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Nano technology plays a vital role in all aspects of scientific advancements and finds its place in various diverse fields of science and technologies. It is therefore called as the cutting edge technology of the new era. One such interesting application of nano technology is the usage of gold nano particles in the detection of latent fingerprints. A nano particle is one with a size of 1-100 nano meters and with an enhanced surface to volume ratio. Gold nano particles are obtained by fine processing of elemental gold to nano size.
In a crime scene, a convict leaves away various forensic evidences out of which fingerprints are most preferred by the law enforcement agencies. This is due to the fact that fingerprints are extremely characteristic to individual and no two individuals can possess the same fingerprint patterns. A fingerprint is featured by ridges, minute loops and whirls which are extremely useful as main forensic evidence.
There are primarily three types of fingerprints- visible prints, impressed prints and latent prints out of which latent fingerprints are the problematic in developing but with high information impinged in them.
In the area of detection of finger marks, various techniques available are physical, chemical, optical and MMD. Though MMD is considered to be highly selective and sensitive, it has various parameters that make it complex. This paper explores the possibility of modification of the conventional MMD technique with the usage of gold nano particles, functionalizing them and applying the luminescence nature of gold nano particles in detection of finger marks. But, the main problem associated with gold nano particles is the diffusive nature of illumination property ie., the wavelength is not having sharp peak.
Conventional MMD techniques and its modification
It is based on the use of colloidal gold (small particle of metallic gold suspended in water along with citrate ions). The optical particle diameter of this colloidal gold is about 14 nm and it gives an intense ruby red color. The process starts with the immersion of the sample containing the latent finger mark in the colloidal gold solution. In certain experimental condition, some components of the residues in the finger marks attract the gold nano particles. As the colloidal gold is of very small size, getting a good contrast becomes difficult. So, it involves the deposition of a second metal on the surface of the gold nano particles. For this purpose, silver (reduced from metallic silver in the presence of a reducing agent like hydro quinine) is used. Each gold aggregate acts as a growing site for silver particles. Because of the lustrous nature of silver the finger marks appear distinctly as dark gray/black prints. Though MMD is highly sensitive and a detailed finger mark is obtained, it has various limitations in the actual investigation process. The most prominent disadvantage is that the MMD process involves too many complex steps like, immersion in number of baths, water rinsing, colloidal gold deposition, second water rinsing, presilver treatment, silver deposition and final wash. Any new modification to the existing MMD process should be designed in such a way that it reduces to complexity without altering sensitivity and efficiency of the process.
Fig. 1.Â Schematic representation of the MultiMetal Deposition technique. Gold nanoparticles are first deposited onto the fingermark residue and are then used as growing sites for the metallic silver deposition.
One such modification is the use of functionalization of gold nano particles and applying the molecular host mechanism of a carrier molecule like cyclo-dextrin and thereby reducing the multi metal deposition steps and making it as a single metal deposition (SMD) with the same sensitivity and efficiency of MMD. Three different CDs were retained, that is 6 - monodeoxy- 6 - monothio Î² - cyclodextrin, per - 6 - thio Î²- cyclodextrin and 6 - monodeoxy - 6 - monothiodecamethylenethio Î² - cyclodextrin. Simplified chemical structures of the Î² cyclodextrins were synthesized during this study.
The actual strategy involves the following process. First, Bare gold nano particles get functionalized by thiolated cyclodextrins. Secondly, a dye(AB25) is added and subsequently packed in CD cavities. Then the solution is used to detect the latent finger marks as dark blue ridges with enhanced contrast. The most prominent feature of this process is that after the immersion of the sample in the solution the detection of latent finger marks is made as a one step process.
Functionalization of the gold nano particles
Gold (Au) is a powerful template, possessing large number of molecular host that binds spontaneously. Out of all available nano particles, colloids of gold were chosen because of their chemical inertness and the property of gold being resistant to oxidation. Gold also binds strongly to the main class of reactants namely the, thiolated molecules. Gold surface is immersed in thiolated molecule solution resulting in the formation of SAM (Self Assembled Mono layers). The chemisorption of the thiolated molecules on to the gold makes it possible to functionalize the gold surface with the molecular layer that is commonly resistant to washes in a relative scale and varying experimental conditions. The colloidal gold is also used in development of bio-sensors on nano scale where Au nano particles play the role of templates for immobilization (arrest) of bio molecules. If the aliphatic chains in the biomolecules are ended by another functional group we can obtain functionalized SAMs.
The modification of MMD involves the binding of an efficient molecular host which would bind to gold and offer chelating abilities in solution level and thereby it could trap some photoactive molecule and dyes, once gold gets deposited on the finger marks. For this purpose modified cyclo-dextrin were chosen because of their ability to change their chemical structure dynamically. Due to the fact that cyclodextrin is a powerful molecular host, it offers effective gold binding. This method is applicable for any surfaces and is independent of previous wetting conditions and age of fingermark.
Fig. 2.Â Illustration of the chemisorption of alkanethiolates on a gold surface, leading to the formation of highly-ordered self-assembled monolayers (SAMs). R can be a methyl ending group (-CH3) or another chemical function, thus providing new properties to the gold surface.
1.3. Cyclodextrin; the molecular host
They are cyclic oligosaccharides composed of a well defined number of "gluco-pyranose" rings linked together by glucosidic bonds to form three dimensional truncated cones. The available CDs are Î±(alpha), Î²(beta) and Î³(gamma) CDs which have six, seven and eight pyranose units respectively. Because of the 3d arrangement of the glucopyranose groups, the outer surface of the cyclo-dextrin is hydrophilic and helps in solubilization in aqueous solution. In contrast, the inner cavity is hydrophobic and forms complex with highly hydrophobic molecules in solution. Therefore, CDs are efficient chelating agents which form stable inclusion complexes with large number of compounds. Out of the three cyclo-dextrins mentioned here, the Î²-CD is the most preferred one, because it is the cheapest of all. It is highly useful as its cavity has got the optimum size to bind a variety of aliphatic and aromatic compounds.
CD modifies their native template with a large variety of chemical functions or groups that can be linked in a regioselective style. This striking feature is also one of the reasons for the preference of CDs as molecular host.
In this actual process, thiolated cyclodextrints were taken because the - SH groups present on the molecule's smaller phase makes them to bind to gold surfaces and thereby play the role of in-situ force.
Production of colloidal gold nano particles:
Tetrachloroauric acid is used as the component from which colloidal gold is produced.
50ml sodium citrate (38.8 mM)
CD - SH - molecular host
AB25 - dye
The methods for the modification of MMD involves
Synthesis of gold nano particles
Synthesis of thiolated CDs
Functionalisation of bare gold nano particles by thiolated CDs
Addition of dye and dye trapping in CD cavities
Detection of fingermarks in a single step and its visualisation
The synthesis of gold nanoparticles in water is commonly performed via the reduction of tetrachloroauric acid (HAuCl4) by a reducing agent in solution. According to the relative quantity of reagents in solution, the nano particle diameters can vary from 1 to 100Â nm. 50Â ml of sodium citrate (38.8Â mM) were added to 500Â ml of boiling HAuCl4 (1Â mM), then kept boiling for 10Â min before cooling to room temperature while stirring, for another 15Â min. Molarities were then obtained by combining the quantity of gold ions converted during the synthesis with the gold density and the size of the obtained nano spheres.
The synthetic route to obtain CD-SH starting from Î²-CD is depicted in following figure. It requires the synthesis of mono (6-O-p-toluenesulfonyl) Î²-cyclodextrin (named CD-Tos), which is a molecular intermediate bearing a toluenesulfonyl group. Briefly, once synthesized, CD-Tos was reacted with thiourea in DMF, at 75Â Â°C for 2 days. The crude product was recrystallized in acetone and then freeze dried. Finally, the isothiouronium salt was converted to the thiol using sodium disulfite (Na2S2O5). At the end, a very light white powder is obtained (yield: 43%)
Fig.3. Synthesis of 6-monodeoxy-6-monothio Î²-cyclodextrin (right, starting from native Î²-cyclodextrin (left) and via mono(6-O-p-toluenesulfonyl) Î²-cyclodextrin (middle).
The functionalization of such colloidal gold solution by CD-SH (and by the other ones) is easily obtained by dissolving 13.29Â mg of CD-SH (0.01Â mmole) in 100Â ml of colloidal gold for 2 days, at room temperature and in the dark. The modified gold nano particles are isolated by the addition of DMSO (mixed again for 48Â h), then CH3CN to initiate their precipitation. The solution is centrifuged to obtain a red solid, which is rinsed by DMSO:CH3CN (1:1, v/v) and finally dissolved in 100Â ml of pure water to give a "CD-SH"-modified colloidal gold solution characterized by a ruby-red color. This solution is stable if preserved at 4Â Â°C until it has to be used for latent fingermark detection.
Just before use, the solution is mixed for 2Â h with a specific dye whose final concentration should be equal to 10âˆ’5Â M, to allow its complexation in the CD cavities. In this case, we chose Acid Blue 25 (AB25), a blue organic dye built on an anthraquinone structure. AB25 has a very high affinity for the CD cavity. Indeed, about 100% of the AB25 molecules in aqueous solution were adsorbed by an immersed cyclodextrin-based polymer, which is used as an efficient depolluting system.
Fig.4. Schematization of the colloidal gold modification strategy. First, the gold nanoparticles are functionalized with thiolated cyclodextrins. A dye is then added in the solution and a sample is finally immersed. The detection of latent fingermarks is then performed in a unique step.
Detection of fingermark is done by dipping the sample in colloidal functionalized gold solution or spraying it over the suspected areas. Within 10-15 minutes, dark blue ridges have been developed against the light background with improved contrast. The dyes can be changed with respect to the background color.
Different sets of latent fingermarks were collected for this study. To verify the efficiency of this modified colloidal gold solution, three kinds of surfaces were chosen for their different behaviour in terms of interactions with latent fingermark or with colloidal gold, that is, a porous one: "M-Office" non-bleached paper; and two non-porous ones: low density polyethylene (LDPE) bags and polypropylene (PP) sheets. In addition to this, six different donors were chosen, i.e., three males and three females, and a well-defined protocol has been set up for the deposition of fingermarks from both hands. After having periodically collected such samples, we were able to possess fresh (few days) and aged fingermarks (one to several months).
4.1 Synthesis of the modified cyclodextrins and functionalization of gold
The modified cyclodextrins were successfully synthesized following the procedures. Those synthesis require no specific glassware or hard experimental conditions. Each final product has been characterized by FT-IR and MALDI-TOF mass spectroscopy to confirm the presence of CD-SH, CD-(SH)7, and CD-S-C10H20-SH.
The functionalization of the colloidal gold by thiolated cyclodextrins was easily performed as this chemisorption is a spontaneous mechanism. No specific experimental conditions were required since the reaction is performed at ambient temperature and pressure, without any catalyst. This is an important point because colloidal gold is very sensitive to environmental changes and may become unstable under certain circumstances. If so, it is easily observed as the gold colloids precipitate in the bottom of the flask, sometimes irreversibly, or the solution color changes from ruby-red to dark-blue. In our case, we observed no instability of the solutions during their functionalization. The addition of DMSO and CH3CN allows the isolation of the modified gold colloids by precipitating them so that they can be purified. After rinsing, the red solid is solubilized again in pure water to obtain the intense ruby-red colored solution, characterizing the presence of 14Â nm gold colloids in solution. Some samples of the cyclodextrin-modified gold nano particles (Au-S-CD) were precipitated and dried to be analyzed by FTIR. The analysis of the resulting spectra revealed the signature of cyclodextrins combined with gold. The modified gold solutions were kept in the fridge and were stable for months. The cyclodextrin-modified gold nano particles with the AB25 encapsulated dye will be referred to as "Au-S-CD (AB25)".
4.2 Detection of fingermarks using the new formulation
Strictly controlled experimental conditions are crucial to a successful interaction with the ridge skin secretions. First, it is necessary to lower the pH to a value of 2.65 to allow the gold nano particles to be deposited on the fingermark components. Moreover, it is necessary to add a surfactant, which greatly helps lower the non-selective deposition of gold nanoparticles on the support background. Schnetz recommends Tween 20. Its quantity must be controlled; as too much Tween 20 leads to a washing effect of the fingermark. A concentration of 1Â Î¼l of Tween 20 per ml of gold solution is the optimal quantity.
In the case of the Au-S-CD(AB25) formulation, a great attention has been paid to the stability of the resulting mixture. Several tests were performed to study the influence of the amount of Tween 20 as well as the pH value on the quality of the detection. The procedure which was finally obtained is the following: a sample is briefly rinsed into distilled water and immersed for 10-15Â min in the modified colloidal gold solution with a pH adjusted to 2.65 and that contains 5Â Î¼l of Tween 20 per ml of gold solution. The sample is finally rinsed in water to remove the non-deposited gold nano particles and dried in the air. As a result, the fingermarks may be observed under ambient light as dark-blue prints and present a good contrast. Thus, the resulting enhancement produces an image that can be directly used for identification.
Fig.5. Experimental procedure of the new proposed MMD formulation.
Fig. 6.Â Illustration of the contrast obtained after having converted in grayscale the picture of a fingermark obtained by immersion of a sample in the new colloidal gold formulation; support: LDPE.
We observed a good stability and efficiency of the solutions with all three CDs, observing no differences between them. This formulation was successful to detect fingermarks in one step for all surfaces: non-bleached paper, low-density polyethylene, and polypropylene. Moreover,
- The pH value of 2.65 remains the best value for a good deposition of the gold nanoparticles onto the fingermarks.
- We increased the quantity of Tween 20 to five times, as our colloidal gold is more concentrated in gold nano particles.
- It is recommended to fix the sample to be developed to the dish containing the colloidal gold solution, while shaking gently (50-100Â rpm for horizontal shakers).
- The same results were obtained regardless of the age of the latent fingermark (up to 6 months).
- Little or no coloration of the paper background was observed. The background darkening is a phenomenon due to the non-selective deposition of gold nanoparticles on the support. This is a minor phenomenon and it does not interfere with the observation of the fingermark since a good contrast is still obtained. However, the silver bath used in the standard MMD method enhances this effect and may cause some loss of contrast.
This research has led to two main improvements of the MMD method: (1) the reduction of the number of immersion baths from six to three (two rinsing baths and one containing the active solution) and, by the same occasion, the obtaining of a solution that efficiently develops latent fingermarks and (2) the modification of the surface of the gold nano particles without interfering with their ability to deposit on the ridge skin components.
The new formulation of the colloidal gold is obtained in three steps: (1) the thiolated cyclodextrins have to be synthesized, (2) a native solution of gold colloids has to be functionalized with those CDs, and (3) before use, a dye is added and the solution is mixed to let the dye be trapped in the CD cavities; then the pH is set to 2.65 and Tween 20 is added. The two first steps are the most time-consuming, because the syntheses require about 3-4 days and the functionalization of colloidal gold another 4 days. However, it does not constitute a drawback since these two steps can be performed well before the use of the new MMD formulation. Once synthesized, the thiolated cyclodextrins are under the form of powders which are dried and thus kept for future uses without any specific storage conditions. Moreover, the quantities that are synthesized in one batch are sufficient to prepare litres of colloidal gold. Then, once the colloidal gold has been functionalized by thiolated cyclodextrins, it is possible to keep the solution in the refrigerator for several months without observing any instability. Another time-consuming step is the solubilization of the dye, just before use. However, this time can be safely reduced. As a matter of fact, other authors reported inclusion times of 7Â min for other organic molecules.
The binding mode of the gold nano particles to the fingermark residues remains misunderstood, even if the electrostatic attraction is the most widely accepted theory. In the case of the modification of the gold nano particles, some citrate ions were logically moved or replaced by thiolated cyclodextrins. However, the nano particles still possess a global negative charge due to the remaining citrate ions at their surface. We can therefore postulate that the deposition is still driven by electrostatic attraction, and that the increased concentration in terms of gold nano particles may compensate the reduction of the negative charge.
While studying the possibility to introduce a luminescent tag in the CD cavity, no luminescent fingermarks were observed using dansyl chloride or 6-p-toluidinylnaphtalene-2-sulfonate, two luminescent molecules known for their affinity for CD cavities. After fluorometric studies, it was concluded that the gold nano particles played a role in this phenomenon since gold in aqueous solution acts as an acceptor of the energy emitted by the luminescent tag through a non-radiative pathway (induced, e.g., by the heavy atom effect). This phenomenon caused the quenching of the fluorescence of the luminescent marker. Further researches will have to be performed to circumvent this problem and to allow a luminescent detection of fingermarks, which should greatly enhance the contrast when working on patterned backgrounds.
This research aimed at modifying and, more specifically, simplifying, the standard MultiMetal Deposition technique as Single Multi Metal technique. Indeed, MMD constitutes a very powerful, efficient and sensitive technique, but is under-exploited due to the tediousness of its experimental procedure. The first step was to sharply reduce the number of immersion baths, so that the detection of fingermarks can be performed in a single step. To reach this goal, gold nano particles were functionalized with thiolated cyclodextrins. Once modified, the CDs offer the ability to be chemically bound to the gold surface and can moreover complex small organic molecules in solution, such as dyes, by trapping them in their cavity. In this study, it was shown that this strategy can successfully lead to the detection of fingermarks in only one immersion bath. The ridge skin appears in the form of dark-blue prints with sufficient contrast compared to the support (on which the gold nano particles do not deposit preferentially). Similarly to the traditional MMD, this technique is very efficient on various surfaces, i.e., porous and non-porous, and is not sensitive to the age of the fingermark, or if it has been previously wetted.
This strategy should be followed by considering gold not as a final component, but rather as a virgin surface on which several molecules can be grafted. In this contribution, molecular hosts, such as cyclodextrins, were used to trap dyes or tentatively luminescent tags, but it is also possible to use molecular linkers which can completely modify the way gold is interacting with the fingermarks. Future research will try to answer the following questions: (1) "Is it possible to propose this formulation as a replacement of the standard MMD or for use at crime scenes?" and (2) "At which level can this new formulation be put in sequence with the existing methods, e.g., physical developer, DFO, ninhydrin, and cyanoacrylate?". Moreover, on-going research is looking into the binding of luminescent biomolecules by following the same strategy.
Exploring its application in real time crime investigation.
Development of a fully automated software for standardizing finger prints.
Creating a standard library for choosing of dye for specific backgrounds.
Designing protocols in such a way that the luminescence property of gold is not interfering in the output detection process.