. Fingerprints were collected on a white copy paper from a 26-year-old male
donor and left 24 hours before treatment. Fingerprints were deposited after the donor was
instructed to wipe his fingers with his nose prior to fingerprint collection. The donor was asked
not to wash his hands within the previous two hours. Fingerprints were obtained using the three
middle fingers holding for 8 seconds. The paper was cut in half before treatment, then the left side
of the paper was developed using derivatives of Nile red or Nile blue and the right side of the paper
was developed using the corresponding parent dye (Nile red or Nile blue) for comparison purposes.
The sample was first immersed in deionized water for 5 minutes, then it was removed and
immersed in the working solution for 30 minutes. The excess solution was removed by immersing
the sample in deionized water for 5 minutes. The sample was left to dry for about one hour on a
paper towel at room temperature in a dark place.
2.6. Sample visualization and evaluation
11

The sample was visualized by the forensic light source (FLS), Mini-CrimeScope 400,
(MCS-400) (Spex Forensics, Edison, NJ) at an excitation wavelength of 515 nm for Nile red
derivatives, CSS for Nile blue derivatives, and white light for all dyes. The two halves for each
sample were placed side by side and photographed together using a digital camera (Nikon D3300)
with a 550 nm long-pass barrier filter. University of Canberra (UC) scale [38] was used to compare
the performance of the two detection methods: A and B in which A is the proposed method and B
is the reference method. In this study, method A is the derivatives of Nile red or Nile blue and
method B is the corresponding parent dye, Nile red or Nile blue. Samples were given scores
between (-2) and (+2) depending on the improvement in detection performance including
fingerprint ridge details and/or contrast between the fingerprint impressions and background
substrate. UC scores: (+2) and (+1) were given when the half fingerprint developed by method A
shows far greater or slightly greater improvement in detection performance, respectively,
compared to the half fingerprint developed by method B; (0) was given when no significant
difference between the two half fingerprints; (-1) and (-2) were given when the half fingerprint
developed by method B shows slightly greater or far greater improvement in detection
performance, respectively, compared to the half fingerprint developed by method A [38].
3. Results and discussion
3.1. Photophysical properties of benzophenoxazine derivatives
The absorbance spectra of Nile red and Nile blue is influenced by the surrounding
environment as shown in Fig. 2. The presence of sodium hydroxide (1:1, v/v) in the working
solution resulted in a red shift of absorbance spectrum of Nile red of about 30 nm. However, the
absorbance spectrum of Nile blue displayed a blue shift of about 100 nm due to the deprotonation
of the amine group at the 5-position (pKa = 11.14) and formation the basic form of the dye. The
12

deprotonation process co-occurs with a color change of Nile blue solution from blue color in pure
ethanol to red color in the working solution, ethanol: sodium hydroxide (1:1, v/v). The high pH of
the working solution was selected as indicated in previous studies [9, 25] and to increase logD
value of benzophenoxazine derivatives. Moreover, improvement of fingerprint detection is
observed for hydrophobic dyes due to interaction with sebaceous secretions in fingerprint residue
as discussed in the following sections. Given the fact that logD is pH dependent, the
hydrophobicity of Nile blue alters by pH changes whereas that of Nile red does not. Therefore,
Nile blue is more hydrophobic in alkaline solution at pH 12.0 (logD = 3.79) compared to neutral
solution at pH 7.4 (logD = 0.60).
The chemical structure and physical properties such as hydrophobicity have an impact on
spectroscopic studies of Nile red and Nile blue derivatives. The best tool to describe the
hydrophobicity is the partition coefficient (logP) or the distribution coefficient (logD). LogP is
defined as the ratio of concentrations of the neutral species of compound in two immiscible phases
such as n-octanol and water at equilibrium. On the other hand, logD takes into account all neutral
and charged species of compound [39]. Regarding the absorption spectra, a general and small blue
shift was observed across Nile red derivatives (Fig. 3a) and the order of shifting was Nile red,
dialkylated derivatives NR1 and NR2, 2-hydroxy-dialkylated derivatives NR3, NR4, and NR5,
and 2-hydroxy-monoalkylated derivatives NR6 and NR7. Similarly, the absorption spectra of Nile
blue and dialkylated derivatives NB1 and NB2 showed a blue shift compared to those
monoalkylated derivatives NB3 and NB4 (Fig. 3b). A similar behavior was observed for Nile blue
derivatives after the addition of sodium hydroxide to ethanol for the preparation of working
solutions. There was a noticeable color change of the working solutions from blue to red color due
to deprotonation of the amine group at the 5-position (pKa = 11.14) and formation the basic form
13

of the dye. Examination the emission spectra (Fig. 4a) indicated that Nile red, dialkylated
derivative and 2-hydroxy-dialkylated derivatives were blue-shifted with low fluorescence intensity
compared to 2-hydroxy-monoalkylated derivatives. In a similar manner, the emission spectra of
Nile blue and dialkylated derivatives showed a blue shift compared to monoalkylated derivatives.
However, the fluorescence intensity of Nile blue derivatives were similar and emission bands were
wider compared to those of Nile red derivatives. The photophysical properties such as quantum
yields (Φf) of some derivatives of benzophenoxazine dyes are summarized in Table 2, and the
complete determination of photophysical properties will be illustrated in Martinez et al. (in
preparation).
3.2. Comparison between Nile red and Nile blue for fingerprint detection
To investigate the detection efficiency of the two benzophenoxazine dyes, the working
solution of Nile red and Nile blue (basic form) was used for developing fingerprints on a porous
surface (paper) as shown in Fig. 5. The sample treated by Nile red produced fingerprints more
luminescent than those with Nile blue at two settings of FLS, 515 nm and CSS, both viewed with
550 nm long-pass barrier filter. Although both dyes have similar hydrophobicity at pH 12.0 (logD
for Nile red and Nile blue is 3.83 and 3.79, respectively), the efficiency of fingerprint detection is
attributed to the difference of chemical structure of the dyes. Both molecules have similar
benzophenoxazine backbone except the attached groups at the 5-position, a carbonyl group and an
amine group for Nile red and Nile blue, respectively. The amine group is a hydrogen bond donor
forming hydrogen bonds with hydroxide ions present in the working solution. The hydrogen bonds
weaken the hydrophobic interaction of Nile blue with sebaceous portion of fingerprint residue
resulting in weak development.
14

FLS setting is an important factor to develop fingerprints with clear ridge details and good
contrast, especially for comparison purposes. Nile red developed luminescent fingerprints and
clear ridges in both settings of FLS as shown in Fig. 5 (left half). However, at CSS which
corresponds to 455 nm the background appeared more bright providing poor contrast. Moreover,
the maximum wavelength of the working solution of Nile red and Nile blue were 576 and 525 nm,
respectively (Fig. 2). Consequently, excitation wavelength at 515 nm is considered the optimum
condition of fingerprint visualization for performance comparison of Nile red and its derivatives.
On the other hand, weak fingerprint detection and poor contrast were observed for Nile blue in
both settings of FLS as shown in Fig. 5 (right half). Moreover, the sample treated by Nile blue at
515 nm seemed more pale due to lack of contrast between fingerprints and the substrate. Therefore,
CSS setting of FLS could be the best for fingerprints visualization for performance comparison of
Nile blue and its derivatives.
3.3. Application of Nile red derivatives for fingerprint devolvement
Fingerprints developed by the dialkylated derivatives NR1 and NR2 were compared to
those developed by Nile red as shown in Fig. 6. Both dyes as well as Nile red showed excellent
ability to detect fingerprint in which luminescent impressions and detailed ridges were observed
at excitation wavelength of 515 nm of the FLS (Fig. 6a), and the results of Nile red are consistent
with work of Braasch et al. [25]. Moreover, the underlying substrate of the sample treated by NR1
was darker compared to Nile red providing more contrast between the prints and background and
thus improvement in fingerprint detection. When using white light of FLS without long-pass filter,
fingerprints appeared as pink impressions with complete ridge details for NR1 and NR2, whereas
the prints were not visible for Nile red (Fig. 6b). However, solid particles were precipitated on the
background of the sample treated by NR2 which could be due to the aqueous nature of the working
15

solution and the great hydrophobicity of the dye. Nonetheless, the precipitation process did not
prevent fingerprint development. The improvement in fingerprint detection is attributed to the fact
that both compounds are more hydrophobic than Nile red as indicated by logD values in Table 3.
It has been hypothesized that Nile red tended to interact with lipids secreted by sebaceous glands
and not with epidermal lipids [25, 28]. Therefore, the hydrophobic interaction of NR1 and NR2
with sebaceous secretions of fingerprint residue is stronger relative to Nile red making the two
derivatives dual-fingerprint reagents developing visible prints seen by the naked eye (pink
impressions) and luminescent prints (yellow-orange impressions) when using FLS at excitation
wavelength of 515 nm.
The use of the 2-hydroxy dialkylated derivatives of Nile red resulted in fingerprints that
produced various degree of luminescence according to their chemical and physical properties (Fig.
7). Blurred and non-luminescent fingerprints were obtained by the less hydrophobic derivative,
NR3 due to the weak interaction with fingerprint components. On the other hand, fingerprints
developed by NR4 and NR5 exhibited comparable results in which fingerprints were luminescent
with clear ridge details. However, fingerprints developed by Nile red displayed a higher degree of
luminescence, ridge details, and clarity. Despite the fact that NR4 and NR5 are hydrophobic and
the latter dye is more hydrophobic than Nile red (Table 3), there was no improvement in the
developing of fingerprints. This could be due to the unwanted interaction of the dye molecules
with the working solution. At pH 12.0, the hydroxyl group at the 2-position is deprotonated (pKa
= 5.37) and the negatively charged hydroxide ion can form a bond with positively charged sodium
ions present in the working solution. Therefore, the hydrophobic interaction of both dyes with
fingerprint secretions is weakened by interference effect. The ability of the dye to develop
fingerprints is mainly influenced by its hydrophobicity and other factors such as type of
16

substituents attached to benzophenoxazine backbone as indicated in the results of dialkylated
derivatives and 2-hydroxy dialkylated derivatives. The chemical structures of Nile red, NR1 and
NR2 are analogous to those for NR3, NR4 and NR5, respectively with the exception that the latter
derivatives contain a hydroxyl group at the 2-position. The presence of hydroxyl group decreases
the hydrophobicity of dyes and has adverse impact on fingerprint development.
Fingerprints developed by the 2-hydroxy monoalkylated derivatives were faint and
appeared as smudged prints compared to those developed by Nile red (Fig. 8). The inefficiency of
NR6 and NR7 is referred to the low hydrophobicity as characterized by logD resulting in weak
interaction with fingerprint residue and poor detection.
It was observed that the fluorescence properties of the final product (fingerprint
impressions) resulting from the interaction between the dye and fingerprint residue were not
influenced by photophysical properties of the dye. For instance, the performance of dialkylated
derivatives NR1 and NR2 were comparable in which fluorescent fingerprints with good contrast
were obtained (Fig. 6) even though the fluorescence intensity of NR2 was higher than NR1 as
shown in Fig. 4a. Moreover, samples treated by 2-hydroxy monoalkylated derivatives NR6 and
NR7 resulted in non-fluorescent fingerprints as shown in Fig. 8 regardless their high fluorescence
intensity compared to other derivatives (Fig. 4a).
3.4. Application of Nile blue derivatives for fingerprint devolvement
Samples treated by the dialkylated derivatives were compared to those treated by Nile blue
(all in the basic form of the dyes) as shown in Fig. 9. The fingerprints developed by NB1 and NB2
appeared more luminescent with clear ridge pattern than those developed by Nile blue at CSS
setting of the FLS. Furthermore, visible fingerprints were obtained as dark blue impressions with
clear definition for dialkylated derivatives samples with the use of white light of FLS. However,
17

no fingerprints were visible for Nile blue sample at the same setting of FLS. Thus, the two
compounds are considered dual-fingerprint reagents due to their ability to reveal fingerprints that
are visible to the naked eye (dark blue impressions) and luminescent when using FLS (yellow-
orange impressions). The enhancement of fingerprint performance for NB1 and NB2 is due to the
great hydrophobicity as characterized by logD (Table 3) resulting in stronger interaction with lipid
components of fingerprint residue.
On the other hand, non-luminescent fingerprints were obtained for the monoalkylated
derivatives samples relative to those for Nile blue sample (all in the basic form of the dyes) as
shown in Fig. 10. The inferior performance of NB3 and NB4 is attributed to the low hydrophobicity
and type of groups attached to benzophenoxazine backbone as previously mentioned. NB3 is less
hydrophobic than Nile blue and thus unsuccessful fingerprint detection was obtained due to the
weak interaction with fingerprint reside. Despite the similarity in hydrophobic properties of NB4
and Nile blue, no development in fingerprint detection was observed. The chemical structure of
NB3 and NB4 is analogous to Nile blue and NB1 respectively with the exception that an extra
alkyl group is attached to the amine at the 9-position in the latter dyes. NB3 and NB4 contain
secondary amine at the 9-position which is considered a hydrogen donor that has the potential to
form hydrogen bonds with hydroxide ions present in the working solution. The hydrogen bonds
suppress the interaction of the dyes with fingerprint residue. Therefore, the use of these dyes to
detect fingerprints is not suitable in forensic detection due to the hydrogen bonds and/or low
hydrophobicity.
It was shown that the photophysical properties of Nile blue derivatives did not have impact
on the intensity of the developed fingerprints. For example, NB2 with the lowest fluorescence
intensity (Fig. 4b) was considered a dual reagent due to its ability to develop fluorescent and visible
18

fingerprints (Fig. 9). On the other hand, NB4 had the highest intensity as indicated in Fig. 4b
despite its poor detection (Fig. 10). Furthermore, the fingerprint performance of NB, NB1, and
NB3 was different regardless their similarity in fluorescence intensity.
3.5. Performance evaluation of benzophenoxazine derivatives
To assess the performance of benzophenoxazine dyes as potential fingerprint reagents, UC
scale [38] was applied for comparison between the two methods A and B, in which method A is
derivatives of Nile red or Nile blue and method B is corresponding parent dye, Nile red or Nile
blue as shown in Table 4. For Nile red derivatives performance, dialkylated derivatives NR1 and
NR2 showed improvement in fingerprint detection relative to Nile red in which they developed
visible and luminescent prints with clear ridge patterns, and NR1 displayed better background
contrast. Consequently, the two dyes can be considered as dual-fingerprint reagents due to their
great hydrophobicity compared to that of Nile red. However, no improvement in fingerprint
detection was observed for the 2-hydroxy dialkylated derivatives and 2-hydroxy monoalkylated
derivatives due to the interference effect and/or the low hydrophobicity. Faint or non-luminescent
fingerprints and poor contrast were obtained by both types of Nile red derivatives.
For Nile blue derivatives detection performance, dialkylated derivatives NB1 and NB2
exhibited enhancement in developing fingerprints relative to Nile blue. Both dyes are considered
dual-fingerprint reagents due to their ability to reveal visible and more luminescent prints with
clear ridge details. However, weak fingerprints were developed by monoalkylated derivatives NB3
and NB4. Therefore, these dyes are not useful reagents for fingerprint detection due to the low
hydrophobicity and/or hydrogen bonds.
Besides the improved results of dialkylated derivatives of Nile red and Nile blue, these
compounds exhibit high sensitivity to produce visible prints at dye concentration of 0.2 mM. This
19

concentration is 5-fold less than the concentration needed for the dual-fingerprint reagent genipin,
to develop visible prints at concentration of 10 mM [33]. In addition, the combination of color and
luminescent properties of the developed prints by a single reagent offer visualization in the field
as well as in the laboratory. Moreover, the fluorescent prints are more practical as they can be
recovered from dark-colored surfaces. Other than immersing the sample in water before and after
the treatment, no pretreatment or post-treatment is needed to produce luminescent prints.
4. Conclusions
The use of two types of benzophenoxazine dyes as developing reagents to reveal latent
fingerprints on porous surfaces was investigated. It was found the most significant property of the
dye to develop fingerprints is hydrophobicity as indicated by logD value. In this work,
hydrophobic dialkylated derivatives of Nile red and Nile blue (in the basic form) showed
enhancement in developing fingerprints. NR1, NR2, NB1, and NB2 detected fingerprints more
effectively than their corresponding parent dyes producing more luminescent and visible prints.
Therefore, these dyes are considered as dual-fingerprint reagents. Furthermore, the application of
the reagents is simple, sensitive, and effective suggesting their potential use in forensic detection.
Besides the significant role of hydrophobicity for fingerprint development, other parameters such
as the functional groups attached to the benzophenoxazine backbone and their ability to form
hydrogen bonds or interact with solvent should be taken into consideration. Therefore, the use of
less hydrophobic derivatives such as NR3, NR6, NR7, and NB3 or hydrophobic derivatives
containing interference substituents such as NR4, NR5, and NB4 as fingerprint reagents was not
sufficient for detection purposes. Further studies are needed to evaluate the implementation of the
modified reagents to develop latent fingerprints on different substrates for a large number of
donors and to reveal aged fingerprints.
20

Author Contribution:
E.A. designed this work and prepared the manuscript. W.A. synthesized and characterized NR1
and NR2. All authors reviewed and edited the manuscript.
Declaration of interests
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The authors would like to thank Mr. Carl Kananda, Georgia State University, USA for
resynthesizing NR5.
This research did not receive any specific grant from funding agencies in the public, commercial,
or not-for-profit sectors.
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(a)
(b)
NR: R1 = R2 = C2H5 , R3 = R4 = H
NB: R1 = R2 = C2H5
NR1: R1 = R2 = C4H9 , R3 = R4 = H
NB1: R1 = R2 = C4H9
NR2: R1 = R2 = (C6H5)C3H6 , R3 = R4 = H
NR3: R1 = R2 = C2H5 , R3 = H, R4 = OH
NR4: R1 = R2 = C4H9 , R3 = H, R4 = OH
NR5: R1 = R2 = (C6H5)C3H6 , R3 = H, R4 = OH
NR6: R1 = C4H9 , R2 = H, R3= CH3 , R4 = OH
NR7: R1 = (C6H5)C3H6 , R3 = H, R3 = CH3 , R4 = OH
NB2: R1 = R2 = (C6H5)C3H6
NB3: R1 = C2H5 , R2 = H
NB4: R1 = C4H9 , R2 = H
Figure 1: Chemical structures of (a) Nile red derivatives and (b) Nile blue derivatives
27

Figure 2: Absorbance spectra of 2.0×10-5 M Nile red (red line) and Nile blue (blue line) in ethanol
(solid line) and in the working solution [ethanol: sodium hydroxide (1:1, v/v)] (dashed line)
28

0.6
0.4
0.2
0.0
(a)
NR
NR1
NR2
NR3
NR4
NR5
NR6
NR7
400
500
600
700
800
Wavelength (nm)
1.0
0.8
0.6
0.4
0.2
0.0
(b)
NB
NB1
NB2
NB3
NB4
400
500
600
700
800
Wavelength (nm)
Figure 3: Absorbance spectra of 2.0×10-5 M of (a) Nile red derivatives and (b) Nile blue
derivatives in the working solution [ethanol: sodium hydroxide (1:1, v/v)]
29