Forensic Science International 244 (2014) e48–e55
Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Rapid Communication
Synthesis and application of an aqueous nile red microemulsion for the development of fingermarks on porous surfaces Mackenzie de la Hunty a, Xanthe Spindler a, Scott Chadwick a, Chris Lennard b, Claude Roux a,* a b
Centre for Forensic Science, University of Technology, Sydney, PO Box 123, Broadway, NSW 2007, Australia School of Science and Health, University of Western Sydney, Richmond, NSW 2753, Australia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 May 2014 Received in revised form 15 August 2014 Accepted 20 August 2014 Available online 4 September 2014
An oil-in-water microemulsion containing a luminescent dye, nile red, has been synthesised using a solvent-diffusion method. This has been demonstrated to be effective in developing fresh latent fingermarks on porous surfaces. The working solution is made using a binary surfactant solution to create a lactescent dual organic–aqueous phase intermediate, which subsequently results in a transparent microemulsion after the organic solvent has evaporated. The solution is non-toxic and performs comparatively with a previously published methanolic formulation but at a much lower cost and with an extended shelf life. The microemulsion outperforms a previously reported aqueous nile blue formulation for the development of both charged and natural fresh fingermarks, and requires lower exposure times for image recording. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Nile red Nile blue Latent fingermarks Physical developer Luminescent lipid dyes
1. Introduction Latent fingermarks occur as a result of the residues that are deposited onto a surface when the friction skin of the hand comes into contact with that surface. There are three major glands in the human body that are responsible for the secretion of bodily fluids through the skin medium: the eccrine glands, the sebaceous glands and the apocrine glands [1]. The secretions from the eccrine and sebaceous glands are most commonly found in fingermark residue; eccrine constituents are endogenous to friction ridge skin, whilst sebaceous constituents are present through contact of friction ridge skin with the face and other regions of the body that have hair follicles. Fingermark residue also contains lipids that originate in the epidermis and are contained within a hydrolipid film (also known as the acid mantle) that is present on the surface of the skin [2–4]. The detection of lipids in latent fingermark investigations is crucial for items that have been wet, as the apocrine and eccrine components are water soluble; however, the water insoluble lipid component of sebum from the sebaceous glands and from the hydrolipid film on the surface of the epidermis, will remain after water exposure and can be targeted for development. The method
* Corresponding author. Tel.: +61 295141718; fax: +61 295141460. E-mail address: [email protected] (C. Roux). http://dx.doi.org/10.1016/j.forsciint.2014.08.028 0379-0738/ß 2014 Elsevier Ireland Ltd. All rights reserved.
of choice to target these lipids is physical developer (PD) [5]. PD works by selectively reducing silver ions to metallic silver on reactive nucleating sites of the water insoluble fraction of fingermark residue [6]. PD is the only technique in routine use for the development of fingermarks on porous surfaces that have been wet. It can also be sequentially used after treatment with amino-acid sensitive techniques if the sample has not been wet, in which case it can develop additional detail or additional fingermarks. Much research has been undertaken to optimise the PD method as the working solution has a short shelf life due to its inherent instability, although this is difficult when the exact mechanism by which the technique operates is still largely unknown; the technique is also time consuming and laborious to apply. PD can interfere with the forensic examination of handwriting, ink, paper, indented impressions and body fluids [7], so planned sequential analyses are important. Due to the inherent difficulties encountered when using PD, the implementation of alternative or complementary techniques is of significant interest. Oil Red O (ORO) is a lysochrome commonly used for biological purposes to colour lipoproteins that have been separated by electrophoresis on cellulose acetate [8]. In recent years it has been shown to be a viable reagent for the development of latent fingermarks on porous surfaces that have been wet, resulting in red ridges on a pink background with no further visualisation techniques needed [4,8–11]. While these studies have collectively shown that ORO presents some practical advantages over PD, and
M. de la Hunty et al. / Forensic Science International 244 (2014) e48–e55
that it can be used in sequence prior to PD treatment, it is less effective on marks older than 4 weeks. Nile red has been used to develop fingermarks on porous surfaces that have been wet, with promising results [12]. Nile red is a luminescent benzophenoxazine dye that has been used as a postcyanoacrylate (CA) stain for the luminescence enhancement of CA developed marks on non-porous surfaces [13,14]. More recently, in a pseudo-operational study on five year old exam booklets, nile red in a methanolic-aqueous working solution has shown great promise as a lipid sensitive fingermark development reagent that develops more marks when used in sequence with PD than can be developed by PD alone [12,15], demonstrating the importance of sequencing nile red with PD. The nile red solution did not appear to enhance previously developed PD marks, but targeted undeveloped fingermarks, indicating that the techniques are complementary. This is possibly due to the two techniques having discrete lipid targets that vary across aged fingermarks from different donors. Despite the demonstrated abilities of the published nile red formulation, improvements were needed to avoid the deposition of nile red particles onto the substrate, which is caused by the hydrophobic nature of nile red in the methanolic-aqueous working solution resulting in precipitation during the treatment of exhibits. This phenomenon also resulted in a very short shelf-life for the working solution. Research has been undertaken to synthesise nile red derivatives with increased water solubility; however, organic solvents are still needed for the complete solubilisation of these compounds in the working solutions [15]. The removal of potentially hazardous organic solvents from the working solution is also of interest from an occupational health and safety perspective. Recently, a new formulation of the nile red working solution has been developed by our research group. This formulation results in a working solution that solubilises the nile red by incorporating the dye into an aqueous microemulsion. Microemulsions can be formed by mixing a primary surfactant with water and an oil (in this case, a hydrophobic compound dissolved in an organic solvent) to create a lactescent emulsion that is then titrated with a secondary surfactant until the mixture becomes clear. The volatile organic solvent is then evaporated from the solution. For this study, the method has been adapted by initially adding the oil (nile red in dichloromethane) to stirring water, and then adding an aqueous solution containing both primary and secondary surfactants. The formulation has been created with a view to ease of operational implementation as the surfactant solution employed is the same as the surfactant solution used in the PD working solution (Tween 20 formulation [16]), with Tween 20 and n-dodecylamine acetate acting as the primary and secondary surfactants, respectively. It is known that nile red targets the lipids found in sebaceous gland secretions, and it has been demonstrated to develop five year old uncharged marks that were previously undeveloped by PD (with PD possibly targeting the epidermal originating lipids) [12,15]. To be effective operationally, the nile red microemulsion needs to be sensitive enough to develop sebaceous lipids that have not been placed there in high concentrations immediately prior to development (which is the case when marks are ‘‘charged’’), but persist in lower concentrations from normal hand to face contact (referred to as ‘‘natural’’ marks). The recently published guidelines by The International Fingerprint Research Group (IFRG) [17] for the evaluation of fingermark development techniques has provided a structural framework for the effective and comprehensive development of fingermark enhancement techniques. It stipulates directives for undertaking an unbiased initial assessment of novel development techniques. The IFRG guidelines [17] encourage the use of both natural and
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charged fingermarks so that the significance of preliminary results is not overstated. It has therefore been deemed important to use a combination of both appropriately charged marks (which provide a positive control) and natural marks (which are more realistic) when assessing and optimising the microemulsion or when comparing it with other development solutions. Frick and co-workers [18] recently published an aqueous nile blue formulation that is effective as a fingermark development method, with this being attributed to the presence of nile red in the solution. The nile red is present as an impurity in nile blue, and may also be formed as a hydrolysis product of nile blue [19]. The formulation was shown to develop fresh, charged fingermarks on paper, and is claimed to outperform the previously published methanolic nile red solution. However, no results were mentioned for the development of natural marks. In this research, a nile red microemulsion has been compared with (1) the methanolic nile red working solution [12] and (2) the nile blue solution [18] to evaluate and compare the effectiveness of each solution on both charged and natural fingermarks. This research is ongoing to satisfy the Phase 1 and Phase 2 directives specified by the IFRG guidelines. At this stage, one fingermark donor, known to produce good natural and charged fingermarks, and one substrate were chosen for the preliminary comparison of the three fingermark development solutions. All experiments were repeated three times and the fingermark collection parameters were meticulously controlled.
2. Materials and method All solvents (AR grade), were obtained from BDH-Prolabo Chemicals (VWR International Pty. Ltd., Australia). Nile red (BioReagent, Sigma–Aldrich, USA), sodium hydroxide (Merck Chemicals, Australia), nile blue (Revector Microscopical Stain, Hopkin & Williams Ltd.), polyoxyethylenesorbitan monolaurate (Tween 20) (Sigma–Aldrich, Australia) and n-dodecylamine acetate (MP Biochemicals Inc., Germany) were used as supplied. 2.1. Surfactant solution (Solution A) 1.5 mL Tween 20 and 1.5 g n-dodecylamine acetate were added to 500 mL deionised water with stirring until dissolved. 2.2. Nile red stock solution (Solution B) 10 mg nile red was added to 1000 mL dichloromethane with stirring until dissolved. 2.3. Nile red microemulsion (Solution 1) 100 mL deionised water was placed in a beaker containing a magnetic stirrer bar. 12 mL of Solution A was added, followed by the slow addition of 8 mL Solution B. The solution was vigorously stirred until the dichloromethane had evaporated; this process was observed by the transformation of a two-phase solution (clear aqueous and bright pink organic phase), to a clear, singlephase, light purple microemulsion. The solution was then stirred for a further 10 min to ensure complete dichloromethane evaporation. 2.4. Nile red methanolic working solution (Solution 2) [12] 170 mL 0.1 mg/mL sodium hydroxide solution was slowly added to 230 mL 0.1 mg/mL nile red in methanol with constant stirring and was used immediately.
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2.5. Nile blue working solution (Solution 3) [18] 5 mg nile blue was added to 100 mL deionised water with constant stirring. 2.6. Fingermark deposition conditions Natural fingermarks were collected from one 24-year-old female donor, known to produce good natural and charged marks, ensuring that the hands had not been washed with soap within the previous 4 h. The donor was requested to rub her hands together, to ensure a uniform coating of secretions across the skin surface, just prior to fingermark collection. A delay of at least 4 h was required in between the collection of sample sets from the same donor. Charged fingermarks were obtained by wiping both hands across the top of the back and then lightly rubbing the hands together to ensure even coverage of sebaceous material. Touching the top of the back, rather than the face, was deemed more appropriate because it avoids possible contamination with cosmetics. Both natural and charged marks were deposited on paper sheets from the same ream of Reflex Ultra White Copy Paper (80GSM A4) using 350 g of force (checked with the use of a laboratory balance) for 8 s using the three middle fingers. Samples were left to cure for 24 h prior to treatment and subsequently visualised within 2 h of development. Each three-finger impression was vertically bisected for comparison purposes immediately prior to treatment. Samples, before and after treatment, were stored in a temperature controlled laboratory setting in a dark cupboard. 2.7. Sample treatment Fingermark samples were treated for 5 min in either Solution 1 or Solution 2, or for 20 min in Solution 3. Samples were removed, briefly immersed in deionised water (1–2 s) and placed on clean paper towel to air dry. 2.8. Sample visualisation For all treatments, samples were visualised using the Rofin
(Fig._1)TD$IG][Poliview system, with a Polilight PL-500 light source operating at
490 nm and with a 555 nm band-pass barrier filter on the camera, which was deemed to be the optimal settings for all treatments. Bisected samples were placed side by side and imaged together. Two images were taken for each fingermark sample using the same angle of incidence and intensity of lighting, sample positioning and camera aperture; only the exposure was altered to obtain the optimal image for each half-impression. Optimal exposure was determined based on an assessment of the best contrast between the luminescent ridges and the dark background. The fingermark development was assessed based on ridge detail, clarity, contrast and the amount of total developed ridges. 3. Results Solution 1 was compared with Solution 2 on both charged (Figs. 1 and 2) and natural fingermarks (Figs. 3 and 4). Preliminary results show that Solution 1 is effective in developing both natural and charged fingermarks. Fig. 1 demonstrates how complete solubilisation of nile red in a microemulsion discourages the precipitation of nile red onto the substrate, and encourages the concentration of nile red into the lipid material present in the fingermark deposit when high lipid concentrations are present. Solution 2 produces ridges that have a greater luminescence intensity and clarity compared to Solution 1. Solubilising the nile red results in decreased contrast, but increased total ridge development and ridge width uniformity, due to the absence of solid nile red. Figs. 3 and 4 show both solutions to be effective on natural fingermarks, with both solutions providing light development of the ridges. Overall, both solutions show comparable development on both charged and natural fingermarks at similar optimal exposure times. Solution 1 was compared with Solution 3 on charged (Figs. 5 and 6) and natural fingermarks (Figs. 7 and 8). Figs. 5 and 6 show that Solution 1 stains more effectively than Solution 3, and does not result in the solid deposition of nile red in heavily sebaceous laden areas of the latent residue particularly present in the furrows, which can result in an inability to visualise certain sections of the developed fingermark as ridge detail is lost. The developed fingermark halves required markedly different exposure times, with Solution 3 needing much longer exposure (>4 times, keeping the other parameters constant) due to decreased staining intensity.
Fig. 1. Charged fingermarks developed by Solution 1 (left) and Solution 2 (right) at 382 ms exposure (optimal exposure for Solution 1).
[(Fig._2)TD$IG]
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Fig. 2. Charged fingermarks developed by Solution 1 (left) and Solution 2 (right) at 163 ms exposure (optimal exposure for Solution 2).
Figs. 7 and 8 show that Solution 3 is less effective than Solution 1 for natural marks, as it stains fingermark ridges less intensely and shows decreased contrast. As with the charged marks, the natural marks developed by Solution 3 required extended exposure times, despite being treated four times longer in the working solution compared to Solution 1. This is most likely attributed to the low concentration of nile red in Solution 3. It should be pointed out that, despite these variations in exposure times, using constant parameters for f-stop and camera settings (Nikon 60 mm micro lens, F2.8.), the exposure times required to obtain optimum results remain in a practical range from an operational viewpoint (i.e. 100 ms to 1.25 s) for Solutions 1 and 2. The problem with weak development, however, is that marks may escape detection if only a simple visual inspection is conducted.
[(Fig._3)TD$IG]
Fig. 9 demonstrates that Solution 1 gives less effective and less intense development one month after solution preparation. The older solution (Fig. 9 – right) shows a darker background with less intense staining of the ridges than is seen with freshly made Solution 1 (Fig. 9 – left). 4. Discussion The comparison of Solution 1 and Solution 2 supports the hypothesis that nile red targets lipids originating in the sebaceous glands and not epidermal originating lipids, due to the weak development seen with the natural marks. However, some degree of development, albeit small, can still be obtained with natural marks and this is most likely attributed to a combination of lipids in the sebaceous glands (which are residually present through
Fig. 3. Natural fingermarks developed by Solution 1 (left) and Solution 2 (right) at 305 ms exposure (optimal exposure for Solution 1).
[(Fig._4)TD$IG]
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M. de la Hunty et al. / Forensic Science International 244 (2014) e48–e55
Fig. 4. Natural fingermarks developed by Solution 1 (left) and Solution 2 (right) at 382 ms exposure (optimal exposure for Solution 2).
[(Fig._5)TD$IG]
normal hand to face contact) and lipids contained in the hydrolipid film (which are endogenous to friction ridge skin). Better ridge continuity is seen in the development on charged marks using Solution 1 when compared to Solution 2, as development has occurred through solubilised nile red diffusion into the lipid material, as opposed to a combination of diffusion and solid nile red precipitation onto the surface of the deposit. Solution 1 results in some dispersion of the stain, making the developed ridges appear slightly blurred compared to the half developed by Solution 2; however, adequate ridge detail is still obtained. Considering that Solution 1 can be made for AU$6.40/L and Solution 2 can be made for just under AU$50.0/L, the microemulsion formulation is significantly more cost effective with little sacrifice to developmental quality.
Solution 1 outperformed Solution 3 on both charged and natural marks, requiring lower exposure times for image capture. Exposure time lengths are a necessary consideration in fingermark visualisation because long exposure times are not operationally viable, and weakly luminescent marks requiring extended exposure times may escape detection when items are screened after treatment. The extended exposure times for Solution 3 are most likely due to the low concentrations of nile red in the nile blue solution; however, this is difficult to confirm as solid nile red has deposited on the substrate in the charged mark experiments. As seen with Solution 2, Solution 3 exhibited precipitated solid nile red on charged marks whereas Solution 1 demonstrates an ability to develop heavily sebaceous laden areas without solid nile red deposition. Solution 3 showed decreased staining intensity and
Fig. 5. Charged fingermarks developed by Solution 1 (left) and Solution 3 (right) at 126 ms exposure (optimal exposure for Solution 1).
[(Fig._6)TD$IG]
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Fig. 6. Charged fingermarks developed by Solution 1 (left) and Solution 3 (right) at 637 ms exposure (optimal exposure for Solution 3).
reduced sensitivity on natural marks when compared to Solution 1, possibly due to the lower concentration of lipids present resulting in incomplete development. Solution 1, when stored for 1 month after preparation, still developed fingermarks but these were significantly reduced in luminescence intensity compared to marks developed with freshly preparation solution. As such, the working solution should not be stored for more than 2 weeks. This is an improvement on the methanolic nile red working solution (Solution 2) as this can only be used on the day it is prepared. In order to obtain stronger luminescence of developed ridges when using Solution 1, various concentrations of nile red in the dichloromethane stock solution were trialled, however concentrations greater than 0.01 mg/mL resulted in precipitation of nile red onto the substrate. Further
work is needed to determine the optimal ratio and concentration of stock and surfactant solutions to obtain a microemulsion that contains more solubilised nile red. The aim of this research is not to replace PD, nor to assume this may be a possibility, as nile red and PD target different fingermark constituents and need to be used in sequence. The aim is to provide a complementary method to PD, that is less hazardous than the current methanolic nile red working solution, cheaper to make and has an extended shelf life. Despite the extended shelf life, lower cost and less toxic nature of the nile red microemulsion solution, at this stage it is still recommended that the existing methanolic formulation be used in sequence after PD until further optimisation of the new method is completed, especially on marks that are older than one week. This research is ongoing in our laboratory.
[(Fig._7)TD$IG]
Fig. 7. Natural fingermarks developed by Solution 3 (left) and Solution 1 (right) at 1.87 s exposure (optimal exposure for Solution 3).
[(Fig._8)TD$IG]
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Fig. 8. Natural fingermarks developed by Solution 3 (left) and Solution 1 (right) at 428 ms exposure (optimal exposure for Solution 1).
[(Fig._9)TD$IG]
preliminary work, the microemulsion formulation was shown to outperform an aqueous nile blue working solution on both fresh charged and natural fingermarks, requiring shorter development times, shorter exposure times for image capture (due to increased staining intensity), and increased overall sensitivity. Further investigation is needed to determine the optimal parameters and formulation for the sequencing of the nile red microemulsion after the use of physical developer and for use on aged marks (older than one month). References
Fig. 9. Fingermark developed using a freshly prepared Solution 1 (left) and one month old Solution 1 (right).
5. Conclusions Nile red has been incorporated into an aqueous microemulsion using a simplified method that is effective in developing both natural and charged fresh fingermarks on porous surfaces that have been wet. The microemulsion produces slightly more diffuse marks than the published methanol-based working solution, but is less toxic, cheaper to make, and has an extended shelf life. In this
[1] C. Champod, C. Lennard, P. Margot, M. Stoilovic, Fingerprint detection techniques, Fingerprints and Other Ridge Skin Impressions, 4, CRC Press, Boca Raton, FL, USA, 2004. [2] S. Shetage, M. Traynor, M. Brown, M. Raji, D. Graham-Kalio, R. Chilcott, Effect of ethnicity, gender and age on the amount and composition of residual skin surface components derived from sebum, sweat and epidermal lipids, Skin Res. Technol. 20 (2014) 97–107. [3] P. Clarys, A. Barel, Quantitative evaluation of skin surface lipids, Clin. Dermatol. 13 (1995) 307–321. [4] R. Ramotowski, Lipid reagents, in: Lee and Gaensslen’s Advances in Fingerprint Technology, third ed., CRC Press, Boca Raton, FL, USA, 2012, pp. 83–96. [5] C. Lennard, Forensic sciencesjfingerprint techniques, in: P. Worsfold, A. Townshend, C. Poole (Eds.), Encyclopedia of Analytical Science, second ed., Elsevier, Amsterdam, The Netherlands, 2005, pp. 414–423. [6] A. Be´cue, A. Cantu´, Fingermark detection using nanoparticles, in: Lee and Gaensslen’s Advances in Fingerprint Technology, third ed., CRC Press, Boca Raton, FL, USA, 2012, pp. 307–380. [7] HOSDB, Manual of Fingerprint Development Techniques: A Guide to the Selection and Use of Processes for the Development of Latent Fingerprints, second ed., Home Office Police Scientific Development Branch, 2004. [8] A. Beaudoin, New technique for revealing latent fingerprints on wet, porous surfaces: oil red O, J. Forensic Ident. 54 (2004) 413–421. [9] A. Rawji, A. Beaudoin, Oil red O versus physical developer on wet papers: a comparative study, J. Forensic Ident. 56 (2006) 33–54. [10] M. Wood, T.O.R.O. James, The Physical Developer replacement, Sci. Justi. 49 (2009) 272–276. [11] J. Salama, S. Aumeer-Donovan, C. Lennard, C. Roux, Evaluation of the fingermark reagent oil red O as a possible replacement for physical developer, J. Forensic Identi. 58 (2008) 203–237. [12] K. Braasch, M. de la Hunty, J. Deppe, X. Spindler, A. Cantu, P. Maynard, C. Lennard, C. Roux, Nile red: alternative to physical developer for the detection of latent fingermarks on wet porous surfaces, Forensic Sci. Int. 230 (2013) 74–80. [13] B. Chesher, J. Stone, W. Rowe, Use of the OmniprintTM 1000 alternate light source to produce fluorescence in cyanoacrylate-developed latent fingerprints stained with biological stains and commercial fabric dyes, Forensic Sci. Int. 57 (1992) 163–168. [14] J. Kempton, W. Rowe, Contrast enhancement of cyanoacrylate-developed latent fingerprints using biological stains and commercial fabric dyes, J. Forensic Sci. 37 (1992) 99–105.
M. de la Hunty et al. / Forensic Science International 244 (2014) e48–e55 [15] M. de la Hunty, An investigation of the techniques for the development of latent fingermarks on porous surfaces that have been wet: Nile red in sequence with physical developer, and the synthesis and novel application of nile red derivatives [Honours], UTS, Australia, 2012. [16] S. Wright, Replacement of synperonic-n within physical developer [Student Placement Report], Home Office Police Scientific Development Branch, 2006.
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[17] IFRG, International Fingerprint Research Group (IFRG) Guidelines for the Assessment of Fingermark Detection Techniques, J. Forensic Identi. 2 (2014) 175–201. [18] A. Frick, F. Busetti, A. Cross, S. Lewis, Aqueous Nile blue: a simple, versatile and safe reagent for the detection of latent fingermarks, Chem. Commun. 50 (2014) 3341–3343. [19] A. Ostle, J. Holt, Nile blue A as a fluorescent stain for poly-beta-hydroxybutyrate, J. Appl. Environ. Microbiol. 44 (1982) 238–241.
Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Rapid Communication
Synthesis and application of an aqueous nile red microemulsion for the development of fingermarks on porous surfaces Mackenzie de la Hunty a, Xanthe Spindler a, Scott Chadwick a, Chris Lennard b, Claude Roux a,* a b
Centre for Forensic Science, University of Technology, Sydney, PO Box 123, Broadway, NSW 2007, Australia School of Science and Health, University of Western Sydney, Richmond, NSW 2753, Australia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 May 2014 Received in revised form 15 August 2014 Accepted 20 August 2014 Available online 4 September 2014
An oil-in-water microemulsion containing a luminescent dye, nile red, has been synthesised using a solvent-diffusion method. This has been demonstrated to be effective in developing fresh latent fingermarks on porous surfaces. The working solution is made using a binary surfactant solution to create a lactescent dual organic–aqueous phase intermediate, which subsequently results in a transparent microemulsion after the organic solvent has evaporated. The solution is non-toxic and performs comparatively with a previously published methanolic formulation but at a much lower cost and with an extended shelf life. The microemulsion outperforms a previously reported aqueous nile blue formulation for the development of both charged and natural fresh fingermarks, and requires lower exposure times for image recording. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Nile red Nile blue Latent fingermarks Physical developer Luminescent lipid dyes
1. Introduction Latent fingermarks occur as a result of the residues that are deposited onto a surface when the friction skin of the hand comes into contact with that surface. There are three major glands in the human body that are responsible for the secretion of bodily fluids through the skin medium: the eccrine glands, the sebaceous glands and the apocrine glands [1]. The secretions from the eccrine and sebaceous glands are most commonly found in fingermark residue; eccrine constituents are endogenous to friction ridge skin, whilst sebaceous constituents are present through contact of friction ridge skin with the face and other regions of the body that have hair follicles. Fingermark residue also contains lipids that originate in the epidermis and are contained within a hydrolipid film (also known as the acid mantle) that is present on the surface of the skin [2–4]. The detection of lipids in latent fingermark investigations is crucial for items that have been wet, as the apocrine and eccrine components are water soluble; however, the water insoluble lipid component of sebum from the sebaceous glands and from the hydrolipid film on the surface of the epidermis, will remain after water exposure and can be targeted for development. The method
* Corresponding author. Tel.: +61 295141718; fax: +61 295141460. E-mail address: [email protected] (C. Roux). http://dx.doi.org/10.1016/j.forsciint.2014.08.028 0379-0738/ß 2014 Elsevier Ireland Ltd. All rights reserved.
of choice to target these lipids is physical developer (PD) [5]. PD works by selectively reducing silver ions to metallic silver on reactive nucleating sites of the water insoluble fraction of fingermark residue [6]. PD is the only technique in routine use for the development of fingermarks on porous surfaces that have been wet. It can also be sequentially used after treatment with amino-acid sensitive techniques if the sample has not been wet, in which case it can develop additional detail or additional fingermarks. Much research has been undertaken to optimise the PD method as the working solution has a short shelf life due to its inherent instability, although this is difficult when the exact mechanism by which the technique operates is still largely unknown; the technique is also time consuming and laborious to apply. PD can interfere with the forensic examination of handwriting, ink, paper, indented impressions and body fluids [7], so planned sequential analyses are important. Due to the inherent difficulties encountered when using PD, the implementation of alternative or complementary techniques is of significant interest. Oil Red O (ORO) is a lysochrome commonly used for biological purposes to colour lipoproteins that have been separated by electrophoresis on cellulose acetate [8]. In recent years it has been shown to be a viable reagent for the development of latent fingermarks on porous surfaces that have been wet, resulting in red ridges on a pink background with no further visualisation techniques needed [4,8–11]. While these studies have collectively shown that ORO presents some practical advantages over PD, and
M. de la Hunty et al. / Forensic Science International 244 (2014) e48–e55
that it can be used in sequence prior to PD treatment, it is less effective on marks older than 4 weeks. Nile red has been used to develop fingermarks on porous surfaces that have been wet, with promising results [12]. Nile red is a luminescent benzophenoxazine dye that has been used as a postcyanoacrylate (CA) stain for the luminescence enhancement of CA developed marks on non-porous surfaces [13,14]. More recently, in a pseudo-operational study on five year old exam booklets, nile red in a methanolic-aqueous working solution has shown great promise as a lipid sensitive fingermark development reagent that develops more marks when used in sequence with PD than can be developed by PD alone [12,15], demonstrating the importance of sequencing nile red with PD. The nile red solution did not appear to enhance previously developed PD marks, but targeted undeveloped fingermarks, indicating that the techniques are complementary. This is possibly due to the two techniques having discrete lipid targets that vary across aged fingermarks from different donors. Despite the demonstrated abilities of the published nile red formulation, improvements were needed to avoid the deposition of nile red particles onto the substrate, which is caused by the hydrophobic nature of nile red in the methanolic-aqueous working solution resulting in precipitation during the treatment of exhibits. This phenomenon also resulted in a very short shelf-life for the working solution. Research has been undertaken to synthesise nile red derivatives with increased water solubility; however, organic solvents are still needed for the complete solubilisation of these compounds in the working solutions [15]. The removal of potentially hazardous organic solvents from the working solution is also of interest from an occupational health and safety perspective. Recently, a new formulation of the nile red working solution has been developed by our research group. This formulation results in a working solution that solubilises the nile red by incorporating the dye into an aqueous microemulsion. Microemulsions can be formed by mixing a primary surfactant with water and an oil (in this case, a hydrophobic compound dissolved in an organic solvent) to create a lactescent emulsion that is then titrated with a secondary surfactant until the mixture becomes clear. The volatile organic solvent is then evaporated from the solution. For this study, the method has been adapted by initially adding the oil (nile red in dichloromethane) to stirring water, and then adding an aqueous solution containing both primary and secondary surfactants. The formulation has been created with a view to ease of operational implementation as the surfactant solution employed is the same as the surfactant solution used in the PD working solution (Tween 20 formulation [16]), with Tween 20 and n-dodecylamine acetate acting as the primary and secondary surfactants, respectively. It is known that nile red targets the lipids found in sebaceous gland secretions, and it has been demonstrated to develop five year old uncharged marks that were previously undeveloped by PD (with PD possibly targeting the epidermal originating lipids) [12,15]. To be effective operationally, the nile red microemulsion needs to be sensitive enough to develop sebaceous lipids that have not been placed there in high concentrations immediately prior to development (which is the case when marks are ‘‘charged’’), but persist in lower concentrations from normal hand to face contact (referred to as ‘‘natural’’ marks). The recently published guidelines by The International Fingerprint Research Group (IFRG) [17] for the evaluation of fingermark development techniques has provided a structural framework for the effective and comprehensive development of fingermark enhancement techniques. It stipulates directives for undertaking an unbiased initial assessment of novel development techniques. The IFRG guidelines [17] encourage the use of both natural and
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charged fingermarks so that the significance of preliminary results is not overstated. It has therefore been deemed important to use a combination of both appropriately charged marks (which provide a positive control) and natural marks (which are more realistic) when assessing and optimising the microemulsion or when comparing it with other development solutions. Frick and co-workers [18] recently published an aqueous nile blue formulation that is effective as a fingermark development method, with this being attributed to the presence of nile red in the solution. The nile red is present as an impurity in nile blue, and may also be formed as a hydrolysis product of nile blue [19]. The formulation was shown to develop fresh, charged fingermarks on paper, and is claimed to outperform the previously published methanolic nile red solution. However, no results were mentioned for the development of natural marks. In this research, a nile red microemulsion has been compared with (1) the methanolic nile red working solution [12] and (2) the nile blue solution [18] to evaluate and compare the effectiveness of each solution on both charged and natural fingermarks. This research is ongoing to satisfy the Phase 1 and Phase 2 directives specified by the IFRG guidelines. At this stage, one fingermark donor, known to produce good natural and charged fingermarks, and one substrate were chosen for the preliminary comparison of the three fingermark development solutions. All experiments were repeated three times and the fingermark collection parameters were meticulously controlled.
2. Materials and method All solvents (AR grade), were obtained from BDH-Prolabo Chemicals (VWR International Pty. Ltd., Australia). Nile red (BioReagent, Sigma–Aldrich, USA), sodium hydroxide (Merck Chemicals, Australia), nile blue (Revector Microscopical Stain, Hopkin & Williams Ltd.), polyoxyethylenesorbitan monolaurate (Tween 20) (Sigma–Aldrich, Australia) and n-dodecylamine acetate (MP Biochemicals Inc., Germany) were used as supplied. 2.1. Surfactant solution (Solution A) 1.5 mL Tween 20 and 1.5 g n-dodecylamine acetate were added to 500 mL deionised water with stirring until dissolved. 2.2. Nile red stock solution (Solution B) 10 mg nile red was added to 1000 mL dichloromethane with stirring until dissolved. 2.3. Nile red microemulsion (Solution 1) 100 mL deionised water was placed in a beaker containing a magnetic stirrer bar. 12 mL of Solution A was added, followed by the slow addition of 8 mL Solution B. The solution was vigorously stirred until the dichloromethane had evaporated; this process was observed by the transformation of a two-phase solution (clear aqueous and bright pink organic phase), to a clear, singlephase, light purple microemulsion. The solution was then stirred for a further 10 min to ensure complete dichloromethane evaporation. 2.4. Nile red methanolic working solution (Solution 2) [12] 170 mL 0.1 mg/mL sodium hydroxide solution was slowly added to 230 mL 0.1 mg/mL nile red in methanol with constant stirring and was used immediately.
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2.5. Nile blue working solution (Solution 3) [18] 5 mg nile blue was added to 100 mL deionised water with constant stirring. 2.6. Fingermark deposition conditions Natural fingermarks were collected from one 24-year-old female donor, known to produce good natural and charged marks, ensuring that the hands had not been washed with soap within the previous 4 h. The donor was requested to rub her hands together, to ensure a uniform coating of secretions across the skin surface, just prior to fingermark collection. A delay of at least 4 h was required in between the collection of sample sets from the same donor. Charged fingermarks were obtained by wiping both hands across the top of the back and then lightly rubbing the hands together to ensure even coverage of sebaceous material. Touching the top of the back, rather than the face, was deemed more appropriate because it avoids possible contamination with cosmetics. Both natural and charged marks were deposited on paper sheets from the same ream of Reflex Ultra White Copy Paper (80GSM A4) using 350 g of force (checked with the use of a laboratory balance) for 8 s using the three middle fingers. Samples were left to cure for 24 h prior to treatment and subsequently visualised within 2 h of development. Each three-finger impression was vertically bisected for comparison purposes immediately prior to treatment. Samples, before and after treatment, were stored in a temperature controlled laboratory setting in a dark cupboard. 2.7. Sample treatment Fingermark samples were treated for 5 min in either Solution 1 or Solution 2, or for 20 min in Solution 3. Samples were removed, briefly immersed in deionised water (1–2 s) and placed on clean paper towel to air dry. 2.8. Sample visualisation For all treatments, samples were visualised using the Rofin
(Fig._1)TD$IG][Poliview system, with a Polilight PL-500 light source operating at
490 nm and with a 555 nm band-pass barrier filter on the camera, which was deemed to be the optimal settings for all treatments. Bisected samples were placed side by side and imaged together. Two images were taken for each fingermark sample using the same angle of incidence and intensity of lighting, sample positioning and camera aperture; only the exposure was altered to obtain the optimal image for each half-impression. Optimal exposure was determined based on an assessment of the best contrast between the luminescent ridges and the dark background. The fingermark development was assessed based on ridge detail, clarity, contrast and the amount of total developed ridges. 3. Results Solution 1 was compared with Solution 2 on both charged (Figs. 1 and 2) and natural fingermarks (Figs. 3 and 4). Preliminary results show that Solution 1 is effective in developing both natural and charged fingermarks. Fig. 1 demonstrates how complete solubilisation of nile red in a microemulsion discourages the precipitation of nile red onto the substrate, and encourages the concentration of nile red into the lipid material present in the fingermark deposit when high lipid concentrations are present. Solution 2 produces ridges that have a greater luminescence intensity and clarity compared to Solution 1. Solubilising the nile red results in decreased contrast, but increased total ridge development and ridge width uniformity, due to the absence of solid nile red. Figs. 3 and 4 show both solutions to be effective on natural fingermarks, with both solutions providing light development of the ridges. Overall, both solutions show comparable development on both charged and natural fingermarks at similar optimal exposure times. Solution 1 was compared with Solution 3 on charged (Figs. 5 and 6) and natural fingermarks (Figs. 7 and 8). Figs. 5 and 6 show that Solution 1 stains more effectively than Solution 3, and does not result in the solid deposition of nile red in heavily sebaceous laden areas of the latent residue particularly present in the furrows, which can result in an inability to visualise certain sections of the developed fingermark as ridge detail is lost. The developed fingermark halves required markedly different exposure times, with Solution 3 needing much longer exposure (>4 times, keeping the other parameters constant) due to decreased staining intensity.
Fig. 1. Charged fingermarks developed by Solution 1 (left) and Solution 2 (right) at 382 ms exposure (optimal exposure for Solution 1).
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Fig. 2. Charged fingermarks developed by Solution 1 (left) and Solution 2 (right) at 163 ms exposure (optimal exposure for Solution 2).
Figs. 7 and 8 show that Solution 3 is less effective than Solution 1 for natural marks, as it stains fingermark ridges less intensely and shows decreased contrast. As with the charged marks, the natural marks developed by Solution 3 required extended exposure times, despite being treated four times longer in the working solution compared to Solution 1. This is most likely attributed to the low concentration of nile red in Solution 3. It should be pointed out that, despite these variations in exposure times, using constant parameters for f-stop and camera settings (Nikon 60 mm micro lens, F2.8.), the exposure times required to obtain optimum results remain in a practical range from an operational viewpoint (i.e. 100 ms to 1.25 s) for Solutions 1 and 2. The problem with weak development, however, is that marks may escape detection if only a simple visual inspection is conducted.
[(Fig._3)TD$IG]
Fig. 9 demonstrates that Solution 1 gives less effective and less intense development one month after solution preparation. The older solution (Fig. 9 – right) shows a darker background with less intense staining of the ridges than is seen with freshly made Solution 1 (Fig. 9 – left). 4. Discussion The comparison of Solution 1 and Solution 2 supports the hypothesis that nile red targets lipids originating in the sebaceous glands and not epidermal originating lipids, due to the weak development seen with the natural marks. However, some degree of development, albeit small, can still be obtained with natural marks and this is most likely attributed to a combination of lipids in the sebaceous glands (which are residually present through
Fig. 3. Natural fingermarks developed by Solution 1 (left) and Solution 2 (right) at 305 ms exposure (optimal exposure for Solution 1).
[(Fig._4)TD$IG]
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Fig. 4. Natural fingermarks developed by Solution 1 (left) and Solution 2 (right) at 382 ms exposure (optimal exposure for Solution 2).
[(Fig._5)TD$IG]
normal hand to face contact) and lipids contained in the hydrolipid film (which are endogenous to friction ridge skin). Better ridge continuity is seen in the development on charged marks using Solution 1 when compared to Solution 2, as development has occurred through solubilised nile red diffusion into the lipid material, as opposed to a combination of diffusion and solid nile red precipitation onto the surface of the deposit. Solution 1 results in some dispersion of the stain, making the developed ridges appear slightly blurred compared to the half developed by Solution 2; however, adequate ridge detail is still obtained. Considering that Solution 1 can be made for AU$6.40/L and Solution 2 can be made for just under AU$50.0/L, the microemulsion formulation is significantly more cost effective with little sacrifice to developmental quality.
Solution 1 outperformed Solution 3 on both charged and natural marks, requiring lower exposure times for image capture. Exposure time lengths are a necessary consideration in fingermark visualisation because long exposure times are not operationally viable, and weakly luminescent marks requiring extended exposure times may escape detection when items are screened after treatment. The extended exposure times for Solution 3 are most likely due to the low concentrations of nile red in the nile blue solution; however, this is difficult to confirm as solid nile red has deposited on the substrate in the charged mark experiments. As seen with Solution 2, Solution 3 exhibited precipitated solid nile red on charged marks whereas Solution 1 demonstrates an ability to develop heavily sebaceous laden areas without solid nile red deposition. Solution 3 showed decreased staining intensity and
Fig. 5. Charged fingermarks developed by Solution 1 (left) and Solution 3 (right) at 126 ms exposure (optimal exposure for Solution 1).
[(Fig._6)TD$IG]
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Fig. 6. Charged fingermarks developed by Solution 1 (left) and Solution 3 (right) at 637 ms exposure (optimal exposure for Solution 3).
reduced sensitivity on natural marks when compared to Solution 1, possibly due to the lower concentration of lipids present resulting in incomplete development. Solution 1, when stored for 1 month after preparation, still developed fingermarks but these were significantly reduced in luminescence intensity compared to marks developed with freshly preparation solution. As such, the working solution should not be stored for more than 2 weeks. This is an improvement on the methanolic nile red working solution (Solution 2) as this can only be used on the day it is prepared. In order to obtain stronger luminescence of developed ridges when using Solution 1, various concentrations of nile red in the dichloromethane stock solution were trialled, however concentrations greater than 0.01 mg/mL resulted in precipitation of nile red onto the substrate. Further
work is needed to determine the optimal ratio and concentration of stock and surfactant solutions to obtain a microemulsion that contains more solubilised nile red. The aim of this research is not to replace PD, nor to assume this may be a possibility, as nile red and PD target different fingermark constituents and need to be used in sequence. The aim is to provide a complementary method to PD, that is less hazardous than the current methanolic nile red working solution, cheaper to make and has an extended shelf life. Despite the extended shelf life, lower cost and less toxic nature of the nile red microemulsion solution, at this stage it is still recommended that the existing methanolic formulation be used in sequence after PD until further optimisation of the new method is completed, especially on marks that are older than one week. This research is ongoing in our laboratory.
[(Fig._7)TD$IG]
Fig. 7. Natural fingermarks developed by Solution 3 (left) and Solution 1 (right) at 1.87 s exposure (optimal exposure for Solution 3).
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Fig. 8. Natural fingermarks developed by Solution 3 (left) and Solution 1 (right) at 428 ms exposure (optimal exposure for Solution 1).
[(Fig._9)TD$IG]
preliminary work, the microemulsion formulation was shown to outperform an aqueous nile blue working solution on both fresh charged and natural fingermarks, requiring shorter development times, shorter exposure times for image capture (due to increased staining intensity), and increased overall sensitivity. Further investigation is needed to determine the optimal parameters and formulation for the sequencing of the nile red microemulsion after the use of physical developer and for use on aged marks (older than one month). References
Fig. 9. Fingermark developed using a freshly prepared Solution 1 (left) and one month old Solution 1 (right).
5. Conclusions Nile red has been incorporated into an aqueous microemulsion using a simplified method that is effective in developing both natural and charged fresh fingermarks on porous surfaces that have been wet. The microemulsion produces slightly more diffuse marks than the published methanol-based working solution, but is less toxic, cheaper to make, and has an extended shelf life. In this
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[17] IFRG, International Fingerprint Research Group (IFRG) Guidelines for the Assessment of Fingermark Detection Techniques, J. Forensic Identi. 2 (2014) 175–201. [18] A. Frick, F. Busetti, A. Cross, S. Lewis, Aqueous Nile blue: a simple, versatile and safe reagent for the detection of latent fingermarks, Chem. Commun. 50 (2014) 3341–3343. [19] A. Ostle, J. Holt, Nile blue A as a fluorescent stain for poly-beta-hydroxybutyrate, J. Appl. Environ. Microbiol. 44 (1982) 238–241.
