WO2017021933A1 - Procédé et kit pour l'échantillonnage micro-invasif d'articles - Google Patents

Procédé et kit pour l'échantillonnage micro-invasif d'articles Download PDF

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WO2017021933A1
WO2017021933A1 PCT/IB2016/054739 IB2016054739W WO2017021933A1 WO 2017021933 A1 WO2017021933 A1 WO 2017021933A1 IB 2016054739 W IB2016054739 W IB 2016054739W WO 2017021933 A1 WO2017021933 A1 WO 2017021933A1
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Prior art keywords
hydrogel
gel
kpa
kit
analysis
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PCT/IB2016/054739
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English (en)
Inventor
Armandodoriano Bianco
Marcella GUISO
Livia LOMBARDI
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Università Degli Studi Di Roma "La Sapienza"
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Publication of WO2017021933A1 publication Critical patent/WO2017021933A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N2001/022—Devices for withdrawing samples sampling for security purposes, e.g. contraband, warfare agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N2001/028—Sampling from a surface, swabbing, vaporising

Definitions

  • the invention relates to a method and kit for the microinvasive sampling of articles.
  • articles are meant any substrate which it is intended to sample, whether objects manufactured by man or present in nature.
  • the invention finds application in all those fields in which it is necessary to carry out the microsampling of organic or inorganic trace substances.
  • the substances are organic substances such as for example dyes, inks, traces of substances of biological origin, explosives or drugs which can be detected by means of analytical techniques suitable for the detection of trace substances such as for example SERS (Surface Enhanced Raman Spectroscopy) and mass spectrometry.
  • Fields of application are for example the field of diagnostics for works of art and archaeology, in which it is essential to sample the object in a non-invasive way, and in the forensic field, in which it is also extremely important to be able to analyse trace organic, inorganic or biological substances in a repeatable way so as not to destroy samples and not have an adverse effect on a scene of crime.
  • XRF is unable to detect signals from light atoms and therefore organic compounds
  • Raman spectroscopy has many limitations associated with the intense fluorescence of these compounds and the fact that they are generally present in very small concentrations
  • infrared spectroscopy is sensitive to interference from binding agents and fillers.
  • it is generally necessary to take relatively large samples (principal dimensions (0.5 - 2 mm), which are needed so that invasive and destructive analytical techniques such as for example HPLC (High Performance Liquid Chromatography) can be performed. Obviously this places a very great limitation on the number of analyses which it is possible to make on a work, not to mention the representativeness of the data obtained.
  • SERS is an advanced spectroscopic technique that has elicited enormous interest in the scientific community, specifically because it is able to overcome the limitations of "conventional" Raman spectroscopy described above.
  • the most widely used techniques are sampling using swabs or adhesive tape or by removing the portion affected by the presence of the trace material from the evidence and taking it away.
  • the trace material is then extracted from the swab or the material in which it has been found so that subsequent analytical investigations can be performed with a view to identifying the substance, or extracting DNA in the case of biological trace materials.
  • the object of the invention is to provide a kit containing what is needed for carrying out a microsampling protocol using a gel matrix which can be associated with a number of analytical techniques such as surface enhanced Raman spectroscopy (SERS), mass spectrometry, the extraction and analysis of nucleic acids or other analytical techniques suitable for the analysis of trace substances.
  • SERS surface enhanced Raman spectroscopy
  • mass spectrometry mass spectrometry
  • the contents of the kit make it possible for a number of samples to be obtained simply and quickly and the gel matrices containing these samples to be stored in an ordered manner and stably over time, thus also allowing analyses to be performed a long time after the time when they were sampled.
  • Another object is to develop a quick, effective and microinvasive method for sampling articles based on the kit according to the invention.
  • Figure 1 shows the UV-VIS absorption spectra of a sample of colloidal Ag in solution prepared according to the Lee-Meisel protocol, diluted 1:3 with ultrapure water, and the same sample after three months storage in the dark in a refrigerator at 4°C (spectral range 300 - 700 nm, scanning rate 200 nm/min).
  • Figure 3 shows a comparison between the ⁇ -Raman spectrum obtained by aiming directly into the layer of paint described as Purple Lake and the SERS spectrum obtained from the sample taken using Ag-gel matrix; both the spectra were obtained with a beam power of 10 mW.
  • Figure 4 shows the overlap between the Raman spectrum obtained by aiming a laser directly onto a banknote stained with Al neutralising ink and the SERS spectrum obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 5 shows the overlap obtained between the Raman spectrum obtained by aiming the laser directly at the banknote stained with A2 neutralising ink and the SERS spectrum obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 6 shows the overlap between the Raman spectrum obtained by aiming a laser directly onto a banknote stained with A3 neutralising ink and the SERS spectrum obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 7 shows the overlap between the Raman spectrum obtained by aiming a laser directly onto a banknote stained with A5e neutralising ink and the SERS spectrum obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 8 shows the SERS spectrum of A7 neutralising ink obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 9 shows the SERS spectrum of A8 neutralising ink obtained from analyses of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 10 shows the overlap between the Raman spectrum obtained by aiming a laser directly onto a banknote stained with A9e neutralising ink and the SERS spectrum obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 11 shows the overlap between the Raman spectrum obtained by aiming a laser directly onto a banknote stained with A10 neutralising ink and the SERS spectrum obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 12 shows the overlap between the Raman spectrum obtained by aiming a laser directly onto a banknote stained with Bl neutralising ink and the SERS spectrum obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 13 shows the overlap between the Raman spectrum obtained by aiming a laser directly onto a banknote stained with B2 neutralising ink and the SERS spectrum obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 14 shows the overlap between the Raman spectrum obtained by aiming a laser directly onto a banknote stained with B3 neutralising ink and the SERS spectrum obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 15 shows the overlap between the Raman spectrum obtained by aiming a laser directly onto a banknote stained with B4 neutralising ink and the SERS spectrum obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 16 shows the overlap between the Raman spectrum obtained by aiming a laser directly onto a banknote stained with B5 neutralising ink and the SERS spectrum obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 17 shows the overlap between the Raman spectrum obtained by aiming a laser directly onto a banknote stained with B6 neutralising ink and the SERS spectrum obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 18 shows the overlap between the Raman spectrum obtained by aiming a laser directly onto a banknote stained with B8e neutralising ink and the SERS spectrum obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 19 shows the overlap between the Raman spectrum obtained by aiming a laser directly onto a banknote stained with B9 neutralising ink and the SERS spectrum obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 20 shows the SERS spectrum of BIO neutralising ink obtained from analyses of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 21 shows the SERS spectrum of C l neutralising ink obtained from analyses of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 22 shows the SERS spectrum of Blue Victoria Base writing ink obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 23 shows the SERS spectrum of Blue Base writing ink obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 24 shows the SERS spectrum of Chrysodine writing ink obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 25 shows the SERS spectrum of Solvent Black 46 writing ink obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 26 shows the SERS spectrum of Rhodamine writing ink obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 27 shows the SERS spectrum of Methyl Violet writing ink obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 28 shows the SERS spectrum of food colouring E 102 (Tartrazine) obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 29 shows the SERS spectrum of food colouring E l 10 (Sunset Yellow) obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 30 shows the SERS spectrum of food colouring E 124 (Ponceau 4R) obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 31 shows the SERS spectrum of food colouring E 133 (Brilliant Blue FCF) obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 32 shows the overlap between the Raman spectrum obtained by aiming a laser directly onto a trace of HMX explosive (concentration 1 mg/ml) and the SERS spectrum obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 33 shows the overlap between the Raman spectrum obtained by aiming a laser directly onto a trace of PENTRITE explosive (concentration 1 mg/ml) and the SERS spectrum obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 34 shows the overlap between the Raman spectrum obtained by aiming a laser directly onto a trace of TNT explosive (concentration 1 mg/ml) and the SERS spectrum obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • Figure 35 shows the SERS spectrum of blood obtained from analysis of a microsample or microsamples obtained using the method and kit according to the invention.
  • This invention relates to a portable kit for carrying out a microsampling protocol for substances that have to be analysed.
  • the protocol is based on contact between a specific gelatinous rigid gel matrix and the substance or material which has to be sampled.
  • the material may be sampled either by direct contact with the water present in the aliquot of rigid gel or may be sampled by dissolving it in suitable organic solvents suitably placed on the surface of the gel, or sampling may take place by physical trapping within the gelatinous matrix.
  • the purpose of the microsampling is for subsequent analytical investigations to be performed.
  • Samplable substances may be of interest either in the field of diagnostics for works of art and archaeology, or in the forensic field. In particular this sampling and analysis procedure is particularly advantageous for identifying fluorescent substances which cannot be analysed using the standard Raman technique.
  • the rigid gels according to this invention may be obtained from a plurality of polymers by procedures known in the literature.
  • the physical and chemical properties of the polymers used in preparation of the matrix are chemical inertness, preferably non-toxicity and good solubility in water at ambient temperatures; polymers having a high gelling power and the ability to form rigid hydrogels in low concentrations are suitable.
  • a rigid gel is defined as a hydrogel, a material, that is, comprising a liquid, typically water, in which a hydrocolloid is dispersed, the latter being defined as a substance which holds the molecules in the liquid state immobile in a specific structure, in particular the structure acquired during the gelling stage.
  • the gel used must be insoluble in ordinary organic solvents and must be sufficiently rigid not to adopt the shape of the surface on which it is placed in contact and not to leave any residue.
  • the rigidity of the rigid gels to which the invention relates is measured using a standard penetrometer according to procedure ASTM D217, a method reported in the literature as being suitable for the measurement of gel rigidities (Walter J. Hamer, An Improved Method for Measurement of Gel Strength and Data on Starch Gels, U.S. Department of Commerce, National Bureau of Standards, Research Paper RP1810 Volume 39, 1947).
  • Rigidity is measured using a standard cone. The tip of the cone is placed in contact with the gel and the depth of penetration is achieved in 5 seconds and is measured in tenths of a millimetre.
  • the Penetration Number a value which expresses penetration within the material by the standard cone in tenths of a millimetre, used to define the rigidity of a material, is determined from this measurement. The higher the penetration number, the deeper the cone penetrates and therefore the less rigid the material.
  • the rigid gels according to the invention are characterised by a penetration number of between 140 and 380, preferably 200 - 300, and even more preferably 260 - 280. Gels having a penetration number higher than 380 cannot be used as they are not sufficiently rigid to be manipulated according to the method according to the invention and may leave residues on the surface sampled.
  • the rigid gels according to the invention are characterised by a elastic energy accumulation modulus within the range 80 - 250 kPa, preferably 100 - 200 kPa, more preferably 130 - 200 kPa, even more preferably 110 - 130 kPa, at a frequency of 1 Hz, measured using the DMA (Dynamic Mechanical Analysis) technique according to the experimental procedure described in "Development of Gellan Gum-Based Microparticles/Hydrogel Matrices for Application in the Intervertebral Disc Regeneration" [D. Ribeiro Pereira et al., Tissue Engineering: Part C, Volume 17, Number 10, (2011)].
  • DMA Dynamic Mechanical Analysis
  • the rigid gels according to the invention must also not be adhesive nor show any peeling effect, that is not be peelable, characteristics which could cause a visible quantity of material to be removed from the surface sampled, thus rendering the sampling no longer microinvasive.
  • the rigid gels should also not contain within them any substances which may potentially react with the substrate being sampled, such as for example catalysts and reaction initiators used in the preparation of many gels, as these could irreversibly and adversely affect the composition and optical characteristics of the surface sampled;
  • a gel which cannot be used is a polyacrylamide gel polymerised using catalysts and reaction initiators.
  • Other gels which cannot be used are gels that are soluble in organic solvents, such as for example cellulose acetate gel.
  • Preparation of the rigid gel is carried out by dispersing a quantity of polymer sufficient to obtain a matrix having a penetration number of between 140 and 380, preferably 200 - 300, even more preferably 260 - 280, in distilled water, preferably ultrapure water; the aqueous dispersion is heated to a temperature which is at least higher than the gelling temperature of the polymer.
  • the still hot solution is poured into moulds and allowed to cool in order to obtain portions of gel having the desired dimensions, or a film of the desired thickness, from the uniform and manipulatable surface.
  • the gel acts by direct contact with the surface being sampled and has the advantage that it is easy and safe to apply because the mechanical force exerted on the surface is limited to the dampening and absorption or incorporation of microfragments of the material being sampled.
  • the gel according to the invention may be one of the following: gellan, agar or agarose-based gel and substances capable of forming the rigid gels according to the invention.
  • Gellan a polysaccharide made of 6-glucose, a-rhamnose and 6- glucuronic acid units capable of forming transparent, thermo- reversible, viscoelastic and rigid gels, also known by the trade name Phytagel® manufactured by Sigma-Aldrich, is particularly preferred. All these properties render it particularly suitable for microsampling.
  • the rigid gel according to the invention may contain metal nan op articles, preferably of gold or silver or mixtures thereof, dispersed within it and is suitable for carrying out subsequent SERS analyses, constituting an active SERS substrate.
  • the purpose of the metal nanoparticles is to allow the gel to act not only as a support for microsampling but also as a substrate ready for the performance of SERS analyses.
  • One method for obtaining the matrix containing nanoparticles is that of mixing an aliquot of polymer in powder form with a colloidal dispersion of a metal.
  • the rigid gel is prepared by adding a quantity of polymer suitable for obtaining a matrix having a penetration number of between 140 and 380, preferably 200 - 300, even more preferably 260 - 280, to the colloidal dispersion, with constant stirring.
  • the dispersion so obtained is heated quickly through the use of microwaves, preferably at 350 W, for the time required to achieve a temperature at least higher than the gelling temperature of the polymer, preferably for 20 - 30 seconds, more preferably for 22 - 26 seconds.
  • the still hot suspension is poured into moulds and allowed to cool to obtain portions of gel of the desired dimensions or a film of the desired thickness, from the uniform and manipulable surface.
  • Rigid gels which cannot be used for the inclusion of nanoparticles are gels gelling in times of more than 30 seconds in microwaves at 350 W, as heating for longer times gives rise to deterioration of the active SERS substrate.
  • a colloidal dispersion of the metal preferably colloidal silver prepared according to the Lee-Meisel protocol (Lee P.C., Meisel D. Adsorption and surface-enhanced Raman of dyes on silver and gold, The Journal of Physical Chemistry 1982; 86 (17): 3391-3395 ) is preferably used in preparation of the matrix.
  • the concentration of nanoparticles in the colloidal dispersion used for preparation of the rigid gel suitable for SERS analysis is 0.11 g/1 (1.1 x 10 - 3 M).
  • the object of this invention is to provide the rigid gel ready for use together with a set of devices or tools required for performing the protocol described below, thus rendering the procedure particularly simple, quick and reproducible.
  • the kit makes it possible to conveniently carry everything necessary for performing the protocol in a case, so that microsampling can be carried out in situ, the samples can be placed in a dedicated container and once in the laboratory the samples obtained can be analysed, even after a long period of time.
  • the kit according to the invention contains materials and tools for the identification of trace compounds according to the method described below, and comprises the following elements:
  • one or more supports on which the gel can be left to dry for example a microscope slide.
  • the kit may further comprise:
  • one or more solvents which may not be necessary if the substances being sampled are water-soluble, as the gel is water-based; dispensers for the one or more solvents;
  • the rigid gelatinous matrix included in the kit is already ready for use and may be provided either as ahquots to be divided up, for example by coring, of dimensions 3 x 3 cm 2 and thickness approximately 1 cm, or in other elongated subdividable formats, preferably cylindrical or flat, packed in an enclosure which preserves them from drying out, for example rigid plastics capable of being inserted into the hand-held device.
  • Packaging in small portions is preferable as this minimises the waste of gelatinous material.
  • the gel may be provided with different contact areas according to specific needs (from 0.03 cm 2 to 0.15 cm 2 ).
  • the gelatinous matrix provided by the kit is packed in such a way that it is stored preventing drying out before use, or already dried and therefore requiring the hydration before use.
  • the gel may also be marketed in a dried form, as it is capable of being rehydrated cold in a short time merely by immersing the dried matrix in a suitable quantity of preferably ultrapure distilled water. Rehydration may be carried out within a container provided in the kit containing a predetermined quantity of preferably ultrapure distilled water.
  • the packaging can be constructed by means of techniques which are in themselves known.
  • the kit may contain one or more solvents, possibly mixed together, for wetting the rigid gel before it is placed in contact with the substrate on which the substance which is to be sampled is present.
  • the said solvent or solvents are preferably provided in containers with droplet counting dispensers.
  • the above solvents are generally organic solvents such as for example ethyl alcohol, acetone or methanol.
  • the support for the gelatinous matrix is in the form of a handheld device which has means for picking up and/or holding a portion of gel such that this portion can be placed in contact with the surface requiring analysis.
  • it may be in the form of a hollow pen or scalpel or brush or corer of the type used in dentistry or a mucotome.
  • the operator is helped in the operations of sampling the matrix, applying the solvent, keeping the surface under investigation in contact and placing the matrix containing the microsample or samples obtained on a slide and then in the sample holder.
  • the sample holder is provided with suitable compartments, preferably numbered, in which a plurality of finds sampled during one or more investigations can be housed.
  • the said sample holder may be made in such a way as to have compartments of suitable size to house the gel units used in microsampling.
  • the sampling holder acts to hold the portions of gel containing the microsamples in a fixed and ordered position.
  • Gloves for one or more operators involved in the operations envisaged in the protocol and instructions for proper use of the devices and tools included may also be provided in the kit.
  • the rigid gel matrix is provided in the kit ready for use or for rehydration in a blister for coring with a corer or for collection using a circular scalpel or in cyhndrical cartridges for insertion in a pen-shaped device.
  • the portion of gel that is to be used to perform the protocol may be picked up with or inserted in the hand-held support.
  • organic solvents these are deposited on the surface of the gel which will be placed in contact with the surface which has to be sampled.
  • the organic solvent can be used in extremely small quantities, below tenths of a ⁇ . Ethanol, methanol, acetone, hexane, ether and mixtures thereof may be used depending upon the nature of the samples which have to be sampled and according to the experience of those skilled in the art.
  • the kit according to the invention allows microsampling to be performed simply and safely, on both horizontal and vertical, rough or irregular or smooth surfaces.
  • the stage of microsampling the surface of the article requiring analysis according to the invention provides for placing the rigid gel matrix in contact with the surface of the object under investigation for a predetermined time, typically a minute or less, preferably within the range 1 - 60 seconds, more preferably within the range 1 - 30 seconds.
  • the time required for microsampling will depend on several factors, in particular the chemical nature of the compounds and their degree of ageing, but as anticipated above it is reasonable to maintain contact between the rigid gel matrix and the surface under examination for the determined time, typically a minute or less, more preferably within the range 1 - 30 seconds.
  • microsampling is complete contact between the portion of the matrix and the article is ended and the portion of gel containing the microsample or samples obtained is separated from the rest of the matrix.
  • the portion removed may be placed on one of the supports provided in the kit and allowed to dry in air. A check can be made that microsampling has taken place through using the portable optical microscope.
  • the drying stage is particularly advantageous in cases where the method according to the invention is intended for SERS analyses.
  • the metal nanoparticles dispersed in the gelatinous matrix have to aggregate. This aggregation takes place mechanically following dehydration of the gel.
  • the matrix containing the sample obtained is ready to undergo subsequent analytical investigations, preferably surface enhanced Raman spectroscopy and mass spectrometry analyses, extraction and analysis of nucleic acids or other analytical techniques appropriate for the analysis of trace substances.
  • the sample so prepared is stable over time.
  • SERS associated with the kit and the method according to the invention are speed of execution, the fact that it is a non-destructive technique and that it allows fluorescent organic compounds which cannot be observed using the "conventional" Raman technique to be identified.
  • the metal nanostructures optionally present within the rigid gel make it possible to amplify the Raman signal of the molecules of the substance under investigation, as a consequence of a plasmon resonance phenomenon. Thanks to the amplification obtained, which may vary by a factor of from 10 2 to 10 12 , the signals from fluorescent molecules present in trace quantities can be detected.
  • the sample can be re-extracted from the gel matrix to carry out other investigations using other analytical techniques, such as for example mass spectrometry, in order to confirm an identification or refine it. This is made possible because the gel matrix is wholly insoluble in common organic solvents.
  • the sampled material can be re- extracted from the gel so that subsequent analytical investigations can be carried out with a view to identifying genetic profile and the gel portion can be used for a SERS analysis, this technique being used for the unequivocal identification of gene sequences;
  • the gel according to the invention is a stable active SERS matrix that can be stored for long periods, unlike colloidal dispersions which are highly unstable as regards aggregation;
  • the gel which optionally contains metal nanop articles dispersed within it makes it possible for these to move closer together through mechanical action during drying out, this process being essential for the purpose of obtaining adequate signal amplification without any risk of aggregation of the nanoparticles;
  • the gel according to the invention is a multipurpose matrix, making it possible to use a single gel matrix for different purposes, and it is suitable for sampling substances of various kinds; in fact the same type of matrix according to the invention can be used to process both biological material and non-biological material without the need to use a different gel for each type of sampling or for each type of analysis which has to be performed subsequently;
  • All the solvents used are RPE (Sigma-Aldrich), analytical silver nitrate (Carlo Erba), sodium citrate (Sigma-Aldrich), gellan gum (Sigma-Aldrich), ultrapure water (Milli-Q).
  • the colloidal solution containing Ag nanoparticles used for preparation of the Ag-gel matrix was synthesised according to the reduction method developed by Lee e Meisel.
  • the solution was kept boiling for 60 minutes in a flask fitted with a reflux condenser to keep the volume of liquid constant.
  • the flask was placed in an ice bath to stop the reduction reaction and obtain a wider distribution of nan op article sizes.
  • colloidal dispersion had the typical yellow-green colour.
  • the nanoparticle concentration was 0.11 g/L (1.1 x 10 - 3 M).
  • the Ag colloids were then stored in a refrigerator at a temperature of +4°C and kept away from sources of light to prevent aggregation and oxidation.
  • the colloid Before use for preparation of the matrix the colloid underwent dialysis for 6 hours in order to remove salts present in the colloidal solution.
  • the prepared colloid was characterised using UV-VIS absorption spectroscopy, using a JASCO UV/VIS v530 spectrophotometer.
  • Figure 1 shows the UV-VIS absorption spectra obtained from a sample of the prepared colloidal solution, diluted 1:3 with ultrapure water. The spectrum obtained using the same procedure after three months' storage in a refrigerator is also shown for the second solution, from which the increase in maximum absorption wavelength due to increase in the average dimensions of the nanoparticles linked to aggregation can be seen.
  • the Ag colloids prepared according to the Lee-Meisel protocol have an optical absorption of between 300 and 700 nm, with a maximum absorption peak generally located around 420 nm, so the colloids prepared are therefore in line with those reported in the literature.
  • the matrices were kept in a cool dry environment, protected from dust and external agents, and were usable for microsampling for approximately the next 8 hours. If kept in well-sealed containers the Ag-gel matrices can be used for a longer time, up to three/four days.
  • the gel obtained had an accumulated elastic energy modulus of approximately 120 ⁇ 13 kPa at a frequency of 1 Hz.
  • the rigidity of the Ag-gel matrix so obtained was measured using a standard penetrometer in accordance with procedure ASTM D217, the method reported in the literature as being suitable for the measurement of gel rigidity (Walter J. Hamer, An Improved Method for Measurement of Gel Strength and Data on Starch Gels, U. S. Department of Commerce, National Bureau of Standards, Research Paper RP1810 Volume 39, 1947). Rigidity was measured using a standard cone. The tip of the cone was placed in contact with the gel and the penetration depth was reached in 5 seconds and expressed as tenths of a millimetre. The penetration number for the Ag-gel matrix, which proved to be 275 ⁇ 5, was determined from this measurement. Microsampling
  • Microsampling was carried out by taking up the Ag-gel matrix in a microcorer (disposable biopsy punch, Kai Medical) of diameter 0.4 cm. The terminal portion of the Ag-gel matrix intended to be placed in contact with the surface to be sampled was then moistened with 10 ⁇ of ethanol and placed on the surface to be sampled.
  • the matrix was held in contact with each sample for 30 s, then the portion placed in contact with the article containing the sampled microsamples was separated from the rest of the Ag-gel matrix using a scalpel and placed on a slide. The portion of Ag-gel matrix containing the microsamples was then observed under the optical microscope in order to ensure that sampling had occurred. The Ag-gel portions were kept in a cool dry environment until they were completely dried out.
  • This stage is of fundamental importance, as contraction of the gel due to loss of water present in the structure causes the silver nanoparticles to move close together.
  • Figure 2 shows a comparison between the ⁇ -Raman spectrum obtained by aiming directly at the layer of paint indicated as Madder Lake and the SERS spectrum obtained from the sample obtained using Ag-gel matrix.
  • the peaks marked with (*), which appear to be strongly amplified in the SERS spectrum, may be due to dyes belonging to the neoflavonoid family, such as brasilein and haematein, the main constituents of Brazil wood and logwood, two organic dyes which are readily available and widely used in the preparation of red lakes, the chemical structures of which are provided here:
  • Figure 3 shows another comparison example between the ⁇ -Raman spectrum obtained by aiming directly at the paint layer indicated as Purple Lake, and the SERS spectrum obtained from the sample obtained using Ag-gel matrix.
  • the peaks marked with (*) may be attributed to the synthetic purple dye PV2, which belongs to the xanthenes family.
  • Peaks attributed to the synthetic purple dye PV 19(v), belonging to the family of quinacridones, and tetrachlorothioindigo PR 88 (r) can also be recognised.
  • Neutralising inks are complex mixtures of pigments (organic and inorganic) and dyes used in Italy and in Europe to neutralise banknotes on the occasion of thefts or assaults on armoured vans, ATMs or ADM (Automatic Deposit Machines).
  • the first provides for removing a portion of the banknote in question of dimensions 1 mm x 1 cm affected by the stain and extracting the dyes using organic solvents so that they can subsequently be analysed by SERS in liquid. Otherwise it is possible to deposit the SERS-active substrate, such as for example colloidal silver, directly on the banknote and record the SERS spectrum. However it is inevitable that in both cases the evidence will be adversely affected and as a consequence the judge and the parties involved must be informed in order for the analyses to be carried out. For this a microsampling system associated with SERS analysis such as that to which this invention relates has elicited appreciable interest in the analysis of this particular type of evidence. In fact the method and kit described make it possible for sampling to be carried out microinvasively without giving rise to any deterioration of the sample and thus speeding up the investigative process and above all making it possible for analyses to be carried out in secrecy.
  • test banknotes were stained with different types of inks used to neutralise banknotes in Italy and Europe.
  • the stained banknotes were then microsampled using Ag-gel matrix.
  • the matrix was cored using a microcorer (disposable biopsy punch, Kai Medical) of diameter 0.4 cm.
  • the terminal portion of the Ag-gel matrix intended to be placed in contact with the surface to be sampled was then moistened with 5 ⁇ of methanol and placed on the surface of the banknote in the location of the ink stain.
  • the matrix was kept in contact with each sample for 30 s. Then the portion placed in contact with the article containing the sampled microsamples was separated from the rest of the Ag-gel matrix using a scalpel and placed on a slide and observed under the optical microscope in order to check that sampling had occurred. It was thus possible to observe the existence of a methanol-soluble fraction which was absorbed by the Ag-gel matrix and an insoluble portion which was visible under the microscope in the form of microfragments trapped in the gel. The Ag-gel portions containing the microsamples were kept in a cool dry environment until they had completely dried out.
  • the stained banknotes were also analysed by conventional Raman spectroscopy directly from the banknote.
  • the overlap between the Raman spectrum obtained by aiming the laser directly onto the stained banknote and that which can be obtained from analysis of the microsamples obtained using the method and kit according to the invention for each security ink analysed is described below (from Figure 4 to Figure 22).
  • the analyses were performed using a micro-Raman Thermo Scientific DXR spectrometer fitted with a 780 nm laser and the measurements were made under the following operating conditions: x20 microscope objective, beam power 5 mW, exposure 1 s and 40 accumulations. The spectra were not subjected to any processing or change of scale.
  • inks in particular were selected: Victoria Blue Base, Basic Blue 7, Chrysodine, Solvent Black 46, Rhodamine, Methyl Violet Base.
  • Rhodamine eibyZ Violet B The inked lines were microsampled using Ag-gel matrix.
  • the matrix was cored using a microcorer (disposable biopsy punch, Kai Medical) of diameter 0.4 cm.
  • the terminal portion of the Ag-gel matrix intended to be placed into contact with the surface to be sampled was then moistened with 5 ⁇ of methanol and placed on the inked line which was to be sampled.
  • the matrix was kept in contact with each sample for 30 s, and the portion placed in contact with the article containing the microsamples obtained was separated from the rest of the matrix using a scalpel and placed on a slide. The portions containing the microsamples were then observed under the optical microscope in order to check that sampling had occurred. The Ag-gel portions were kept in a cool dry environment until they had completely dried out.
  • the analyses were performed using a micro-Raman Thermo Scientific DXR spectrometer fitted with a 780 nm laser and the measurements were carried out under the following operating conditions: x20 microscope objective, beam power 10 mW, exposure 1 s and 40 accumulations.
  • the applicability of the Ag-gel matrix to the sampling and analysis of food colouring traces was evaluated.
  • four of the most widely used food colourings (E102, El 10, E124, E 133) were dissolved in water in a concentration of 1 mM.
  • each solution 10 ⁇ ⁇ of each solution were deposited on microscope slides and allowed to dry in air. Once dry, each trace was sampled using the Ag- gel matrix without using microsampling solvents, as these are water- soluble substances.
  • the matrix was cored using a microcorer (disposable biopsy punch, Kai Medical) of diameter 0.4 cm and kept in contact with each trace of dye for 10 s, then the portion placed in contact with the article containing the samples obtained was separated from the rest of the Ag- gel matrix using a scalpel and placed on a slide.
  • the Ag-gel portions were kept in a cool dry environment until they were completely dry.
  • the analyses were performed using a micro-Raman Thermo Scientific DXR spectrometer fitted with a 780 nm laser and the measurements were made under the following operating conditions: x20 microscope objective, beam power 10 mW, exposure 1 s and 40 accumulations. The spectra illustrated did not undergo any processing or change of scale.
  • HMX Three explosives (HMX, pentrite and TNT) were selected for this purpose and these were dissolved in methanol in a concentration of 1 mg/mL.
  • the matrix was cored using a microcorer (disposable biopsy punch, Kai Medical) of diameter 0.4 cm and kept in contact with each trace of dye for 10 s, then the portion placed in contact with the article containing the samples obtained was separated from the rest of the Ag- gel matrix using a scalpel and placed on a slide.
  • the Ag-gel portions were kept in a cool dry environment until they were completely dry.
  • the analyses were performed using a micro-Raman Thermo Scientific DXR spectrometer fitted with a 780 nm laser and the measurements were made under the following operating conditions: x20 microscope objective, beam power 10 mW, exposure 1 s and 40 accumulations.
  • the analyses were performed using a micro-Raman Thermo Scientific DXR spectrometer fitted with a 780 nm laser and the measurements were made under the following operating conditions: x20 microscope objective, beam power 10 mW, exposure 1 s and 40 accumulations.

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Abstract

La présente invention concerne un procédé de micro-échantillonnage de substances à l'état de trace à partir d'articles en vue de réaliser des examens analytiques ultérieurs. La procédure est basée sur l'utilisation d'un kit contenant les outils nécessaires pour la réalisation d'échantillonnage micro-invasif à l'aide d'une matrice de gel rigide qui, lorsqu'elle est placée en contact avec la surface de l'article qui doit être échantillonné, permet d'échantillonner des parties extrêmement petites de matière. Au moyen de ce kit, il est par conséquent possible de transporter tout ce qui est nécessaire afin de réaliser le protocole mentionné ci-dessus in situ de manière commode, de stocker les échantillons de manière stable et, une fois dans un laboratoire, d'analyser les échantillons obtenus, même après une certaine période de temps.
PCT/IB2016/054739 2015-08-05 2016-08-05 Procédé et kit pour l'échantillonnage micro-invasif d'articles WO2017021933A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5163441A (en) * 1988-06-09 1992-11-17 Becton, Dickinson And Company Polyurethane biological sample collection and transport device and its use
US20110256531A1 (en) * 2008-09-30 2011-10-20 3M Innovative Properties Company Biodetection articles
US20120088233A1 (en) * 2010-10-12 2012-04-12 Life Technologies Corporation Method of Preparing Quality Control Material for FFPE
US20120329081A1 (en) * 2008-05-13 2012-12-27 Nerys Bennion Sampling devices and methods of use
WO2015026969A1 (fr) * 2013-08-23 2015-02-26 Elwha Llc Système, méthodes et dispositifs d'évaluation des microbiotes de la peau

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5163441A (en) * 1988-06-09 1992-11-17 Becton, Dickinson And Company Polyurethane biological sample collection and transport device and its use
US20120329081A1 (en) * 2008-05-13 2012-12-27 Nerys Bennion Sampling devices and methods of use
US20110256531A1 (en) * 2008-09-30 2011-10-20 3M Innovative Properties Company Biodetection articles
US20120088233A1 (en) * 2010-10-12 2012-04-12 Life Technologies Corporation Method of Preparing Quality Control Material for FFPE
WO2015026969A1 (fr) * 2013-08-23 2015-02-26 Elwha Llc Système, méthodes et dispositifs d'évaluation des microbiotes de la peau

Non-Patent Citations (1)

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Title
FUHRER ROLAND ET AL: "Crosslinking metal nanoparticles into the polymer backbone of hydrogels enables preparation of soft, magnetic field-driven actuators with muscle-like flexibility", SMALL, WILEY - VCH VERLAG GMBH & CO. KGAA, DE, vol. 5, no. 3, 1 March 2009 (2009-03-01), pages 383 - 388, XP009145959, ISSN: 1613-6810, DOI: 10.1002/SMLL.200801091 *

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