ArticlePDF Available

Abstract and Figures

In a crime scene, the collection of evidence and a subsequent laboratory analysis compose the fundamental steps to allow the expert to reveal the truth for the final verdict in a jury and to bring back the comfort to the victim’s family. Bloodstains are usually found and sent to laboratories as a vestige to unravel the origin of the material. However, some scenes are modified in order to conceal the real culprit for the criminal act. For these cases, the luminol reagent can be useful. This test is very often used to visualize occult blood. Luminol is considered the most sensitive test once it can identify the blood presence in scale of nanograms. When this reagent comes into contact with blood, the light emission occurs through a phenomenon known as chemiluminescence. This luminescence can be produced by other interfering compounds, leading to a misinterpretation for the presence of blood. Despite this shortcoming, the present review article highlights the indispensability of the reagent luminol on a crime scene.
Content may be subject to copyright.
172
J. Biotec. Biodivers. v. 3, N.4: pp. 172-177, November 2012
Vol. 3, N. 4: pp. 172-177, November 2012
ISSN: 2179-4804 Journal of Biotechnology
and Biodiversity
Luminol in the forensic science
Luminol na ciência forense
Rafaela Rogiski da Silva1, Bruna Carla Agustini2, André Luís Lopes da Silva3, Henrique
Ravanhol Frigeri3*
1Graduate Degree in Chemistry; Polytechnic School; Pontifical Catholic University of Parana; 80215-901;
Curitiba - PR - Brazil, 2Laboratory of Enzymology and Fermentation Technology; 80210-170; Curitiba - PR -
Brazil, 3Health and Biosciences School; Pontifical Catholic University of Parana; 80215-901; Curitiba - PR -
Brazil.
ABSTRACT
In a crime scene, the collection of evidence and a subsequent laboratory analysis compose the fundamental steps to
allow the expert to reveal the truth for the final verdict in a jury and to bring back the comfort to the victim’s family.
Bloodstains are usually found and sent to laboratories as a vestige to unravel the origin of the material. However,
some scenes are modified in order to conceal the real culprit for the criminal act. For these cases, the luminol
reagent can be useful. This test is very often used to visualize occult blood. Luminol is considered the most sensitive
test once it can identify the blood presence in scale of nanograms. When this reagent comes into contact with blood,
the light emission occurs through a phenomenon known as chemiluminescence. This luminescence can be produced
by other interfering compounds, leading to a misinterpretation for the presence of blood. Despite this shortcoming,
the present review article highlights the indispensability of the reagent luminol on a crime scene.
Key words: luminol, chemiluminescence, crime scene, occult blood, criminalistics, blood stain
INTRODUCTION
The place where a crime has taken place should be
well preserved and investigated in order to provide
a reliable report from laboratory analysis and
support the defendant judgment. Criminalistics,
through a junction of sciences, analyzes the
material collected by specialists in the crime scene
to identify the source of the evidence. It does not
have the role of judge but to seek the truth without
deleting the particularity of justice, serving as
expert aid in elucidating a crime.
THE BLOOD
Blood is responsible for gas exchange, transport of
hormones and nutrients, pathogenic defense,
coagulation, homeostasis and regulation of
osmotic pressure. In appearance can be described
as a red liquid with an alkaline character that can
be divided into two parts: (i) hematocrit (elements
or figurative bodies, such as leukocytes,
erythrocytes and platelets) and (ii) the liquid
portion, composed of approximately 90% water,
referred to as the plasma (Dorea, 1995)
Erythrocytes
Erythrocytes (Red Blood Cells, RBC) are
anucleate cells originated from bone marrow and
are constituted mainly by a protein called
hemoglobin (Hb) (Gartner & Hiatt, 2007; Baynes
& Dominiczak, 2007). The main function of this
cell is to transport gas. When erythrocytes are
situated in the alveoli, this phenomenon is called
hematosis in which the exchange of CO2 and O2 is
verified. CO2 molecule binds to the heme group of
the hemoglobin and is transported to the cells
throughout the body in order to generate energy
for maintenance of life in a process known as
cellular respiration. The Hb molecule have a
tetrahedral structure formed by four polypeptide
chains, two alpha and two beta, constituted with
heme group – and its structure was proposed in
1959 by Max Perutz and collaborators. The heme
________________________________________________________________________
Author for correspondence: henrique.frigeri@gmail.com
173
J. Biotec. Biodivers. v. 3, N.4: pp. 172-177, November 2012
group is formed by the union of the porphyrin IX
ring which has four nitrogen atoms with free
ligands which bind with the ion Fe2+ to the center
by simple covalent and coordinate bounds
(Moreira et al.; 2011; Henry, 1999; Baynes &
Dominiczak, 2007). The Figure 1 shows the
chemical structure of Protoporphyrin IX and the
heme group.
Figure 1. Chemical structure of protoporphyrin IX and heme group. This illustration shows two chemical structures:
(A) It represents Protoporphyrin IX molecule and (B) ferro-protoporphyrin IX, also called as heme group. Nitrogen
atoms form simple covalent (in black) and covalent coordinate bonds (in blue) with Fe2+ ions (in red) in the (B)
structure.
Leukocytes
Leukocytes (White Blood Cells, WBC) are
responsible for the body's defense and they are
classified in two general groups: i) granulocytes
and ii) agranulocytes. The first group has specific
cytoplasmic granules and is classified as
neutrophils, eosinophils and basophils. The second
group has specific granules and is divided into
lymphocytes and monocytes (Gartner & Hiatt,
2007). Among several complex mechanisms of
defense, diapedesis is a very common one. In this
process, monocytes from blood cross through the
cells junctions and transform themselves in
phagocytes when they find a foreign or pathogenic
agent (Dorea, 1995; Baynes & Dominiczak, 2007).
Platelets
Platelets are constituted of megakaryocytes
fragments and do not present a nucleus as part of
their constitution. These cells participate in blood
coagulation by preventing a hemorrhagic process.
When a vascular event occurs, the platelets are
activated and the mechanism of coagulation starts.
In the end of this process, fribrinogen (factor I) is
converted to fibrin (factor Ia) which contributes
for the clot formation on the injury site (Dorea,
1995; Baynes &; Dominiczak, 2007).
BLOOD AS CRIME EVIDENCE
The collection of material from a crime scene from
the recovery technique to its storage should be
performed very carefully, following a standard
protocol. The procedure must be error-free to
assure all legal requirements. Another important
fact of a criminalistics study is the reconstruction
of the event, because, this way is possible to have
a greater veracity linking the evidence to the
crime.
Evidence can be originated from Biological and
Non Biological Source (BS, NBS). Impressions, or
fingerprints, with waxes and rust are examples of
NBS. Bloodstains, saliva and semen constitute
examples of BS. For criminalistics, the interesting
in a spot of blood is its nature and its form. It can
be identified through reagents that act on
hemoglobin and its derivatives (proteins and
amino acids). Due to its immutable and non-
transferable characteristics these components are
capable of identifying the origin of evidence.
174
J. Biotec. Biodivers. v. 3, N.4: pp. 172-177, November 2012
The crime scene can be changed by the defendant
to hide any evidence left behind, puzzling the
investigation. Advances in forensic science had
provided some reagents to support specialists’
analysis. These reagents can unravel traces
invisible to naked eye. The reagents used by the
experts are part of the identification tests. One
example is the benzidine test, which identifies the
presence of hemoglobin in the sample by the
appearance of a blue color. This result
demonstrates the oxidation of the hemoglobin
molecule by hydrogen peroxide present in the
reaction medium. A disadvantage of this method is
the DNA degradation within forty-eight hours later
use (Tobe et al., 2007). Another test, known as
Kastle-Meyer, also reveals a change in medium
color in the presence of hemoglobin. It goes from
colorless to pink due to the presence of
phenolphthalein. Luminol test is a third assay and
has been considered one of the most well-known
test in the field of forensic science. Luminol reacts
with blood promoting light emission.
The luminol test is based on a chemiluminescence
reaction (emission of light through a chemical
reaction) due to the breakage of a bond containing
energy stored. The intensity of the emitted light
varies with pH, temperature and concentration of
reagents involved. The light emission can cease if
there is a critical change in these parameters
(Barni et al., 2007).
Among the conditions required for a
chemiluminescent reaction to occur, stand out: (i)
the reaction must be spontaneous (exothermic
process), due to the breakage of the bound; (ii) the
direction of the reaction should be favorable to the
formation of the excited electronic state and (iii),
after the light emission, the excited state is
deactivated, causing the electrons back to their
ground energy state (Kai et al., 2001).
LUMINOL
Luminol (5-amino-2,3-dihydro-1,4-phthalazine
dione), shown in Figure 2, was first synthesized in
1908 (Barni et al., 2007). Its use as an adjuvant at
a crime scene has been reported only in 1937, after
experiments with hydrogen peroxide and
derivatives from the heme group. Advances on
researches with luminol have pointed out
inhibitors and interferences that might promote the
variation of light emitted by this reagent (Barni et
al., 2007).
Figure 2. Chemical Structure of the Luminol. This illustration shows four representations of luminol structure. (A)
It represents a simple structure. (B) It shows the structure in balls and sticks. The atoms are showed by different
colors (grey/carbon, white/hydrogen, blue/nitrogen and red/oxygen). (C) It reports, in blue and red colors, points in
the molecule with right polarity and electronegativity and (D) Evidences the planar structure of Luminol.
This compound belongs to acyl hydrazide class
and is found in crystal form (Barni et al., 2007).
Luminol solution is sensitive to acids and strong
bases, oxidizing agents and light. Although being
considered the most popular assay, Luminol does
not have specificity for human blood as well as the
other tests mentioned above (Blum et al., 2006).
Luminol solution
The medium where the test is carried out is
composed of sodium hydroxide (NaOH), soda
(Na2CO3.nH2O), hydrogen peroxide (H2O2) and
sodium perborate (NaBO3). These components are
considered irritating to the eyes and mucous, thus
the use of personal protective equipment is
essential. Federal departments do not control the
sale of this product and only a few studies about
its toxicity are available in the literature.
175
J. Biotec. Biodivers. v. 3, N.4: pp. 172-177, November 2012
Considering that pH values between ten and
thirteen enable optimal conditions to luminol
reaction, the medium pH must remain alkaline in
order to increase the assay’s efficiency. Prior
experiments have demonstrated that pH 11.5 is
ideal to identify blood spots (Blum et al., 2006).
The intensity of the light depends on the nature
and concentration of the catalyst used, may present
and this effect can be photographed and lasting
approximately one minute (Barni et al., 2007;
Tobe et al., 2007).
The test requires the application of luminol
solution in the blood stain, whether visible or not
to the naked eye, to prove the result. When
compared to other screening tests, Luminol shows
higher sensibility, making feasible the detection in
nanogram scale. For this reason, depending on the
sample concentration it is the best alternative for
the analysis. Another variable to luminol assay lies
on the surface area in which the reaction will take
place. Surfaces of absorbent material, even after a
cleaning process or a long period of time from the
crime occurrence, will enable a positive result.
Luminol solution presents also the advantage of
maintaining the integrity of the genetic material
after the analysis.
Mechanism of action
The light emission is associated with the
conversion of 3-aminophthalic to 3-
aminophthalate (Albertin et al., 1998). The first
and second steps of the reaction mechanism of
Luminol is represented in figures 3 and 4,
respectively.
Figure 3. Luminol’s mechanism of reaction (Step 1). In solution, hydrogen peroxide oxidizes luminol (I)
giving rise to compounds diazoquinone (II) and peroxide ions with the aid of a catalyst that, in the presence of
blood, is the cation iron II. Due to its high reactivity, the diazoquinone does not need of catalyst or oxidizing agent
and when it reacts with the peroxide ions, there is the formation of the endoperoxide (III). Adapted from Albertin et
al. (1998) with modifications.
Figure 4. Luminol’s mechanism of reaction (Step 2). In this second stage, the endoperoxide (III) after losing
molecular nitrogen is in its excited state, characterized by 3-amino phthalic (IV). When it decays, in terms of energy,
occurs the release of light that is emitted and is able to measure its intensity and formation of the 3-aminophthalate
(V). Adapted from Albertin et al. (1998) with modifications.
176
With the cleanup of the place as an attempt to
disappear with the evidences, interfering
compounds can be added to the local modifying
the outcome of Luminol’s chemiluminescence,
concerning to color, intensity and form of
distribution. These changes enable the
specialists to clearly differentiate a place that has
been altered from another that has not.
Interferents
The result of luminol’s assay may be altered by
a broad range of interfering compounds,
commonly found in home environment, such as
iron salts, copper, iodine, potassium
permanganate, animal hemoglobin, plant
peroxidases and - hypochlorite ion. The latter
represents the principal cause of a false positive
result.
The clearance of a bloodstain upon washing with
hypochlorite solution removes more hemoglobin
compared to water alone. As the hemoglobin
concentration decreases, the emission of light by
Luminol is reduced. However, a successive
wash with hypochlorite solution promotes the
accumulation of this ion which increases the
intensity of light emission by Luminol. In
summary, the hemoglobin is replaced by
hypochlorite ion leading to a light emission
equivalent as the one emitted by a blood stain
that has only been washed with water (Creamer
et al., 2005; Barni et al., 2007, Castelló et al.,
2009).
Beyond the crime scene
In addition to the role of luminol in revealing
hidden blood on a crime scene, it can also be
useful in protecting patients and staff inside a
hospital. The identification of blood traces on
clothes and other hospital utensils helps on
disease control, for example, in a hemodialysis
unit, preventing any transference of biological
material. Luminol’s assay has another
interesting feature while estimating the age of a
bone. This test is capable of identifying traces of
hemoglobin after 400 years postmortem
(Bergervoet et al., 2008; Ramsthaler et al., 2009;
Ramsthaler et al., 2011). The intensity of the
light emitted is inversely proportional to the
bone’s age due to the degradation of hemoglobin
as time goes by (Creamer & Buck, 2009).
CONCLUSION
The relevance of Luminol for forensic
specialists is immeasurable due to its high
sensitivity and easy handling. Besides its higher
sensitivity, Luminol, as the other used tests, is
not specific for human blood. Thus we
encourage new researches focusing in the
improvement of Luminol´s specificity, since
there are scarce studies in this area.
ACKNOWLEDGEMENTS
We would like to thank Pontifical Catholic
University of Parana for all support.
RESUMO
Em uma cena de crime, a coleta de evidências e a
posterior análise laboratorial compõem etapas
fundamentais que permitem ao perito revelar a
verdade quanto ao veredito final, em um júri, e trazer
de volta o conforto para a família de uma vítima de
crime. Manchas de sangue são, normalmente,
encontradas e enviadas para laboratórios como
vestígio para desvendar a origem do material. No
entanto, algumas cenas são modificadas a fim de
ocultar o verdadeiro culpado pelo ato criminoso.
Para estes casos, o reagente luminol pode ser útil.
Este teste é muito utilizado para visualizar o sangue
oculto. O teste do luminol é considerado o mais
sensível, uma vez que pode identificar a presença de
sangue na escala de nanogramas. Quando este
reagente entra em contato com o sangue, a emissão
de luz ocorre através de um fenômeno conhecido
como quimioluminescência. Esta luminescência pode
ser produzida por outros compostos interferentes,
levando a uma interpretação quanto à presença
de sangue. Apesar dos achados recentes, o presente
artigo de revisão destaca a indispensabilidade do
reagente luminol em uma cena de crime.
Palavras-chave: luminol, quimioluminescência, cena
do crime, sangue oculto, criminalística, mancha de
sangue
REFERENCES
ALBERTIN, R.; ARRIBAS, M. A. G.; BASTOS, E.
L.; RÖPKE, S.; SAKAI, P. N.; SANCHES, A. M.
M.; STEVANI, C. V.; UMEZU, I. S.; YU, J.;
BAADER, W. J. Quimiluminescência orgânica:
alguns experimentos de demonstração para a sala de
aula. Química Nova, v. 21, n. 6, p. 772-779, 1998.
BARNI, F.; LEWIS, S. W.; BERTI, A.;
MISKELLY, G. M.; LAGO, G. Forensic application
of the luminol reaction as a presumptive test for
latent blood detection. Talanta, v. 72, 896-913, 2007.
177
BAYNES, J. W.; DOMINICZAK, M. H. Bioquímica
Médica, 2 ed. Rio de Janeiro: Elsevier, 2009, 566p.
BERGERVOET, P. W. M.; RIESSEN, N. V.;
SEBENS, F. W.; ZWET, W. C. V. D. Application of
the forensic Luminol for blood in infection control.
Journal of Hospital Infection, v. 68, p. 329-333,
2008.
BLUM, L. J.; ESPERANÇA, P.; ROCQUEFELTE,
S. A new high-performance reagent and procedure
for latent bloodstain detection based on luminol
chemiluminescence. Canadian Society of Forensic
Science, v. 39, n. 3, p. 81-110, 2006.
CASTELLÓ, A.; FRANCÉS, F.; VERDÚ, F. Bleach
interference in forensic luminol test on porous
surfaces: More about the drying time effect. Talanta,
v. 77, p. 1555-1557, 2009.
CREAMER, J. I.; QUICKENDEN, T. I.;
CRICHTON, L. B.; ROBERTSON, P.; RUHAYEL,
R. Attempted cleaning of bloodstains and its effect on
the forensic test. Luminescence, v. 20, p. 411-413,
2005.
CREAMER, J. I.; BUCK, A. M. The assaying of
haemoglobin using luminal chemilumencence and its
application to the dating of human skeletal remains,
Luminescence, v. 24, p. 311-316, 2009.
DOREA, L. E. As manchas de sangue como indício
em local de crime. 2.ed. Porto Alegre: Sagra-
Luzzato, 1995, 96p.
GARTNER, L. P.; HIATT, J. L. Tratado de
histologia. Rio de Janeiro (RJ): Guanabara Koogan;
2007.
HENRY, J. B. Diagnósticos clínicos e tratamentos
por métodos laboratoriais. 18th edition. Mande:
São Paulo, 1999.
KAI, M.; OHTA, K.; KURODA, N.; NAKASHIMA,
K. Chemiluminescence and Bioluminescence in DNA
Analysis. In: CAMPAÑA, A. M. G.; BAEYENS, W.
R. G. Chemiluminescence in analytical chemistry,
p. 551-566, 2001.
MOREIRA, L. M.; MORAES, P. C. G.;
MENDONÇA, J. P. R. F.; GUIMARÃES, L.; LYO,
J. P.; AIMBIR, F.; POLI, A. L.; IMASAT, H.
Hemoglobina extracelular gigante de glossoscolex
paulistus: um extraordinário sistema supramolecular
hemoproteico, Química Nova, v. 34, p. 119-130,
2011.
RAMSTHALER, F.; KREUTZ, K.; ZIPP, K.;
VERHOFF, M. A. Dating skeletal remains with
luminol-chemiluminescence. Validity, intra-and-
interobserver error, Forensic Science International,
v. 187, p. 47-50, 2009.
RAMSTHALER, F.; EBACH, S. C.;
BIRNGRUBER, C. G.; VERHOFF, M. A.
Postmortem interval of skeletal remain through the
detection of intraosseal hemin traces. A comparison
of UV-fluorescence luminol, Hexagon-OBTI, and
Combur test, Forensic Science International, v. 36,
p. 153 -160, 2011.
TOBE, S. S.; WATSON, N. L.; DAIÊD, N. N.
Evaluation of six presuntive tests for blood, their
specificity, sensitivity and effect on high molecular-
weight DNA. Journal Forensic Science, v. 52, p.
102-109, 2007.
... Sin embargo, esta oxidación no es espontánea pues requiere de la participación de un catalizador (Bustos et al., 2001). Dentro de la química forense, ciencia que analiza evidencia encontrada en escenas de crimen, el catalizador empleado es la sangre humana (Silva et al., 2012); tal como retrata Hollywood. Pero, ¿cómo exactamente cataliza la sangre una reacción quimioluminiscente? ...
... El luminol produce quimioluminiscencia por la oxidación con peróxido de hidrógeno, en medio acuoso básico, y con acción del catalizador sangre; específicamente por su contenido en hemoglobina (Silva et al., 2012). Esta metaloproteína se estructura en base a un anillo porfirínico, en cuyo centro se encuentra coordinado un ión Fe 2+ (Püntener & Schlesinger, 2000). ...
... Simultáneamente a la reducción del peróxido de hidrógeno, el luminol se oxida hacia diazoquinona, un compuesto intermedio en la reacción quimioluminiscente. Esta diazoquinona se oxida espontáneamente hacia endoperóxido, el cual pierde nitrógeno en forma de gas, formando el anión 3-aminofalato, entrando en consecuencia en un estado de excitación molecular, como se ilustra en la Figura 1. Cuando este regresa a su estado energético basal emite un fotón de luz azul de longitud de onda 431 nm (Silva et al., 2012). ...
Article
Full-text available
La quimioluminiscencia consiste en la emisión de luz visible como resultante de reacciones químicas, en las cuales reactivos, como el luminol, son oxidados por agentes como el peróxido de hidrógeno produciendo fotones de luz. Sin embargo, esta oxidación no es espontánea pues requiere de la participación de un catalizador (Bustos et al., 2001). Dentro de la química forense, ciencia que analiza evidencia encontrada en escenas de crimen, el catalizador em-pleado es la sangre humana (Silva et al., 2012); tal como retrata Hollywood. Pero, ¿cómo exactamente cataliza la sangre una reacción quimioluminiscente?
... The results of the analysis in the previous steps of this study were integrated with available information in literature obtained through text mining [18][19][20]. The mechanism was fine-tuned with existing organic chemistry reactions, and an elaborated mechanism of chemiluminescence of luminol was proposed. ...
Article
Objective This study aimed to discover chemiluminescent analogues of luminol, predict their molecular binding to hemoglobin of bloodstains in household crime, and expound the mechanism of chemiluminescence of luminol. Methods Similarity and clustering analyses of luminol analogues were conducted, and molecular dockingwas carried out using hemoglobin from Homo sapiens and four domestic organisms namely Gallus gallus, Drosophila melanogaster, Rattus norvegicus, and Canis familiaris. Results The results show the order of overall binding score as D. melanogaster > H. sapiens > C. familiaris > R. norvegicus > G. gallus. Seven compounds namely ZINC16958228, ZINC17023010, ZINC19915427, ZINC34928954, ZINC19915369, ZINC19915444, and ZINC82294978, were found to be consistently stable in binding to diverse hemoglobin and possibly have chemiluminescence than luminol in this in silico study. The amino acid residues involve in the interaction of human hemoglobin with 30 chemical compounds which include luminol, showed that His45, Lys61, Asn68, Val73, Met76, Pro77, Ala79, Ala82, Leu83, Pro95, Phe98, Lys99, Ser102, Ser133, Ala134, and Thr134 were possibly significant in the mechanism of action of presumptive test compounds. It was hypothesized that the improved mechanism of chemiluminescent for identification of blood was based on peroxidase-like reaction, that produce nitric oxide which bind to hemoglobin (Hb) and inhibits Hb degradation without yield of fluorescent products. The compound 2,3-benzodioxine-1,4,5(6H)-trione was formed which possibly emit light. Conclusion This study provides novel insight on the luminol and its expanded mechanism for broader possible applications of luminol with careful development of new methodologies.
... Coelenterazine is derived from a protein called coelenterate, which has been synthesized by several methods described elsewhere (Dodeigne et al., 2000). This compound possesses a superoxide anion in its structure, which is responsible for causing coelenterazine to give out CL. Unlike luminol, coelenterazine does not require any catalyst to trigger CL (Silva et al., 2012). CLA (2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one) and specifically MCLA probe (2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one) which is more efficient are some of the several coelenterazine analogs that have been prepared and used (Dubuisson et al., 2005;Wang et al., 2012;Diaz et al., 2018). ...
Article
Full-text available
Tissue imaging has emerged as an important aspect of theragnosis. It is essential not only to evaluate the degree of the disease and thus provide appropriate treatments, but also to monitor the delivery of administered drugs and the subsequent recovery of target tissues. Several techniques including magnetic resonance imaging (MRI), computational tomography (CT), acoustic tomography (AT), biofluorescence (BF) and chemiluminescence (CL), have been developed to reconstruct three-dimensional images of tissues. While imaging has been achieved with adequate spatial resolution for shallow depths, challenges still remain for imaging deep tissues. Energy loss is usually observed when using a magnetic field or traditional ultrasound (US), which leads to a need for more powerful energy input. This may subsequently result in tissue damage. CT requires exposure to radiation and a high dose of contrast agent to be administered for imaging. The BF technique, meanwhile, is affected by strong scattering of light and autofluorescence of tissues. The CL is a more selective and sensitive method as stable luminophores are produced from physiochemical reactions, e.g. with reactive oxygen species. Development of near infrared-emitting luminophores also bring potential for application of CL in deep tissues and whole animal studies. However, traditional CL imaging requires an enhancer to increase the intensity of low-level light emissions, while reducing the scattering of emitted light through turbid tissue environment. There has been interest in the use of focused ultrasound (FUS), which can allow acoustic waves to propagate within tissues and modulate chemiluminescence signals. While light scattering is decreased, the spatial resolution is increased with the assistance of US. In this review, chemiluminescence detection in deep tissues with assistance of FUS will be highlighted to discuss its potential in deep tissue imaging.
... In short, along with the synthesis of many lactic derivatives that reduced the synthesis of chemical luminol and did not change the maximum emission wavelength, then measured the basic physical properties of luminol and diphthalateamipin, and found that the results shown through the search is identical with theoretical calculations. Two types of chemiluminescence , Fluorescence and Phosphorescence [16][17]. ...
Article
Full-text available
DESPITE the development and progress in the detection of the significance of crimes, and after more than eighty years of discovery is a luminol is the basic chemical in the detection of crimes previously and now, was addressed the theoretical aspects of the compound of the luminol preparation methods and some of its chemical reactions as well as discuss the phenomenon of scintillation and transfer Energy, and then addressing some of the applications of chemical flash and receptor tests, DNA investigations were discussed, and the sensitivity of these reactions.
... In short, along with the synthesis of many lactic derivatives that reduced the synthesis of chemical luminol and did not change the maximum emission wavelength, then measured the basic physical properties of luminol and diphthalateamipin, and found that the results shown through the search is identical with theoretical calculations. Two types of chemiluminescence, Fluorescence and Phosphorescence [16,17]. ...
Article
Full-text available
DESPITE the development and progress in the detection of the significance of crimes, and after more than eighty years of discovery is a luminol is the basic chemical in the detection of crimes previously and now, was addressed the theoretical aspects of the compound of the luminol preparation methods and some of its chemical reactions as well as discuss the phenomenon of scintillation and transfer Energy, and then addressing some of the applications of chemical flash and receptor tests, DNA investigations were discussed, and the sensitivity of these reactions.
... The CL property of luminol has been used as an analytical tool for the detection of the inorganic substrate and free radicals, monitoring metabolic activities, immune assay as well as forensic identification [8,9]. The chemiluminescence based luminol test remains the most famous test in the forensic science field [5,11]. ...
Article
The presence of electron donating substituents altered the electronic properties of the luminol and this phenomenon was evaluated in relation to the chemiluminescence potential of the studied disubstituted luminol derivatives using three different theoretical calculation methods: Restricted Hartree Fork (RHF), Density Functional Theory (DFT) with hybrid function of Becke three-parameters Lee, Yang and Parr (B3LYP), i.e. (DFT-B3LYP), and Møller-Plesset Perturbation Theory (MP2). Different behavior was observed for the studied molecular structures in different phases (gas, water, and dimethylformamide) to emphasize the influence of molecular environment on the chemiluminescence property, regardless of the theoretical methods employed. Time-dependent DFT with correction terms enabled accurate prediction of frontier orbital energies such that calculated energy gaps could reasonably correlate with the lowest excitation energies. Besides, the electron donating moiety induced better chemiluminescence property depending on the donating power, position as well as the number of such substituents. Dimethylamino-substituted luminol showed better quantum chemical parameters and spectra pattern to have much-enhanced chemiluminescence properties possibly. Specifically, its positional isomer named 6,8-bis(dimethylamino)-5-amino-2,3-dihydro-1,4-phthalazinedione showed a greater potential of being a better chemiluminescence reagent in various analytical assays.
Article
The accurate and fast diagnosis of infectious diseases play a critical role in the timely and tailored treatment of the patients and preventing the infections from the spread. This is especially necessary in areas with low- and middle-incoming of the world. According to the smartphone-based sensory applications such as the inimitable ability to obtain portable, user-friendly, accurate, and effective functions, that in turn, converts lab-on-hardware an interesting area of new investigations. In the present review, original research articles published in English were collected from various databases during January 2011-July 2021. Also, the reference lists of the articles have been searched. In between 121 electronically searched citations, 35 articles had the inclusion criteria. The maximum potential and extended utilization of smartphone-based biosensors in the diagnosis of human infectious agents belonged to colorimetric smartphone biosensors. Also, because of their unique futures in easy visualization, comfortable analyzing and processing by smartphone, they are easier to use for point-of-care products. Based on the results, smartphone-based biosensors are rapid, precise, and low-cost diagnostic methods, and increasing their quality in limited-resource settings is of major importance. In addition, their abilities to connect to the “cloud” data storage systems and the Internet of Things (IoT) networks for collecting and sharing medical analytical data provide a trending novel research field in medical information technology.
Article
Bloodstains can provide important information about a criminal act. These biological traces, when analyzed at murder sites, for example, can determine the dynamics of a criminal offense, the identity of a suspect, and the time at which a crime was committed. Determine the time since deposition (TSD) of these blood traces may be the first clue for the police investigators to estimate the time-lapse of a murder. During a criminal attack, the blood spilled from an injury begins the process of degradation and aging from the moment it leaves the human body and comes into contact with the physical environment. The biophysical properties (morphology and elasticity) of red blood cells (RBCs) undergo several changes when outside the human body, which can be analyzed using microscopic techniques such as atomic force microscopy (AFM). Aiming to apply the AFM/force spectroscopy techniques in the analysis of criminal traces, the present study investigated the TSD for blood smears by analyzing possible changes in the RBCs of a group of voluntary donors. Also, we investigated whether there was any difference in TSD analysis after blood smears deposition onto three different surfaces (glass, metal, or ceramic); and finally, we evaluated force × distance curves obtained from deformation of the membrane surface of RBCs as a function of time. The qualitative results apparently showed that there is no perceptible difference in the structure of RBCs when AFM images were analyzed by simple visual comparison over 28 days (T0–T5). Nevertheless, our quantitative results, measured by AFM, demonstrated the increasing trend of the measurements, such as average height (μm), perimeter (μm), area (μm²) and volume (μm³) of these cells during that period. Additionally, the type of surface of bloodstain deposition should be considered during analyses for the TSD, and the results obtained on glass, metal, or ceramic supports showed significant differences. Therefore, the use of force spectroscopy to obtain force × distance curves for the forensic science approach has been shown to have applicability for the calculation of TSD in the RBCs present in the blood smears. In spite of the promising observations obtained, the use of AFM in crime scenes still requires the expansion and development of more studies for a definitive evaluation of the TSD for blood spots.
Article
Full-text available
A aplicação do luminol se destaca como um dos testes presuntivos para sangue mais importantes, sendo amplamente utilizado por investigadores forenses em locais de crime. Ao ser pulverizado sobre uma mancha de sangue, o luminol reage emitindo luz quimiluminescente, permitindo a avaliação ocasional dessas manchas, e possibilitando a reconstrução dos eventos de um crime. Porém, existem diversas substâncias que são capazes de afetar ou alterar a quimiluminescência do luminol induzida pelo sangue, entre essas substâncias estão alimentos e bebidas antioxidantes. Dentre os alimentos funcionais, o chá é uma bebida amplamente utilizada, perdendo apenas para a água como a bebida mais consumida no mundo. As propriedades antioxidantes das catequinas presentes nos chás verde e preto estão relacionadas com sua estrutura química. Produtos alimentares que contém antioxidantes (como o chá verde e o chá preto) podem ser utilizados em atividades criminosas para ocultar manchas de sangue e evitar resultados em exames de cenas de crime, uma vez que atuam inibindo a reação quimiluminescente do luminol, gerando resultados falso-negativos.
Article
The aim of this paper is to present the approach to the problem of collecting the forensic evidence within the CBRN contaminated crime scene with the use of teleoperated mobile robot. The idea of the whole system is presented first. While presenting a holistic approach to the problem is not feasible within a single paper, the article focuses on two selected solutions elaborated in R&D project entitled “Design of mobile platform for supporting forensic surveys in places where CBRN threats may occur”. The first solution is intended to gather chemical samples with the use of the SPME (Solid Phase Microextraction) adsorber. The second solution is designed to reveal the forensic evidence with the use of luminol solution deployed by the mobile robot. Conclusions, major issues addressed by described system and development trends are presented at the end of the paper.
Article
Full-text available
The generation of "cold light", visible to the human eye, by chemical reactions has attracted the attention of the scientific community since the beginning of this century. Besides the academic interest in the elucidation of the mechanisms of excited state formations, many chemiluminescence reactions have found widespread analytical applications. Moreover, the phenomenon of chemiluminescence can also be used as a tool in undergraduate and college teaching. In this article, we describe several known chemiluminescence demonstrations, which are suitable for school teaching. The main objective of this work is to produce didactic material in Portuguese to stimulate Brazilian secondary and high school teachers to use these experiments in the classroom. The demonstrations include singlet oxygen emission, the luminol reaction, oscillating chemiluminescence and the peroxyoxalate reaction.
Article
Full-text available
Luminol, leuchomalachite green, phenolphthalein, Hemastix, Hemident, and Bluestar are all used as presumptive tests for blood. In this study, the tests were subjected to dilute blood (from 1:10,000 to 1:10,000,000), many common household substance, and chemicals. Samples were tested for DNA to determine whether the presumptive tests damaged or destroyed DNA. The DNA loci tested were D2S1338 and D19S433. Leuchomalachite green had a sensitivity of 1:10,000, while the remaining tests were able to detect blood to a dilution of 1:100,000. Substances tested include saliva, semen, potato, tomato, tomato sauce, tomato sauce with meat, red onion, red kidney bean, horseradish, 0.1 M ascorbic acid, 5% bleach, 10% cupric sulfate, 10% ferric sulfate, and 10% nickel chloride. Of all the substances tested, not one of the household items reacted with every test; however, the chemicals did. DNA was recovered and amplified from luminol, phenolphthalein, Hemastix, and Bluestar, but not from leuchomalachite green or Hemident.
Article
Full-text available
Transmission of hepatitis C virus occurs frequently in haemodialysis units. A possible route of transmission is indirectly via the hospital environment although this has never been recorded. We investigated the haemodialysis unit in Deventer Hospital, Deventer, The Netherlands, with the forensic Luminol test. With this test, invisible traces of blood can be visualised based on the principle of biochemiluminescence. We demonstrated extensive contamination of the environment with traces of blood. The aim of this article is to introduce this method to infection control professionals, so it can be used to monitor cleaning and disinfection procedures, and alert healthcare workers to the possibility of contamination of the hospital environment with blood.
Article
The forensic luminol test has long been valued for its ability to detect trace amounts of blood that are invisible to the naked eye. This is the first quantitative study to determine the effect on the luminol test when an attempt is made to clean bloodstained tiles with a known interfering catalyst (bleach). Tiles covered with either wet or dry blood were tested, and either water or sodium hypochlorite solution (bleach) was used to clean the tiles. As expected, the chemiluminescence intensity produced when luminol was applied generally decreased with the number of times that a tile was cleaned with water, until the chemiluminescence was neither visible nor detectable. However, when the tiles were cleaned with bleach there was an initial drop in chemiluminescence intensity, followed by a rise to a consistently high value, visibly indistinguishable from that of blood. Examination of bleach drying time suggested that any interfering effect becomes negligible after 8 h.
Article
A new occult blood revealing agent was designed such that DNA material would be preserved and a DNA profile could be obtained. Indeed, there were uncertainties about previous chemicals that might alter blood DNA content and have adverse effects on DNA typing. BlueStar® is a chemiluminescent bloodstain detecting agent resulting from our research. It combines ease-of-use with minimum health risks and does not have any harmful effects on DNA profile determination.
Article
With the goal of obtaining additional practically applicable methods for estimating the PMI of skeletal remains, 39 samples of human and 5 samples of domestic animal long bones with known PMI (PMI=1 to approximately 2000 years) were tested with two established methods (UV-fluorescence of a freshly sawn cross-section and the luminol test) and two screening tests (Hexagon-OBTI® test and Combur® test) that were being tried out in this context for the first time. The hypothesis underlying this experiment was the supposition that the PMI-related chemiluminescence of the luminol reaction for bone is based on the presence of persisting hemin from hemoglobin molecules in bone. Our results showed that lack of luminescence and reduced UV-fluorescence were more meaningful results for estimating PMI and excluding forensic relevance than a positive luminol reaction or strong UV-fluorescence, as both of the latter findings revealed the limitations of these methods in this particular context. Particularly for cases showing a positive luminol reaction, the use of additional absolute dating methods may be indicated. Against our expectations, both the Combur® test strips and the Hexagon-OBTI® test, which were both devised to demonstrate blood, delivered negative results for all samples. They are thus not suitable for estimating the PMI of skeletal remains. Future research will be necessary to elucidate whether the negative results obtained for these tests may be due to the poor solubility of potentially present hemoglobin or hemoglobin breakdown products in the Tris buffer used in this experiment.
Article
The luminol chemiluminescence reaction has, for some time, been used as a tool for the detection of haemoglobin at crime scenes. More recently, the luminol test has been suggested as a possible tool for estimating the post-mortem interval (PMI) of skeletal remains. The preliminary results from the following study indicate that the chemiluminescent luminol test is a relatively easy and economical method for distinguishing between remains of medico-legal (< or =100 years) and historical (>100 years) interest. The femur was the preferred bone for PMI measurements using the luminol test, due to its robustness and relative resistance to diagenesis. Initial results suggest that bone that was historical in nature, produced a demonstrably weaker reaction than that of medico-legal interest. These results suggest that the luminol test is a promising technique, albeit with some limitations, for the assessment of skeletal material that may be potentially of medico-legal interest.
Article
As criminals try to avoid leaving clues at the scene of a crime, bloodstains are often washed away, but fortunately for investigators, they are difficult to eliminate completely. Porous surfaces easily retain blood traces, which are sometimes invisible to the naked eye. The reagent of choice for detecting latent blood traces on all types of surfaces is luminol, but its main disadvantage is a high degree of sensitivity to oxidising contaminants in the blood sample. If household bleach is used to clean bloodstains, presumptive tests are invalidated. Hypochlorites, however, are known to be unstable and deteriorate over time, and this feature could be of help in preventing household bleach-induced interference. Previous studies have evaluated the effect of the drying time on nonporous surfaces, but nothing has as yet been published about this effect on porous surfaces. Consequently, this paper reports on hypochlorite interference with luminol reagents used on this type of surface, evaluating the effects of drying time on the household bleach-luminol reaction, and ascertaining whether the drying procedure could be applied to prevent household bleach interference on bloodstained porous surfaces. The results indicate that the drying method may very well overcome household bleach interference in luminol reaction tests, if the investigation allows for an appropriate waiting time.
Article
The forensic application of the luminol chemiluminescence reaction is reviewed. Luminol has been effectively employed for more than 40 years for the presumptive detection of bloodstains which are hidden from the naked eye at crime scenes and, for this reason, has been considered one of the most important and well-known assays in the field of forensic sciences. This review provides an historical overview of the forensic use of luminol, and the current understanding of the reaction mechanism with particular reference to the catalysis by blood. Operational use of the luminol reaction, including issues with interferences and the effect of the luminol reaction on subsequent serological and DNA testing is also discussed.