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Technical note: latent fingermarks, colloidal gold and multimetal deposition (MMD)
Forensic Science International 118 (2001) 21±28
Technical note: latent ®ngermarks, colloidal gold and multimetal deposition (MMD) Optimisation of the method Bertrand Schnetza, P. Margotb,* a
Police cantonale du Jura, Identite Judiciaire, DeleÂmont, Switzerland Institut de Police Scienti®que et, Universite de Lausanne, Criminologi UNIL-BCH, 1015 Lausanne, Switzerland
b
Received 27 June 2000; accepted 26 July 2000
Abstract Operational details and optimisation of the colloidal gold or multimetal deposition technique (MMD) for the detection of latent ®ngermarks on non porous and porous surfaces demonstrate the power of the method. Control of particulate size, pH, reagent, handling are shown to be essential. A dif®cult case example illustrates the potential of MMD. # 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Fingerprints; Physical developer; Particle size; pH
1. Introduction The use of colloidal gold into the domain of forensic sciences was introduced by Saunders [1], as a method for the detection of latent ®ngermarks. He called the technique multi metal deposition (MMD). The treatment comprises two stages: the ®rst is the immersion of the object to be treated in a solution containing colloidal gold as the active component; and the second is the enhancement of the detected ®ngermarks by the use of a physical developer. This technique is applicable for the detection of prints on both porous and non-porous supports. Several forensic science laboratories have tested this technique [2,3], and concluded that colloidal gold is a particularly effective reagent. It is their opinion that the development of latent ®ngermarks by MMD on smooth surfaces is of equal, if not superior, ef®ciency when compared to the well known methods of cyanoacrylate fuming and vacuum metal deposition. However, despite these positive conclusions the colloidal gold technique has been tested, evaluated and subsequently abandoned by a majority of forensic laboratories throughout the world. No obvious *
Corresponding author.
reasons have been given for such an outcome but the authors have gathered that one main reason is the lack of controlled activity, i.e. inconsistency of results combined to the operational dif®culty of working according to tight laboratory conditions (cleanliness, etc.). Following optimisation, MMD was used on the basis of biochemical enhancement techniques intended for a multiplication of the signal given by weak ®ngermarks [4]. The process of multiplication should by-pass whatever improvements that could be obtained by changing single reagent structures, such as observed with the intensive research activity around ninhydrin analogues. Structural modi®cations only offer marginal improvements, whereas a multiplication of the signal should allow a quantum leap in the improvement. It is not intended here to discuss and explain the scienti®c basis of the process described by Schnetz [4], but the operational optimisation of the technique for ®ngermark detection. 2. MMD: methods and materials According to the optimisation proposed by Allman et al. [3], using data presented by Saunders [1], the parameters
0379-0738/01/$ ± see front matter # 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 9 - 0 7 3 8 ( 0 0 ) 0 0 3 6 1 - 3
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B. Schnetz, P. Margot / Forensic Science International 118 (2001) 21±28
were set as follows: during the ®rst stage of the detection procedure, the object to be treated was immersed in an acidi®ed colloidal gold solution (adjusted to pH 3 by the addition of citric acid). The quantity of sodium citrate that is added enabled the formation of gold particles with an average diameter of 30 nm as suggested by Frens [5] and Goodman et al. [6]. Five ml of Tween 80 was also added to the solution. The second step of the technique was an ampli®cation of the developed prints with physical developer (PD) by immersion in a solution of silver nitrate and a ferric/ferrous redox system. Various parameters of the method were evaluated: particle size and homogeneity, the effect of the pH of the gold solution, the development temperature; the respective concentrations of the gold and Tween solutions; and the in¯uence of the development time on background interference. The most favourable formulation and conditions are reported hereafter. 2.1. Effect of particle size and homogeneity The ®rst modi®cation to the former technique concerns the reduction of the diameter of the gold particle, in an attempt to increase the methods' sensitivity because the solution proposed by Saunders [1] is not homogeneous and the average particle diameter (APD) and coef®cient of variation (CV) are too high (Fig. 1) and do not respect the properties of colloidal particles needed for our application. Particles with diameters varying from 6±14 nm were synthesised according to Slot and Geuze [7]. This method permits homogeneous particle formation, with a range of diameters between 3±17 nm (Fig. 2). Here, the deposition of gold onto the latent mark is superior to that obtained when larger particles are employed (20 nm diameter). 2.2. Effect of pH (from 1 to 10) Colloidal gold is being used at different pH depending on applications in biochemistry, usually in basic conditions. Saunders [1] proposed pH 3, but it soon became obvious that
Fig. 2. Homogeneous gold particles, size 14 nm prepared according to Slot and Geuze [7].
slight variations in this parameter could affect results tremendously. A slight change between pH 3 and 2.5 demonstrates a dramatic increase in sensitivity of detection (Fig. 3). 2.3. Effect of gold concentration (0.005±0.2% w/v) Concentration effects are also marked within narrow ranges, multiple experiments have allowed to focus on optimal operational concentrations (Figs. 4 and 5) 2.4. Effect of Tween 20 concentration (0.05±0.5% w/v) The detergent Tween 20 plays a role in stabilising the colloidal gold solution as well as avoiding a certain background noise. It is particularly important with large inhomogeneous particles, less so with homogeneous and small particles. The 0.5% concentration given by Saunders is therefore much too high and an optimum concentration was found at 0.1% w/v. No Tween gives a high background and too much Tween 20 has a wash effect. 2.5. Effect of temperature of development The temperature for the development (48C, 108C or room temperature) does not affect the ®nal results. Therefore room temperature work is selected for convenience. 2.6. Cleanliness and glassware preparation
Fig. 1. Gold particles (30 nm) prepared according to Saunders [1] under the TEM.
The use of deactivated (siliconised) glassware and bidistilled water for all solutions is imperative. Gold is deposited by nucleation from the colloidal gold solution and any nucleation site can create an imbalance, or a background development. This is also true for the physical developer treatment that follows. It emerges from the biochemical literature, that this factor has been widely affecting the use of colloidal gold. The negative effect from untreated glassware
B. Schnetz, P. Margot / Forensic Science International 118 (2001) 21±28
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Fig. 3. Left side pH 3; right side pH 2.5 (®ngermark after the colloidal gold treatment only, without further development with physical developer).
and poor cleanliness can be dramatic as described by many authors. ``. . .since the nature of glass surface is an important factor in initiating the process of colloid formation and in determining the reproducibility of the colloid preparations, care must be taken to have very clean siliconized vessels.'' (Horisberger [8]).
``. . .since the properties of glass surface are important factors in initiating the reduction process and in determining the reproducibility of the colloidal gold, special care should be taken in preparation of glassware used. Furthermore, double distilled water should be used in preparation of different solutions.'' (Roth [9]). This is experimentally demonstrated in Figs. 6 and 7.
Fig. 4. Left side 0.01% w/v; right side 0.005% w/v (®ngermark after colloidal gold treatment only).
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B. Schnetz, P. Margot / Forensic Science International 118 (2001) 21±28
Fig. 5. Left side 0.01% w/v; right side 0.02% w/v (®ngermark after the colloidal gold treatment).
3. Secondary treatment: physical developer When considering ampli®cation by a physical developer, there are four potential sources of silver ions: silver lactate, silver acetate, silver bromide (from photographic emulsion) and silver nitrate [10±13]. After testing several reducing agents, hydroquinone emerged as the best reagent and the formulation with silver acetate as the most sensitive. The silver lactate is also sensitive but has the disadvantage of being strongly photosensitive.
Treatment in dark rooms is impossible and impractical for our application. 4. Proposal for an optimised method 4.1. Glassware deactivation (siliconised) 4.1.1. Reagents EXTRAN, MAO 01 Alcalin, MERCK No 75553-aminopropyltriethoxysilane 98%, SIGMA No A-3648
Fig. 6. Effect of siliconized vessels on glass for the preparation of the gold solution: left, not siliconized; right, siliconized vessels.
B. Schnetz, P. Margot / Forensic Science International 118 (2001) 21±28
25
vials) and the appropriate volume of bidistilled water is added to achieve the concentration of 0.1 g/ml. The source and grade of the tannic acid is important (it works well with this particular brand). 4.2.2. Gold bath (particles of 14 nm diameter) It is recommended that a maximum volume of 500 ml is prepared at any one time. 1. To 400 ml bidistilled water, add 500 ml of the solution A. 2. In a separate recipient mix 75 ml bidistilled water and 20 ml solution B and 100 ml solution D (tannic acid). Heat both of the above solutions separately to 608C; when this temperature is reached pour the second solution rapidly into the first, stirring vigorously. The resulting mixture is heated to its boiling point, whereupon the solution becomes orange.
Fig. 7. On the left, distilled water was used; on the right, bidistilled water (®ngermark after the colloidal gold treatment).
Acetone 100% Active solution: 4 ml of 3-aminopropyltriethoxysilane in 200 ml of acetone (100%) Procedure Immerse glassware overnight in 10% solution of Extran. Rinse well with hot water, followed by a second rinse with cold water. Dry in oven at 1008C. Remove from oven and let cool to ambient temperature. Immerse in active solution for 5 seconds. Rinse twice with 100% acetone. Rinse twice with distilled water. Dry overnight in oven at 428C. 4.2. Preparation of the colloidal gold 4.2.1. Reagents Solution A: dissolve 1 g of tetrachloroauric acid (MERCK No. 1.01582.0001) in 10 ml of bidistilled water Ð conserve in the refrigerator. Solution B: 1% aqueous sodium citrate Ð conserve at ambient temperature (MERCK No. 644810000). Solution C: 0.1 M citric acid Ð conserve at ambient temperature (MERCK No. 2441000). Solution D: 1% EM grade tannic acid Ð conserve in refrigerator (Polyscience No. 04459-100) The solutions B, C, D, are stable under the stated conditions for 1 month. Solution A is stable for several months in the refrigerator. The concentration is important and must be 0.1 g/ml. For optimal reagent, the glass vial should be broken when preparing this solution (avoid using old opened
This solution is stable for several months when refrigerated and conserved in a polypropylene bottle. Directly before use the solution should be brought to room temperature and is prepared as follows Add 500 ml Tween 20 to the above solution while stirring continuously. Adjust the pH to 2.5±2.8 by addition of solution C (citric acid). The reagent is ready to be used. The sample should be washed with distilled water before being dipped and gently moved with the colloidal gold solution. 4.3. Preparation of the physical developer All procedures can be undertaken in ambient light conditions, however the developer will turn dark grey after the necessary development time of 18 min. This phenomenon does not affect the ®nal results. Prepare the following solutions for a silver bath of 200 ml. Solution A: dissolve 200 mg silver acetate (FLUKA No. 85140) in 100 ml bidistilled water, stirring continuously, in a recipient covered with aluminium foil. Silver acetate dissolves slowly in water, therefore this solution should be prepared 30 mib in advance. Solution B: dissolve 1 g hydroquinone in 200 ml of pH 3.8 buffer solution. Buffer solution (pH 3.8): (conserve respective solutions at ambient temperature) Citric acid (255 g l 1):sodium citrate (235 g l 1):bidistilled water (24:22:50): (citric acid: MERCK No. 2441000, sodium citrate: MERCK No. 64481000) 4.3.1. Procedure Add 100 ml bidistilled water to 100 ml of solution B; soak the object to be treated in the resulting solution for 2±5 min.
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B. Schnetz, P. Margot / Forensic Science International 118 (2001) 21±28
Fig. 8. Example of a ®ngermark developed on a porous surface (here white paper) using the proposed optimised method (on the right) against the old (left).
Transfer the object to a silver bath, consisting of 100 ml solution A and 100 ml solution B (this solution should be freshly prepared directly before use); leave the sample to soak for approximately 18 min at room temperature. Rinse the object with distilled water. Soak the developed object for 2±5 min with photographic fixing solution (1:9 dilution with water). Rinse the object well with tap water.
5. Results 5.1. New formulation of MMD The following photographs show the ®nal results of the detection of latent ®ngermarks on porous (Fig. 8) and nonporous (Fig. 9) surfaces. The left half of the ®ngermark was developed using the method proposed by Saunders [1], and the right half using the method proposed above.
Fig. 9. Example of a ®ngermark detected on a non-porous surface (white polythene plastic bag) with the optimised technique on the right.
B. Schnetz, P. Margot / Forensic Science International 118 (2001) 21±28
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Fig. 10. Common background noise observed after physical developer treatment on plastic using the traditional physical developer (left) or the optimised recipe (right).
The same results are obtained regardless of the age of the latent ®ngermark. On ®rst observation, the right half of the prints appear sharper and, for the second example (Fig. 9), there is less background interference and no markings due to an uncontrolled ampli®cation of the silver as illustrated above (a phenomenon which is also seen with the treatment using physical developer Ð Fig. 10). It is to be noted that chemicals from different producers sometimes showed marked differences in quality of the ®nal treatment. It must be emphasised that tetrachloroauric acid should be obtained in sealed vials, under inert conditions and tannic acid should be EM (electron microscopy) grade. 6. Discussion and conclusion The tests undertaken using the new MMD formulation highlight three improvements: an increased reactivity due to the homogeneity of the solution and the controlled pH; an improved resolution, due to the smaller gold particle size; and a greater ampli®cation selectivity (due to an improved targeting of the ®ngermarks developed with colloidal gold by the silver species), resulting in decreased background interference. Despite an apparent complexity, the preparation of this MMD formulation is relatively easy: the stock solutions should be prepared following the proposed methods, and the appropriate products. After having prepared large quantities of the aforementioned solutions, the authors would like to stress that although these methods are simple, the slightest deviation of procedure results in defective solutions. For example, the controlled pH of the solutions is very important: at a pH > 3:1 the secretions that constitute the latent
®ngermarks loose their reactivity towards gold particles. It is recommended to work at a pH slightly below pH 2.8 (i.e. 2.5±2.8) rather than one slightly above. The laboratories having tested Saunders' method, at pH 3, most probably overlooked this parameter thus resulting in varying quality (a measure with pH paper is not satisfactory). This may indeed explain the decision by many laboratories to abandon Saunders' method. The authors want to highlight the importance, from the beginning, of the care and attention needed when preparing the solutions, and to expose the high sensitivity of the solutions to certain parameters. During the development of latent ®ngermarks, the object to be treated must be held in the gold bath (always using plastic tweezers), and shaken/moved gently (neither too slow nor too fast). Before submerging the object to be treated, it should be pre-washed with distilled water (a rinse for non-porous supports, or a 2±5 min wash for porous surfaces). It was found that a better, more uniform and faster development was obtained if the treated sample was moving together with the treatment dish (®xed tight to it) rather than left swimming freely in the dish. The incubation time is short (2±15 min). The procedure of ampli®cation with silver (physical developer) does not need discussion (always use clean tweezers). Time of incubation can be controlled optically by visualisation. The new formulation of MMD can be used as a routine practice in laboratories. At ®rst it appears to be a complex procedure and certainly a minimum amount of experience is necessary for the successful application of this technique. It has been introduced routinely in identi®cation bureaux in Switzerland with successful outcomes. Fingermarks on banknotes are notoriously dif®cult to visualise and the following example shows a successful development on a
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Fig. 11. Case example: successful detection of a mark on a Swiss banknote that resulted in an identi®cation.
Swiss banknote which resulted in an identi®cation, using the proposed MMD (Fig. 11). This article is part of the Ph.D. thesis of one author (BS), which is further considering biochemical ampli®cations of the colloidal gold treatment [1]. References [1] G. Saunders 1989. Multimetal deposition technique for latent ®ngerprint development, International Association for Identi®cation, 74th Annual Educational Conference, June, Pensacola, USA. [2] F. Irrausch 1991. DeÂtection des empreintes digitales au reÂactif aÁ base d'or colloõÈdal, SeÂminaire, Institut de Police Scienti®que et de Criminologie, Universite de Lausanne, Suisse.
[3] D.S. Allman, S.J. Maggs, C.A. Pounds 1992. The use of colloidal gold/multi-metal deposition for the detection of latent ®ngerprints Ð a preliminary evaluation, Central Research and Support Establishment, Home Of®ce Forensic Science Service, Aldermaston, UK, Report 747, pp. 1±24. [4] B. Schnetz 1999. La reÂveÂlation des empreintes digitales par l'or colloõÈdal: l'ampli®cation du signal par des techniques biochimiques, seÂrie criminalistique, Doctoral thesis, Institut de police scienti®que et de criminologie, Universite de Lausanne. [5] G. Frens, Controlled nucleation for the regulation of the particle size in monodisperse gold solutions, Nature (London) Phys. Sci. 241 (1973) 20±22. [6] S.L. Goodman, G.M. Hodges, L.K. Trejdosiewicz, D.C. Livingston, Colloidal gold markers and probes for routine application in microscopy, J. Microsc. 123 (1981) 201±213. [7] J.W. Slot, H.J. Geuze, A new method of preparing gold probes for multiple-labelling cytochemistry, Eur. J. Cell Biol. 38 (1985) 87±93. [8] M. Horisberger 1985. The gold method as applied to lectin cytochemistry in transmission and scanning electron microscopy, in: G.R. Bullock, P. Petrusz (Eds.), Techniques in Immunocytochemistry, Academic Press, New York, Vol. 3, pp. 155±178. [9] J. Roth 1982. The protein A-gold (pAg) technique Ð a qualitative and quantitative approach for antigen localization on thin sections, in: G.R. Bullock, P. Petrusz (Eds.), Techniques in Immunocytochemistry, Academic Press, New York, Vol. 1, pp. 107±133. [10] G. Danscher, Histochemical demonstration of heavy metals. A revised version of the sulphide silver method suitable for both light and electron microscopy, Histochemistry 71 (1981a) 1. [11] G. Danscher, Localization of gold in biological tissue: a photochemical method for light and electron microscopy, Histochemistry 71 (1981) 81±88. [12] G. Danscher, J.O.R. Norgaard, Ultrastructural autometallography: a method for silver ampli®cation of catalytic metals, J. Histochem. Cytochem. 33 (1985) 706±710. [13] G. Danscher, G.W. Hacker, L. Grimelius, J.O.R. Norgaard, Autometallographic silver ampli®cation of colloidal gold, J. Histotech. 16 (1993) 201±207.
Technical note: latent ®ngermarks, colloidal gold and multimetal deposition (MMD) Optimisation of the method Bertrand Schnetza, P. Margotb,* a
Police cantonale du Jura, Identite Judiciaire, DeleÂmont, Switzerland Institut de Police Scienti®que et, Universite de Lausanne, Criminologi UNIL-BCH, 1015 Lausanne, Switzerland
b
Received 27 June 2000; accepted 26 July 2000
Abstract Operational details and optimisation of the colloidal gold or multimetal deposition technique (MMD) for the detection of latent ®ngermarks on non porous and porous surfaces demonstrate the power of the method. Control of particulate size, pH, reagent, handling are shown to be essential. A dif®cult case example illustrates the potential of MMD. # 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Fingerprints; Physical developer; Particle size; pH
1. Introduction The use of colloidal gold into the domain of forensic sciences was introduced by Saunders [1], as a method for the detection of latent ®ngermarks. He called the technique multi metal deposition (MMD). The treatment comprises two stages: the ®rst is the immersion of the object to be treated in a solution containing colloidal gold as the active component; and the second is the enhancement of the detected ®ngermarks by the use of a physical developer. This technique is applicable for the detection of prints on both porous and non-porous supports. Several forensic science laboratories have tested this technique [2,3], and concluded that colloidal gold is a particularly effective reagent. It is their opinion that the development of latent ®ngermarks by MMD on smooth surfaces is of equal, if not superior, ef®ciency when compared to the well known methods of cyanoacrylate fuming and vacuum metal deposition. However, despite these positive conclusions the colloidal gold technique has been tested, evaluated and subsequently abandoned by a majority of forensic laboratories throughout the world. No obvious *
Corresponding author.
reasons have been given for such an outcome but the authors have gathered that one main reason is the lack of controlled activity, i.e. inconsistency of results combined to the operational dif®culty of working according to tight laboratory conditions (cleanliness, etc.). Following optimisation, MMD was used on the basis of biochemical enhancement techniques intended for a multiplication of the signal given by weak ®ngermarks [4]. The process of multiplication should by-pass whatever improvements that could be obtained by changing single reagent structures, such as observed with the intensive research activity around ninhydrin analogues. Structural modi®cations only offer marginal improvements, whereas a multiplication of the signal should allow a quantum leap in the improvement. It is not intended here to discuss and explain the scienti®c basis of the process described by Schnetz [4], but the operational optimisation of the technique for ®ngermark detection. 2. MMD: methods and materials According to the optimisation proposed by Allman et al. [3], using data presented by Saunders [1], the parameters
0379-0738/01/$ ± see front matter # 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 9 - 0 7 3 8 ( 0 0 ) 0 0 3 6 1 - 3
22
B. Schnetz, P. Margot / Forensic Science International 118 (2001) 21±28
were set as follows: during the ®rst stage of the detection procedure, the object to be treated was immersed in an acidi®ed colloidal gold solution (adjusted to pH 3 by the addition of citric acid). The quantity of sodium citrate that is added enabled the formation of gold particles with an average diameter of 30 nm as suggested by Frens [5] and Goodman et al. [6]. Five ml of Tween 80 was also added to the solution. The second step of the technique was an ampli®cation of the developed prints with physical developer (PD) by immersion in a solution of silver nitrate and a ferric/ferrous redox system. Various parameters of the method were evaluated: particle size and homogeneity, the effect of the pH of the gold solution, the development temperature; the respective concentrations of the gold and Tween solutions; and the in¯uence of the development time on background interference. The most favourable formulation and conditions are reported hereafter. 2.1. Effect of particle size and homogeneity The ®rst modi®cation to the former technique concerns the reduction of the diameter of the gold particle, in an attempt to increase the methods' sensitivity because the solution proposed by Saunders [1] is not homogeneous and the average particle diameter (APD) and coef®cient of variation (CV) are too high (Fig. 1) and do not respect the properties of colloidal particles needed for our application. Particles with diameters varying from 6±14 nm were synthesised according to Slot and Geuze [7]. This method permits homogeneous particle formation, with a range of diameters between 3±17 nm (Fig. 2). Here, the deposition of gold onto the latent mark is superior to that obtained when larger particles are employed (20 nm diameter). 2.2. Effect of pH (from 1 to 10) Colloidal gold is being used at different pH depending on applications in biochemistry, usually in basic conditions. Saunders [1] proposed pH 3, but it soon became obvious that
Fig. 2. Homogeneous gold particles, size 14 nm prepared according to Slot and Geuze [7].
slight variations in this parameter could affect results tremendously. A slight change between pH 3 and 2.5 demonstrates a dramatic increase in sensitivity of detection (Fig. 3). 2.3. Effect of gold concentration (0.005±0.2% w/v) Concentration effects are also marked within narrow ranges, multiple experiments have allowed to focus on optimal operational concentrations (Figs. 4 and 5) 2.4. Effect of Tween 20 concentration (0.05±0.5% w/v) The detergent Tween 20 plays a role in stabilising the colloidal gold solution as well as avoiding a certain background noise. It is particularly important with large inhomogeneous particles, less so with homogeneous and small particles. The 0.5% concentration given by Saunders is therefore much too high and an optimum concentration was found at 0.1% w/v. No Tween gives a high background and too much Tween 20 has a wash effect. 2.5. Effect of temperature of development The temperature for the development (48C, 108C or room temperature) does not affect the ®nal results. Therefore room temperature work is selected for convenience. 2.6. Cleanliness and glassware preparation
Fig. 1. Gold particles (30 nm) prepared according to Saunders [1] under the TEM.
The use of deactivated (siliconised) glassware and bidistilled water for all solutions is imperative. Gold is deposited by nucleation from the colloidal gold solution and any nucleation site can create an imbalance, or a background development. This is also true for the physical developer treatment that follows. It emerges from the biochemical literature, that this factor has been widely affecting the use of colloidal gold. The negative effect from untreated glassware
B. Schnetz, P. Margot / Forensic Science International 118 (2001) 21±28
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Fig. 3. Left side pH 3; right side pH 2.5 (®ngermark after the colloidal gold treatment only, without further development with physical developer).
and poor cleanliness can be dramatic as described by many authors. ``. . .since the nature of glass surface is an important factor in initiating the process of colloid formation and in determining the reproducibility of the colloid preparations, care must be taken to have very clean siliconized vessels.'' (Horisberger [8]).
``. . .since the properties of glass surface are important factors in initiating the reduction process and in determining the reproducibility of the colloidal gold, special care should be taken in preparation of glassware used. Furthermore, double distilled water should be used in preparation of different solutions.'' (Roth [9]). This is experimentally demonstrated in Figs. 6 and 7.
Fig. 4. Left side 0.01% w/v; right side 0.005% w/v (®ngermark after colloidal gold treatment only).
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B. Schnetz, P. Margot / Forensic Science International 118 (2001) 21±28
Fig. 5. Left side 0.01% w/v; right side 0.02% w/v (®ngermark after the colloidal gold treatment).
3. Secondary treatment: physical developer When considering ampli®cation by a physical developer, there are four potential sources of silver ions: silver lactate, silver acetate, silver bromide (from photographic emulsion) and silver nitrate [10±13]. After testing several reducing agents, hydroquinone emerged as the best reagent and the formulation with silver acetate as the most sensitive. The silver lactate is also sensitive but has the disadvantage of being strongly photosensitive.
Treatment in dark rooms is impossible and impractical for our application. 4. Proposal for an optimised method 4.1. Glassware deactivation (siliconised) 4.1.1. Reagents EXTRAN, MAO 01 Alcalin, MERCK No 75553-aminopropyltriethoxysilane 98%, SIGMA No A-3648
Fig. 6. Effect of siliconized vessels on glass for the preparation of the gold solution: left, not siliconized; right, siliconized vessels.
B. Schnetz, P. Margot / Forensic Science International 118 (2001) 21±28
25
vials) and the appropriate volume of bidistilled water is added to achieve the concentration of 0.1 g/ml. The source and grade of the tannic acid is important (it works well with this particular brand). 4.2.2. Gold bath (particles of 14 nm diameter) It is recommended that a maximum volume of 500 ml is prepared at any one time. 1. To 400 ml bidistilled water, add 500 ml of the solution A. 2. In a separate recipient mix 75 ml bidistilled water and 20 ml solution B and 100 ml solution D (tannic acid). Heat both of the above solutions separately to 608C; when this temperature is reached pour the second solution rapidly into the first, stirring vigorously. The resulting mixture is heated to its boiling point, whereupon the solution becomes orange.
Fig. 7. On the left, distilled water was used; on the right, bidistilled water (®ngermark after the colloidal gold treatment).
Acetone 100% Active solution: 4 ml of 3-aminopropyltriethoxysilane in 200 ml of acetone (100%) Procedure Immerse glassware overnight in 10% solution of Extran. Rinse well with hot water, followed by a second rinse with cold water. Dry in oven at 1008C. Remove from oven and let cool to ambient temperature. Immerse in active solution for 5 seconds. Rinse twice with 100% acetone. Rinse twice with distilled water. Dry overnight in oven at 428C. 4.2. Preparation of the colloidal gold 4.2.1. Reagents Solution A: dissolve 1 g of tetrachloroauric acid (MERCK No. 1.01582.0001) in 10 ml of bidistilled water Ð conserve in the refrigerator. Solution B: 1% aqueous sodium citrate Ð conserve at ambient temperature (MERCK No. 644810000). Solution C: 0.1 M citric acid Ð conserve at ambient temperature (MERCK No. 2441000). Solution D: 1% EM grade tannic acid Ð conserve in refrigerator (Polyscience No. 04459-100) The solutions B, C, D, are stable under the stated conditions for 1 month. Solution A is stable for several months in the refrigerator. The concentration is important and must be 0.1 g/ml. For optimal reagent, the glass vial should be broken when preparing this solution (avoid using old opened
This solution is stable for several months when refrigerated and conserved in a polypropylene bottle. Directly before use the solution should be brought to room temperature and is prepared as follows Add 500 ml Tween 20 to the above solution while stirring continuously. Adjust the pH to 2.5±2.8 by addition of solution C (citric acid). The reagent is ready to be used. The sample should be washed with distilled water before being dipped and gently moved with the colloidal gold solution. 4.3. Preparation of the physical developer All procedures can be undertaken in ambient light conditions, however the developer will turn dark grey after the necessary development time of 18 min. This phenomenon does not affect the ®nal results. Prepare the following solutions for a silver bath of 200 ml. Solution A: dissolve 200 mg silver acetate (FLUKA No. 85140) in 100 ml bidistilled water, stirring continuously, in a recipient covered with aluminium foil. Silver acetate dissolves slowly in water, therefore this solution should be prepared 30 mib in advance. Solution B: dissolve 1 g hydroquinone in 200 ml of pH 3.8 buffer solution. Buffer solution (pH 3.8): (conserve respective solutions at ambient temperature) Citric acid (255 g l 1):sodium citrate (235 g l 1):bidistilled water (24:22:50): (citric acid: MERCK No. 2441000, sodium citrate: MERCK No. 64481000) 4.3.1. Procedure Add 100 ml bidistilled water to 100 ml of solution B; soak the object to be treated in the resulting solution for 2±5 min.
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B. Schnetz, P. Margot / Forensic Science International 118 (2001) 21±28
Fig. 8. Example of a ®ngermark developed on a porous surface (here white paper) using the proposed optimised method (on the right) against the old (left).
Transfer the object to a silver bath, consisting of 100 ml solution A and 100 ml solution B (this solution should be freshly prepared directly before use); leave the sample to soak for approximately 18 min at room temperature. Rinse the object with distilled water. Soak the developed object for 2±5 min with photographic fixing solution (1:9 dilution with water). Rinse the object well with tap water.
5. Results 5.1. New formulation of MMD The following photographs show the ®nal results of the detection of latent ®ngermarks on porous (Fig. 8) and nonporous (Fig. 9) surfaces. The left half of the ®ngermark was developed using the method proposed by Saunders [1], and the right half using the method proposed above.
Fig. 9. Example of a ®ngermark detected on a non-porous surface (white polythene plastic bag) with the optimised technique on the right.
B. Schnetz, P. Margot / Forensic Science International 118 (2001) 21±28
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Fig. 10. Common background noise observed after physical developer treatment on plastic using the traditional physical developer (left) or the optimised recipe (right).
The same results are obtained regardless of the age of the latent ®ngermark. On ®rst observation, the right half of the prints appear sharper and, for the second example (Fig. 9), there is less background interference and no markings due to an uncontrolled ampli®cation of the silver as illustrated above (a phenomenon which is also seen with the treatment using physical developer Ð Fig. 10). It is to be noted that chemicals from different producers sometimes showed marked differences in quality of the ®nal treatment. It must be emphasised that tetrachloroauric acid should be obtained in sealed vials, under inert conditions and tannic acid should be EM (electron microscopy) grade. 6. Discussion and conclusion The tests undertaken using the new MMD formulation highlight three improvements: an increased reactivity due to the homogeneity of the solution and the controlled pH; an improved resolution, due to the smaller gold particle size; and a greater ampli®cation selectivity (due to an improved targeting of the ®ngermarks developed with colloidal gold by the silver species), resulting in decreased background interference. Despite an apparent complexity, the preparation of this MMD formulation is relatively easy: the stock solutions should be prepared following the proposed methods, and the appropriate products. After having prepared large quantities of the aforementioned solutions, the authors would like to stress that although these methods are simple, the slightest deviation of procedure results in defective solutions. For example, the controlled pH of the solutions is very important: at a pH > 3:1 the secretions that constitute the latent
®ngermarks loose their reactivity towards gold particles. It is recommended to work at a pH slightly below pH 2.8 (i.e. 2.5±2.8) rather than one slightly above. The laboratories having tested Saunders' method, at pH 3, most probably overlooked this parameter thus resulting in varying quality (a measure with pH paper is not satisfactory). This may indeed explain the decision by many laboratories to abandon Saunders' method. The authors want to highlight the importance, from the beginning, of the care and attention needed when preparing the solutions, and to expose the high sensitivity of the solutions to certain parameters. During the development of latent ®ngermarks, the object to be treated must be held in the gold bath (always using plastic tweezers), and shaken/moved gently (neither too slow nor too fast). Before submerging the object to be treated, it should be pre-washed with distilled water (a rinse for non-porous supports, or a 2±5 min wash for porous surfaces). It was found that a better, more uniform and faster development was obtained if the treated sample was moving together with the treatment dish (®xed tight to it) rather than left swimming freely in the dish. The incubation time is short (2±15 min). The procedure of ampli®cation with silver (physical developer) does not need discussion (always use clean tweezers). Time of incubation can be controlled optically by visualisation. The new formulation of MMD can be used as a routine practice in laboratories. At ®rst it appears to be a complex procedure and certainly a minimum amount of experience is necessary for the successful application of this technique. It has been introduced routinely in identi®cation bureaux in Switzerland with successful outcomes. Fingermarks on banknotes are notoriously dif®cult to visualise and the following example shows a successful development on a
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Fig. 11. Case example: successful detection of a mark on a Swiss banknote that resulted in an identi®cation.
Swiss banknote which resulted in an identi®cation, using the proposed MMD (Fig. 11). This article is part of the Ph.D. thesis of one author (BS), which is further considering biochemical ampli®cations of the colloidal gold treatment [1]. References [1] G. Saunders 1989. Multimetal deposition technique for latent ®ngerprint development, International Association for Identi®cation, 74th Annual Educational Conference, June, Pensacola, USA. [2] F. Irrausch 1991. DeÂtection des empreintes digitales au reÂactif aÁ base d'or colloõÈdal, SeÂminaire, Institut de Police Scienti®que et de Criminologie, Universite de Lausanne, Suisse.
[3] D.S. Allman, S.J. Maggs, C.A. Pounds 1992. The use of colloidal gold/multi-metal deposition for the detection of latent ®ngerprints Ð a preliminary evaluation, Central Research and Support Establishment, Home Of®ce Forensic Science Service, Aldermaston, UK, Report 747, pp. 1±24. [4] B. Schnetz 1999. La reÂveÂlation des empreintes digitales par l'or colloõÈdal: l'ampli®cation du signal par des techniques biochimiques, seÂrie criminalistique, Doctoral thesis, Institut de police scienti®que et de criminologie, Universite de Lausanne. [5] G. Frens, Controlled nucleation for the regulation of the particle size in monodisperse gold solutions, Nature (London) Phys. Sci. 241 (1973) 20±22. [6] S.L. Goodman, G.M. Hodges, L.K. Trejdosiewicz, D.C. Livingston, Colloidal gold markers and probes for routine application in microscopy, J. Microsc. 123 (1981) 201±213. [7] J.W. Slot, H.J. Geuze, A new method of preparing gold probes for multiple-labelling cytochemistry, Eur. J. Cell Biol. 38 (1985) 87±93. [8] M. Horisberger 1985. The gold method as applied to lectin cytochemistry in transmission and scanning electron microscopy, in: G.R. Bullock, P. Petrusz (Eds.), Techniques in Immunocytochemistry, Academic Press, New York, Vol. 3, pp. 155±178. [9] J. Roth 1982. The protein A-gold (pAg) technique Ð a qualitative and quantitative approach for antigen localization on thin sections, in: G.R. Bullock, P. Petrusz (Eds.), Techniques in Immunocytochemistry, Academic Press, New York, Vol. 1, pp. 107±133. [10] G. Danscher, Histochemical demonstration of heavy metals. A revised version of the sulphide silver method suitable for both light and electron microscopy, Histochemistry 71 (1981a) 1. [11] G. Danscher, Localization of gold in biological tissue: a photochemical method for light and electron microscopy, Histochemistry 71 (1981) 81±88. [12] G. Danscher, J.O.R. Norgaard, Ultrastructural autometallography: a method for silver ampli®cation of catalytic metals, J. Histochem. Cytochem. 33 (1985) 706±710. [13] G. Danscher, G.W. Hacker, L. Grimelius, J.O.R. Norgaard, Autometallographic silver ampli®cation of colloidal gold, J. Histotech. 16 (1993) 201±207.