RU2417749C2 - Mass spectrometry method of imprint analysis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine. A matrix mass spectrometry technique is used to detect the presence of a residue in an imprint. A powder containing hydrophobic particles of silicon dioxide, as well as a metal, metal nitride, a metal oxide or carbon is applied on the imprint. An average particle diametre of said metal, metal nitride, metal oxide or carbon introduced in the hydrophobic particles of silicon dioxide is 65 to 90 mcm or 400 to 500 nm preferentially. The investigated imprint is lifted from the application area with using a lifting tape and brought in contact with a sample substrate of a mass spectrometre, then the powder is applied on the imprint. The powder is magnetic or paramagnetic and additionally contains a fluorescent or pigmented dye molecule. The mass spectrometry is conducted with using a matrix specified in a group including MALDI-TOF-MS-MS or SALDI-TOF-MS-MS and their combinations. The residue represents an endogenous residue, particularly endogenous and/or exogenous metabolites and a contact residue. The endogenous metabolite represents squalene, while the exogenous metabolite is nicotine metabolite, e.g. cotinine, and the contact residue is the drug cocaine.

EFFECT: method allows executing the imprint residue analysis on a human imprint directly that eliminates it to be replaced.

21 cl, 14 dwg

Description

Technical field

The present invention relates to a method for determining the presence of a residue in a fingerprint using a mass spectrometry technique.

State of the art

Latent prints contain various compounds, such as natural bodily compounds, for example, cholesterol, squalene and fatty acids [1-3], or compounds that can remain in a latent print due to contact, for example, with cocaine or other drugs. To study such residues, Raman spectroscopy has been used up to now [4, 5]. When conducting such analyzes, there is a difficulty associated with the difficulty of visualizing the localization of drugs for analysis, and this method has low sensitivity and is relatively non-specific. The most common way to analyze latent fingerprints is by gas chromatography - mass spectrometry (GC-MS). It has already been shown previously that residues from latent prints can be extracted into a solvent and analyzed using GC-MS [2, 3]. Such compounds include squalene and cholesterol, but their levels in latent imprints vary, not only between subjects (individuals), but also in time for the same subject [3]. GC-MS is also already used to detect contact residues, such as cocaine from enriched fingerprints, with a detection limit of about 300 μg [6], and to detect drugs and metabolites from serial sweat stains, to nanogram levels per sweat stain [7], and from saliva [8]. However, all of the above methods require the use of complex extraction procedures prior to analysis.

Laser desorption / ionization using a time-of-flight mass spectrometry (MALDI-TOF-MS) was developed in the late 1980s by researchers at Karas and Hillenkamp, and began to be used as a technique for analyzing and determining the exact molecular weight of large macromolecules, such as proteins, polysaccharides , nucleic acids and synthetic polymers, with high accuracy of mass measurement and with very high sensitivity. MALDI is a “soft” ionization process that creates minimal fragmentation, and in which the energy from the laser is spent more on the evaporation of the matrix, rather than the destruction of the macromolecule. However, MALDI-TOF-MS was not previously used to identify residues present in latent prints. MALDI-TOF-MS is also carried out using surface laser desorption / ionization (SALDI), when graphite, titanium dioxide, or silicon dioxide are used as suspension matrices for MALDI [9, 11].

SUMMARY OF THE INVENTION

In accordance with the present invention, various materials are provided that can be used in the detection and / or imaging of prints. These materials can typically also be used as matrix agents in various mass spectrometry techniques. Thus, these materials have special properties that allow them to be used as “dual-use” materials.

In accordance with a first aspect of the present invention, there is provided a method for determining the presence of a residue in a fingerprint, said method including the following operations:

i) applying to the fingerprint a powder containing material that is capable of (1) acting as a matrix agent or material in a mass spectrometry technique using a matrix; and (2) to facilitate the detection and / or visualization of the print in order to form a print with particles deposited thereon; and then

ii) mass spectrometry of the material forming the imprint with particles deposited thereon to detect the presence or absence of a residue.

In accordance with one of the embodiments, the method involves the use of materials such as, for example, metals, metal oxides, metal nitrides and carbon, which can be used (1) as agents for imaging prints, alone or in combination with a carrier or when introduced into a carrier, for example, a carrier of silicon dioxide, and (2) as a matrix for fingerprint analysis using a mass spectrometry technique using a matrix. The mass spectrometry technique is used to determine the presence or absence of substances such as one or more endogenous compounds or metabolites, exogenous compounds or metabolites and / or contact residues that the fingerprint contains. According to one embodiment, the mass spectrometry technique is selected from the group consisting of (1) MALDI-TOF-MS and (2) SALDI-TOF-MS and (3) combinations thereof.

The imprint to which the proposed method is applied may be an imprint raised from the surface using a lifting tape.

According to one embodiment, the powder is hydrophobic in order to facilitate the application of the powder to the fingerprint and the contact of the powder with it.

It should be borne in mind that the term "fingerprint" includes a partial fingerprint and / or fingerprint, and not the other part of the body, and, for example, the portion of the fingerprint on which the powder was applied, can be subjected to mass spectrometry. Typically, an imprint is raised from the underlying surface before mass spectrometry, the term “imprint” includes such elevated imprints. In accordance with various embodiments, the imprint is raised prior to application of the powder. The present invention includes methods in which step ii) involves mass spectrometry of a powder that has an acquired fingerprint analyte. The present invention also includes methods in which step (ii) involves performing mass spectrometry of both the print material and the powder.

It should be borne in mind that the terms "sample" and / or "analyte" in the context of the present invention may mean a fingerprint, a sample taken from a fingerprint, and / or the remainder present on the fingerprint or contained in the fingerprint.

In accordance with some of the methods provided herein, the fingerprint is lifted from the surface and powder is applied thereon (processing agent), before or after lifting, and then the raised fingerprint (at least the material contained in the fingerprint) is placed in a mass spectrometry device. In accordance with other methods, the imprint is made (applied) directly to the sample substrate, and, after applying the processing agent to the print, the sample substrate is placed in a mass spectrometry device.

According to one embodiment, the method further comprises localizing and / or visualizing the fingerprint and analyzing the fingerprint using the mass spectrometry technique described above, for example, MALDI-TOF-MS and / or SALDI-TOF-MS.

In accordance with a first aspect of the present invention, there is provided a method for determining the presence of a residue in a print located on a surface, said method including the following operations:

i) applying to the fingerprint a processing agent, for example, a powder containing a material selected from the group consisting of particles of metal, metal oxide, metal nitride, carbon, and combinations thereof; and

ii) mass spectrometry of the fingerprint to detect the presence or absence of the specified residue.

As already mentioned above, the present invention relates to detection, which possibly includes the quantification of residues in prints. The term "residue" refers to any material that you want to detect, and in particular, refers to pre-selected compounds. The remainder is contained or may be contained in the print, so that the material that forms the print contains or may contain a residue.

According to one embodiment, the powder is hydrophobic. In accordance with one class of methods, the powder can be used in conjunction with other matrix agents and / or with other fingerprint detection agents.

In accordance with the present invention, a method is provided which comprises detecting (1) endogenously produced substance, for example, proteins, lipids, DNA, peptides and / or endogenously obtained metabolites, which are present as a residue contained in a fingerprint; (2) an exogenous compound or metabolite that is present as a residue contained in the print; and / or (3) a contact residue that is present on the print or in the print. Examples of types of residues are discussed below, and they include, for example (1) squalene and cholesterol; (2) cocaine and its metabolites and nicotine and its metabolite; and (3) ballistic residues, for example, from firearms and / or explosives, and residues from drugs, for example cocaine.

According to one embodiment, the method also allows contact residues that have been deposited on the surface together with endogenously produced compounds (i.e., endogenous metabolites and / or exogenous metabolites) to be detected.

The method described here typically does not require complex extraction procedures prior to analysis, as previously known methods, and also provides lower detection thresholds.

In one class of methods, they seek to determine the presence or absence of a given substance. In this case, examine the mass spectrum for the presence of one or more peaks associated with a known substance. In another class of methods, one seeks to identify one or more substances in a fingerprint by comparing peaks in the mass spectrum with a database or with a library of peaks. Both classes of methods can be implemented in combination with each other.

In accordance with one embodiment, the method comprises detecting and / or identifying a residue contained in an imprint that has been directly applied to a MALDI-TOF-MS or SALDI-TOF-MS sample substrate (plate) prior to application of the powder. According to an alternative embodiment, the method comprises detecting the presence or absence of a residue in a print that has been lifted from a surface using a lifting means, such as a lifting tape. The surface may be the place of deposition of the fingerprint, for example, at the crime scene. The method may include contacting the raised fingerprint with a MALDI-TOF-MS or SALDI-TOF-MS sample substrate, before applying the powder.

The method may alternatively include contacting the print with the powder previously applied to the print of a lifting means, such as a lifting tape. According to one embodiment, the surface is first sprinkled with powder to localize the latent imprint. Then a powdery imprint is lifted using a lifting tape. The imprint located on the lifting tape is then applied to the target plate (on the sample substrate) MALDI or SALDI, after which MS (mass spectrometric) analysis is carried out.

Typically, a MALDI-TOF-MS or SALDI-TOF-MS sample substrate is a plate, for example, a stainless steel plate, which is used in an MS system. The plate may have a recess or a plurality of recesses into which the sample is introduced (i.e., an imprint, for example, a raised imprint). In accordance with one of the embodiments, the prints are semi-solid deposits (sediment) on the sticky surface of the lifting tape.

The method can be used qualitatively to determine the presence or absence of a residue and / or quantitatively to determine the amount of residue. Moreover, in accordance with one of the embodiments, the method can be used to visualize or image the print. This imaging or imaging is important because it allows you to identify the "owner" of the print. To facilitate imaging of the print, the particles preferably contain a dye, for example a fluorescent or color (dyed) dye. Suitable dyes are known to those skilled in the art, the dye being, for example, rhodamine 6G.

According to an embodiment of the present invention, it is proposed to use a powder that contains metal, metal oxide and / or carbon to detect a residue or residues in a print using a matrix mass spectrometry technique.

In accordance with a first aspect of the present invention, it is proposed to use a mass spectrometry technique using a matrix to identify residues contained in a fingerprint. In particular, it is proposed to use the MALDI-TOF-MS and / or SALDI-TOF-MS technique to detect a residue in the print. The residue can be an endogenous residue (for example, an endogenous substance or metabolite, for example, it can be squalene, or an exogenous metabolite, for example a drug or drug metabolite), and / or a “contact” residue, for example, ballistic residues from explosives or firearms. According to one embodiment, the MALDI-TOF-MS technique and / or the SALDI-TOF-MS technique can be used using fingerprint imaging agents to detect (and especially visualize) fingerprints. Typical examples of imaging agents (imaging) include aluminum, Magneta Flake, and commercial white powder. According to one embodiment, suitable matrix agents are used in the MALDI-TOF-MS / SALDI-TOF-MS technique to facilitate the desorption / ionization process. Typical examples of matrix agents include 2,5-dihydroxy benzoic acid (DHB or DHBA) and α-cyano-4-hydroxycinnamic acid (α-CHCA).

According to an alternative embodiment, the use of MALDI-TOF-MS and / or SALDI-TOF-MS involves the use of a powder that contains a material selected from the group consisting of (1) metal; (2) metal oxide; (3) metal nitride; and (4) carbon, as a matrix agent. The powder may have the additional characteristics described here. The powder can typically also be used as an agent for detecting and / or imaging an imprint.

In accordance with one embodiment, the imprint has a residue contained therein. According to one embodiment, the powder may be used in combination with other matrix agents or materials (sometimes referred to as matrix support agents and / or matrix enhancement agents).

In accordance with one embodiment, use may further include identification of the residue.

DETAILED DESCRIPTION OF THE INVENTION

In the following description of the invention and in the claims, the words “includes” and “comprise” and their variations, for example, “comprising” and “including”, mean “includes, but are not limited to” and are not intended to limit ( and do not limit) the use of other parts, additives, components, integers or operations.

In the following description of the invention and in the claims, the singular includes the plural, unless otherwise specifically indicated. In particular, when the indefinite article is used in the English text, the description of the invention provides for the use of both the singular and the plural, unless otherwise specified in the context.

It should be borne in mind that the signs, integers, characteristics, compounds, chemical components or groups described in accordance with a specific aspect, option or example of the present invention are applicable in any other aspect, option or example described here, if there is no incompatibility with it .

In the description of the present invention, various aspects thereof are set forth in a specific sequence. However, it should be borne in mind that this sequence is provided for convenience only and is not restrictive. Thus, the description range includes all possible sub-ranges and individual numbers .. For example, a description in the range from 1 to 6 includes specific sub-ranges from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers in this range, for example, 1, 2, 3, 4, 5 and 6. This is applicable regardless of the width of the range. It should also be borne in mind that the ranges in the description include a specifically disclosed combination of endpoints.

Way

The present invention relates to the use of mass spectrometry in (in) analysis of the imprint residue. In particular, the present invention relates to the use of mass spectrometry techniques using a matrix, for example, MALDI-TOF-MS and / or SALDI-TOF-MS, in the analysis of imprint residues.

As already mentioned above, the term "residue" refers to any material that you want to detect, and in particular, to predefined compounds. The remainder is, or may be, within the print, that is, the material that forms the print contains or may contain a residue.

In accordance with one aspect of the present invention, there is provided a method for determining the presence of a residue on a fingerprint, the method comprising (i) applying a powder to the fingerprint, the powder having the following properties: (1) it is suitable for use as a matrix agent in the mass spectrometry technique using a matrix, and (2) it is suitable for use as an agent for detecting and / or imaging an imprint; and (ii) imprint mass spectrometry to detect the presence or absence of said residue.

Preferably, the powder is hydrophobic. The present invention also provides methods for analyzing a hydrophobic substrate using mass spectrometry using a matrix, characterized in that the matrix is hydrophobic. In accordance with one of the embodiments, the method provides, for example, before the operation (ii), the preparation of at least one calibration standard for use in the calibration of mass spectrometry techniques. According to one embodiment, the method comprises analyzing the results of operation (ii), for example, to determine that a specific residue (e.g. nicotine) is present in the fingerprint.

As already mentioned above, specialists will easily understand that the term "fingerprint" includes a partial fingerprint and / or fingerprints, not other parts of the body, and, for example, the area of the fingerprint on which the powder was applied can be used to the implementation of mass spectrometry. Typically, an imprint is raised from the surface prior to mass spectrometry, wherein the term “imprint” accordingly includes raised imprints. In accordance with embodiments, the imprint is raised prior to application of the powder. The present invention provides methods in which step ii) comprises selecting a powder for mass spectrometry. The present invention also provides methods in which step (ii) involves performing mass spectrometry of both the fingerprint and the powder. In one class of methods, operation (i) involves immersing an object on which there is or may be imprinted in a liquid medium containing powder. Diving time is not critical and can range from about 15 minutes to 12 hours or more. The fingerprint can then be lifted from the subject using a lifting tape.

It should be borne in mind that the terms "sample" and / or "analyte" in the context of the present invention may mean a fingerprint and / or a residue present on the fingerprint or contained in the fingerprint deposited directly or, alternatively, lifted from the surface using lifting means , for example, a lifting tape.

According to one embodiment, the mass spectrometry technique is selected from the group consisting of MALDI-TOF-MS and SALDI-TOF-MS. Briefly, MALDI-TOF-MS requires mixing samples with matrix molecules and applying matrix material to the sample. The MALDI target is introduced into the ion source of the mass spectrometer, which is under high vacuum. A strong electric field is created between the sample and the extraction plate (s). The laser beam is sent to the sample, resulting in desorption due to the absorption of laser energy by the molecules of the matrix.

Thus, in accordance with the present invention, methods are provided that use a material that can be used as matrix material in the MALDI-TOF-MS and / or SALDI-TOF-MS technique. Not wanting to be bound by any particular theory, one can still say that the powder contains material that can absorb laser energy and transmit it to the analyte contained in the sample. In accordance with the present invention, the analyte is typically a substance forming a residue or residues in a fingerprint. Energy transfer to the analyte leads to ionization of the analyte and to acceleration through the mass spectrometer. When MALDI-TOF-MS is used in this manner, that is, with ion transfer to a sample (analyte), this is called positive ion detection.

According to an embodiment, electron transfer from analyte to powder may occur. In this embodiment, MALDI-TOF-MS (or SALDI-TOF-MS) operates in negative ionization mode.

If it is believed that a sample (for example, a residue or a suspected residue on or in a fingerprint) has functional groups that easily accept a proton (H ⁺ ), then positive ion detection can be used. If it is believed that a sample (for example, a residue or a suspected residue on or in a fingerprint) has functional groups that easily lose a proton, then negative ion detection can be used.

The method allows to determine the presence of (i) endogenous residues, for example, endogenous metabolites and exogenous metabolites and (ii) contact residues contained in the print. Endogenous metabolites and “contact” residues can be co-precipitated in the print. The method may further comprise identifying the residue.

One class of methods can be used to detect and / or identify endogenous residues, for example, residues resulting from the metabolism of a substance by the human body. Endogenous residues may contain endogenous metabolites (e.g., metabolites of molecules produced by the human body) or exogenous metabolites (e.g., metabolites of molecules swallowed or transferred to the body and then metabolized by the body).

Other examples of endogenous residues that can be identified using the proposed method are, for example, endogenous substances (e.g. squalene, cholesterol, paraffins and esters, steroids, e.g. estrogens and testosterone, and sex and health markers) that can be secreted through the pores. skin and precipitate along with other chemicals in the print. The method can also be used to detect metabolites and conjugates of the above substances. Examples of endogenous residues are also exogenous metabolites, for example, a drug and its metabolites, and a drug and its metabolites, prescribed drugs and metabolites and compounds derived from food sources or their breakdown products. The method can also be used for proteomic (proteomic) or genomic analysis of cells (eg, skin cells) or DNA, respectively, located in the developed imprint. The method can also be used to detect other contact residues, such as drugs, explosive material, such as material used in the manufacture of bombs, and the remainder from the use of firearms.

In accordance with another aspect of the present invention, the use of the MALDI-TOF-MS and / or SALDI-TOF-MS technique for detecting and / or identifying a residue that is contained in a fingerprint is provided. The residue may be an endogenous residue and / or a “contact” residue, for example, ballistic residues, for example, from explosives or firearms. According to one embodiment, the residue may be a “contact” residue, that is, a residue that has been transferred to a person’s hand by contact with a substance and then transferred to the surface by fingerprint. The discovery of such residues is of particular interest to law enforcement agencies and can serve as critical evidence in a judicial review. According to one embodiment, a powder that contains a material selected from the group consisting of metal, metal oxide, carbon, and combinations thereof, is used to contact a fingerprint, and it acts as a matrix in a MALDI-TOF-MS device.

According to an alternative embodiment, the fingerprint may be contacted with a fingerprint developing agent that is used to detect fingerprints. Examples of suitable fingerprinting agents include aluminum, Magneta Flake, and commercial white powder. The method then typically uses a MALDI-TOF-MS matrix agent, for example, DHBA (2,5-dihydroxy benzoic acid) or α-cyano-4-hydroxycinnamic acid (α-CHCA), or another matrix agent, which may be, for example the powder described here.

The method in accordance with the present invention can be used to analyze various residues that can be found on the print. So, in accordance with one embodiment, the method can be used to develop and analyze latent prints left by smokers. It has been established that nicotine is highly metabolized to cotinine in vivo (in a living organism) [22], and it is known that both nicotine and cotinine are excreted together with sweat [8]. 11 and 12 show that the powder described here, and in particular hydrophobic particles of silicon dioxide, containing a substance selected from the group consisting of metal, metal nitrides, metal oxides and carbon, can be used as developers for visualizing latent fingerprints obtained from smokers, and such fingerprints can be analyzed using MALDI-TOF-MS, directly on a suitable surface or after lifting from the surface to detect nicotine and / or its metabolites.

In accordance with one embodiment, the method described herein can be used to detect or determine that a person has been involved in drugs or has consumed drugs, such as cocaine. In accordance with other embodiments, the method described herein can be used as part of a personal screening to determine that a person is, for example, a smoker or taking drugs, for example cocaine.

Although the method described here can be used to identify the presence of a specific residue on or in a person’s fingerprint, these results do not have the highest level of accuracy and therefore may not be acceptable in accordance with standards applied, for example, in criminal courts. In such a case, for example, when the method is carried out to obtain evidence for the court, it may further include tandem mass spectrometry, that is, the implementation of an additional mass spectrometry technique, typically to obtain structural information regarding the compound present in the residue in the print.

Thus, in accordance with one embodiment, the method comprises conducting MALDI-TOF-MS-MS and / or SALDI-TOF-MS-MS for a print and / or powder. When carrying out MALDI-TOF-MS-MS / SALDI-TOF-MS-MS, ions of a specific sample are typically fragmented inside the mass spectrometer and thereby provide additional structural information regarding the residue. Thus, in accordance with one of the embodiments, the method provides for the identification of the obtained fragment ions. This structural information can be used in some situations, for example, as evidence in court.

The method and uses described herein can be used in various applications. In accordance with one embodiment, the methods and uses may be used as part of a personal screening process for employees, for example, to determine if a person is smoking or taking drugs. In accordance with one embodiment, the methods and uses may be used as part of the “drug screening” process, for example, in professional or recreational sports.

In accordance with one embodiment, the method described herein can be used to detect contact residues or endogenous residues that contain prohibited substances and / or their metabolites. In particular, the method can be used to test athletes in the field of professional or amateur sports and / or in random tests for the presence or absence of prohibited substances in a residue taken from a person’s fingerprint. The advantage of the proposed method in comparison with the methods currently used is that the analysis is carried out directly on the fingerprint of a person and therefore the replacement of the sample is not possible, unlike the methods currently used in which urine and saliva samples are examined.

MALDI-TOF-MS and / or SALDI-TOF-MS devices have not previously been used to detect and / or identify a residue on a fingerprint, so using these techniques to detect and / or identify a residue on a fingerprint is part of the present invention. This use may include contacting the powder with the fingerprint, as already indicated above. Alternatively, use may include contacting a conventional detection agent with a fingerprint. In accordance with this embodiment, a matrix agent that is commonly used in mass spectrometry can additionally be used.

Alternatively, the previously described powder can be used as a matrix agent in mass spectrometry.

The methods and uses in accordance with the present invention can also be used at border points to check travelers for residues that may indicate drug use or handling (contact) with them, as well as contact with firearms and ammunition.

As already mentioned here above, the proposed methods can be used as evidence in court, for example, as evidence of contact with drugs or ammunition in a criminal court.

Powder

In accordance with the present invention, it is proposed to use a powder that is brought into contact with a fingerprint, which can then be used in mass spectrometry to determine the presence of a residue on the fingerprint.

The powder mainly contains a material selected from the group consisting of metal, metal nitride, metal oxide, carbon, and combinations thereof.

In accordance with one of the embodiments of the present invention, the powder may contain hydrophobic particles of silicon dioxide. One class of hydrophobic particles of silicon dioxide is obtained in accordance with the following method:

A method is proposed (designated method A) for the preparation of hydrophobic particles of silicon dioxide, which involves reacting in a single operation a mixture of (1) silane ester monomers, for example, alkoxysilane, and (2) organic substitution of silane ester monomers, for example, phenyl-modified silicate, with an agent hydrolysis, for example, with alkali.

Thus, the method typically involves the use of alkoxysilane monomers. The method may include the use of tetraalkoxysilanes (TAOS). TAOS is selected from TEOS (tetraethoxysilane) or TMOS (tetramethoxysilane).

According to one embodiment, the mixture further comprises a water miscible solvent, for example ethanol, as well as water. The method can be carried out at ambient temperature. The duration of the reaction is not critical. The reaction between TAOS monomers and PTEOS monomers can proceed overnight or for an equivalent period of time, which is approximately 12 to 18 hours. The reaction time affects the size of the obtained particles of silicon dioxide. It can be assumed that the sooner the reaction is stopped, the smaller the formed particles will be. Therefore, the reaction should be carried out for a period of less than 12 hours, for example, for a period of 6 to 12 hours. The reaction alternatively can last more than 18 hours. Optionally, the temperature can be increased (or decreased) and the reaction time reduced (or increased).

The hydrolysis agent, which is typically an alkali, acts as a catalyst during the reaction. Advantageously, this catalyst is a hydroxide, for example ammonium hydroxide. The catalyst instead may be acid. Inorganic acids, for example hydrochloric acid, can be cited as examples of suitable acids. In this method, the reaction is an acid-excited hydrolysis.

Silane ester monomer, for example TAOS, and organically substituted silane ester monomer, for example PTEOS, can be used, for example, in ratios (PTEOS: TAOS) of from 2: 1 to 1: 2, for example, from 4: 3 to 3: 4 , and, in particular, from 1.2: 1 to 1: 1.2. In one class of methods, the ratio is at least about 1: 1, for example, up to 1: 5, for example, 1: 2. In one class of methods, the PTEOS: TAOS ratio is preferably 1: 1 by volume. It should be borne in mind that when replacing one or both TAOS and PTEOS with other reagents, a similar relationship can be used.

The hydrophobic particles of silicon dioxide obtained by this method are mainly nanoparticles, that is, have an average diameter of approximately 200 nm to 900 nm, and typically approximately 300 nm to 800 nm, in particular 400 nm to 500 nm. These nanoparticles can then be processed to form microparticles that can be considered fused nanoparticles. Microparticles can be obtained using a method that, for example, includes the following operations:

i) centrifuging a suspension of particles;

ii) transferring a suspension of hydrophobic particles of silicon dioxide into the aqueous phase;

iii) extracting the suspension from the aqueous phase into the organic phase;

iv) evaporation of the organic phase; and

v) grinding and sieving the product obtained in operation (iv).

The organic phase is predominantly an organic solvent that is non-polar or has a low polarity. The organic phase may be dichloromethane or another organic solvent, for example alkanes such as hexane, toluene, ethyl acetate, chloroform and diethyl ether.

Alternatively, hydrophobic particles of silicon dioxide can be obtained from a reaction product containing hydrophobic nanoparticles of silicon dioxide, using a method that includes the following operations:

(a) centrifuging the reaction product;

(b) washing the reaction product with a liquid.

The method may include repeating steps (a) and (b) many times. Preferably, the liquid is a mixture of a solvent with water, and typically an aqueous-organic mixture of a solvent. Typically, the organic solvent is ethanol. Advantageously, the starting liquid contains a mixture of water and an organic solvent in a ratio of from 60 (water): 40 (solvent) to 40:60, by volume. In accordance with other embodiments, the solvent may be, for example, dimethylformamide, n-propanol or iso-propanol.

Typically, the proportion of solvent in the mixture is increased between the initial washing (b) and the final washing of the suspension. To obtain microparticles, which are fused nanoparticles, the prepared suspension is dried. Microparticles can then be sieved. After sieving, the microparticles are ready to be applied to the fingerprint as a developing agent, for example, in step (i) of the method proposed here, to detect a residue in the fingerprint.

Microparticles can be considered aggregates of smaller silicon dioxide nanoparticles. In accordance with this embodiment, the microparticles are of a sufficiently large size, allowing them to effectively capture using face masks and, therefore, not to swallow. Thus, in accordance with one embodiment, the silica microparticles have an average diameter of at least 10 μm, and typically at least 20 μm. Typically, the microparticles have an average diameter of about 30-90 microns. In accordance with some variants of implementation, the microparticles have an average diameter of about 45-65 microns or approximately from 65 to 90 microns. In particular, the powder containing microparticles may be a dry powder.

In one class of methods, the powder contains hydrophobic silica nanoparticles. Hydrophobic silica nanoparticles can be isolated using a method that involves centrifuging the reaction product from method A and suspending it in a mixture of water and a solvent. The mixture of water and solvent is the first mixture and is preferably a 50:50 mixture. The method may further include removing the reaction product from the first mixture of water and solvent, centrifuging it and suspending it in a second mixture of water and solvent. Advantageously, the second mixture of water and solvent has a proportion of solvent and water similar to that of the first mixture. The solvent in the mixture of water and the solvent is, for example, a water-miscible solvent, for example ethanol. Alternatively, dimethylformamide, n-propanol or iso-propanol may be used.

The operation of suspending the reaction product in a mixture of water and a solvent can be repeated many times. Advantageously, the composition of the mixture of water and the solvent is changed in order to increase the proportion of the solvent in the mixture during repeated suspending operations. Advantageously, the method comprises, in the final operation, suspending the reaction product in a mixture of water and a solvent that contains about 0% water and 100% solvent. The total number of suspension operations is typically from 3 to 10, for example, 4, 5, 6, 7, 8 or 9. Typically, after each suspension operation, in addition to the final suspension operation, the suspension is centrifuged. Nanoparticles can be stored in the final ethanol suspension. It should be borne in mind that centrifugation is only one exemplary method for separating nanoparticles from a mixture of water and solvent, so the use of other suitable nanoparticle isolation technologies is not ruled out.

According to one embodiment, the powder comprises hydrophobic silicon dioxide nanoparticles. One class of powder is a suspension of hydrophobic silica nanoparticles in a fluid. The fluid may be an ethanol aqueous suspension. Alternatively, other organic solvents, for example, dimethylformamide, n-propanol or iso-propanol, can be used in suspension in place of ethanol.

The physical nature and size of the particles forming the powder can be determined using SEM and TEM microscopes. Particles have the form of amorphous particles of silicon dioxide, which are usually used as a non-stick additive for various food products, as well as a non-stick additive and as a filler in pharmaceuticals in the preparation of various drugs and vitamins [12].

Thus, in accordance with a preferred embodiment, a powder that contains nanoparticles is applied to a fingerprint or surface in an appropriate liquid medium. Typically, the liquid medium is a mixture of an aqueous component with a solvent. The solvent may be a water miscible solvent. According to one embodiment, the water component is water. The solvent may be, for example, a water-miscible solvent, that is, a solvent 100% miscible with water in all proportions. In one embodiment, the solvent is ethanol. The ratio of water to solvent lies in the range of approximately from 99.9: 0.1 to 96: 4. The solvent content is preferably approximately not more than 4%, since higher solvent levels can lead to dissolution of prints or to a decrease in resolution. Preferably, at least trace amounts of solvent are used so that the nanoparticles remain discrete and do not stick together to form aggregates.

Alternatively, a powder containing hydrophobic particles of silica can be obtained using conventional techniques (see, for example, Tares et al., NanoSci. Nanotech. 2002. Vol.2. No.3 / 4 pp.405-409. ERMenzel, SM Savoy, SJUlvick, KH Cheng, RH Murdock and MR Sudduth, Photoluminescent Semiconductor Nanocrystals for Fingerprint Detection, Journal of Forensic Sciences (1999) 545-551; and ERMenzel, M. Takatsu, RH Murdock, K.Bouldin and KH Cheng, Photoluminescent CdS / Dendrimer Nanocomposites for Fingerprint Detection, Journal of Forensic Sciences (2000) 770-773).

According to one embodiment, the powder comprises hydrophobic silica particles into which a dye is incorporated. According to one embodiment, the dye that is incorporated into the particles is, for example, a color or fluorescent dye. Examples of suitable dyes include, but are not limited to, fluorescein derivatives, for example, Oregon Green, Tokyo Green, SNAFL and carboxy naphtho fluorescein, rhodamine (e.g. rhodamine B and rhodamine 6G) and their analogues, thiazole orange, oxazine perchlorate, methylene blue , base yellow 40, base red 28, as well as a violet crystal and its analogs. Not wanting to be bound by any specific scientific theory, it can still be assumed that dyes that are positively charged, for example, rhodamine, can be better incorporated into particles when using PTEOS in the process than dyes that contain an anionic or cationic group, such as carboxyl groups. As examples of other dyes that can be used in accordance with the present invention, dyes that have a planar aromatic substructure and positively charged functional groups (for example, ethidium bromide and other agents embedded in DNA) can be cited.

Particles can mainly be magnetic or paramagnetic. For example, magnetizable microparticles can be easily poured onto prints using a magnetic probe or other suitable tool. Thus, in accordance with a preferred embodiment of the present invention, magnetic or paramagnetic subparticles are introduced into hydrophobic particles of silicon dioxide. According to an embodiment of the present invention, the particles are magnetizable, for example, magnetic or paramagnetic. Magnetic or paramagnetic particles can contain any magnetic or paramagnetic component, for example, metals, metal nitrides, metal oxides and carbon. Iron may be given as examples of magnetic metals, while magnetites and hematite can be metal oxides.

In accordance with another preferred embodiment of the present invention, the carbon is carbon black, carbon nanoparticles, fullerene compound or graphite, as well as their analogues. A fullerene compound contains at least 60 carbon atoms (e.g., C ₆₀ ). Preferably, carbon has the form of carbon nanoparticles. Carbon nanoparticles can be in the form of, for example, carbon nanotubes (derivatives or non-derivatives). Carbon nanotubes can be multiple-walled or single-walled nanotubes.

According to one embodiment, the metal oxide is selected from the group consisting of titanium dioxide (TiO ₂ ), magnetite, hematite, and combinations thereof. In accordance with one embodiment, the metal is selected from the group consisting of aluminum, iron, and combinations thereof. However, it should be borne in mind that in alternative embodiments, those skilled in the art may use other metal oxides and / or metal nitrides that facilitate the desorption / ionization process of MALDI-TOF-MS and / or SALDI-TOF-MS. Similarly, those skilled in the art can use other metals and / or carbon forms in the powder that contribute to the ionization process. Metal, metal oxide, metal nitride or carbon may be incorporated into the powder particles. The powder particles preferably have an average diameter of ≤100 μm, for example a diameter of ≤1 μm. In accordance with one of the embodiments, the particles have an average diameter of approximately from 10 nm to 100 microns.

The hydrophobicity of the silica particles enhances the adhesion of the particles to the fingerprint. Thus, in accordance with one embodiment, metal, metal oxide and carbon are introduced and / or incorporated into hydrophobic particles of silicon dioxide.

In those methods that use an imprint, the powder and the imprint are brought into contact with each other. It should be borne in mind that the term "fingerprint" in this context can mean, for example, a fingerprint deposited on the surface, or, alternatively, an "indirect" fingerprint that has been lifted from the surface using a conventional lifting means, for example, using a lifting tape .

The application of the powder can be carried out using a magnetic probe, and in this embodiment, the powder is magnetic or paramagnetic. This is safer because it reduces the effect of the powder on a person who can inhale it. Alternatively, the imprinted object may be immersed in a liquid medium (i.e., a suspension of nanoparticles). Diving time is not critical and can range from about 15 minutes to 12 hours or more.

In accordance with one of the embodiments, the powder contains hydrophobic particles of silicon dioxide, which contain magnetizable particles, as mentioned above.

One class of powder contains hydrophobic particles of silicon dioxide. The particles may be nanoparticles or microparticles, or a combination thereof. According to one embodiment, the silica microparticles have an average diameter of at least 10 μm, and typically at least 20 μm. Typically, the microparticles have an average diameter of about 30-90 microns. In accordance with some variants of implementation, the microparticles have an average diameter of approximately from 45-65 microns to 65-90 microns.

It is believed that nanoparticles that have an average diameter of approximately 200 nm to 900 nm can be used in the proposed methods.

Preferably, the nanoparticles have an average diameter of approximately 400 to 500 nm. However, it is believed that a powder containing nanoparticles having an average diameter of approximately 200 nm to 900 nm, for example, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, can be used in the method proposed here. , 750, 800, 850 or 900 nm.

The term "average diameter" refers to the average diameter of particles typically formed by the methods in accordance with the present invention. The term "average" is a statistical term that is obtained by dividing the sum of all measured diameters by the number of particles used in such measurements. The diameters of nanoparticles are determined by SEM images using scales (rulers), and the diameters of microparticles are determined using a combination of sieve size, measurement results of particle size distribution and SEM images. One way to determine the average diameter is to determine using the Malvern Mastersizer (Malvern Instruments Ltd.).

According to one embodiment, the powder may comprise a mixture of (1) hydrophobic silica particles described hereinabove and (2) magnetic or paramagnetic particles, for example iron particles.

In accordance with one of the embodiments, the powder contains hydrophobic particles of silicon dioxide, which additionally contain a molecule that helps in imaging and / or imaging of the print. According to one embodiment, the hydrophobic silica particle comprises a dye molecule. Examples of dyes include rhodamine, for example, rhodamine 6G and its derivatives. The fingerprint visualization operation can be carried out using various known techniques. For example, optical methods can be used, including a scanner with a UV search beam, an optical scanner with a flat scan, a fluorescent scanner and a scanner in the UV and visible spectral regions.

Examples

Embodiments of the present invention will now be described by way of example with reference to the accompanying drawings.

Figure 1 shows the results of a fluorescence scan of prints deposited on a slide and lifted from it using rhodamine 6G (Rh.6G) as a contact agent (λex 543 nm, λem 590 nm).

On figa shows latent fingerprints on a microscope slide. Above and below prints with rhodamine 6G, in the center there is an empty control.

FIG. 1 b shows the fingerprints of FIG. 1 a shown using Sunderland White powder (hydrophobic silica particles with TiO ₂ incorporated therein).

On figs shows the remainder (traces) of the prints of fig.1b after lifting them using a commercially available tape for lifting prints.

On fig.1d shows the raised prints of fig.1b on a commercially available lifting tape.

Figure 2 shows the mass spectra of rhodamine 6G precipitated from ethanol (higher) and rhodamine 6G from a raised print (lower), both spectra being analyzed on a MALDI tape (bottom).

Figure 3 shows the response of the MALDI-TOP-MS system to silicon dioxide particles with carbon black introduced into them, in the presence and absence of cocaine hydrochloride.

Figure 4 shows a comparison of the matrix material for the detection of cocaine in enriched prints deposited directly on a MALDI-TOF-MS metal plate using 2,5-dihydroxy benzoic acid (DHB) at a concentration of 10 mg / ml, with the upper curve obtained from using magnetic hydrophobic particles of silicon dioxide.

Figure 4a compares matrix material for detecting cocaine in enriched prints deposited directly on a MALDI-TOF-MS metal plate. The lower curve was obtained using DHB at a concentration of 10 mg / ml, and the upper curve was obtained using magnetic hydrophobic particles of silicon dioxide.

Fig. 4b shows spectral intensities at m / z 304.5 for prints deposited on metal plates of targets and sprinkled with three powders in the presence and absence of cocaine.

Figure 5 shows the mass spectra of the raised latent prints sprinkled with hydrophobic particles of silicon dioxide with introduced TiO ₂ to detect contact with cocaine. Positive contact is shown on the upper curve, and lack of contact with cocaine is shown on the lower curve.

Figure 6 shows a photograph of a single recess / unit cell on a surface having 96 recesses of a MALDI plate located in a mass spectrometer, where you can see the protrusion part (light bars) of a raised print, previously sprinkled with hydrophobic particles of silicon dioxide with introduced TiO ₂ , up to analysis using MALDI-TOF-MS.

Figure 7 shows the relative intensities at m / z 433.55 (molecular ion for squalene plus sodium adduct) for the raised prints sprinkled with 10 compositions of hydrophobic particles (1-10), and for controls (11 and 12). No peaks were observed at m / z 386 due to cholesterol.

Fig. 8 shows the relative intensities at m / z 433.55 (squalene, weight of the formula 410.72, plus sodium adduct (22.99)) for squalene standards in the presence of eight hydrophobic powder formulations (1-8) and for two controls (9, 10).

Figure 9 shows the relative intensities at m / z 386.37 (weight of the formula 386.65 for cholesterol) for squalene cholesterol standards in the presence of eight hydrophobic powder formulations (1-8) and for two controls (9, 10).

Figure 10 (a) shows the mass spectra of MALDI-TOF-MS for the standard squalene.

Figure 10 (b) shows the mass spectra of MALDI-TOF-MS for cholesterol standards.

11 shows the results of the MALDI-TOF-MS analysis of latent fingerprints for a non-smoker (FR) on a stainless steel plate and latent fingerprints enriched with 10 μl of a solution containing 1 μg / ml of a mixture of nicotine (RMM 163) and cotinine (RRM 176 ) Upwards:

a) - enriched latent prints not sprinkled with hydrophobic particles or treated with DHB;

b) enriched latent fingerprints treated with DHB;

c) - enriched latent prints pre-sprinkled with hydrophobic particles;

d) - not enriched latent imprint, pre-sprinkled with hydrophobic particles.

12 shows MALDI-TOF-MS latent prints for a former smoker (24 hours after the last cigarette), on a stainless steel plate pre-sprinkled with hydrophobic particles:

a) m / z range 155-180, b) m / z range 180-205, c) m / z range 205-450.

13 shows MALDI-TOF-MS latent fingerprints for a smoker, sprinkled with powder. The peaks at 163, 148, 133, 119, 105, 91, 84 and 79 are characteristic of nicotine and serve as clear evidence of its presence.

On Fig shows MALDI-TOF-MS latent prints for the smoker, sprinkled with powder. The peaks at 177, 161, 147, 135, 119, 105, 97, 91 and 79 are characteristic of cotinine and serve as clear evidence of its presence.

Materials and methods

A Tecan LS300 scanner from Tecan UK., Reading, Berkshire, and the Kratos Axima-CFR MALDI-TOF-MS system (Shimadzu Biotech, Manchester UK) with Shimadzu target plates were used in this study. As a serial matrix, 2,5-dihydroxy benzoic acid (DHB) was used (10 mg / ml in a 50:50 solution of acetonitrile: deionized water [dH ₂ O]). The carbon black suspension was kindly provided by Cabot Corp., Cheshire UK. All other chemicals were purchased from Sigma-Aldrich, Dorset UK., Including anatase titanium dioxide. Serial dusting agents, finger brushes, magnetic probes and serial lifting tape were obtained from Crime Scene Investigation Equipment Ltd., 10 (formerly known as K9 Scenes of Crime Ltd.) Northampton, UK.

A solution was prepared for calibrating the MALDI-TOF-MS system. Solutions of papaverine hydrochloride (10 mg / ml in dH ₂ O) and reserpine (5 mg / ml in dimethylformamide, DMF) were prepared. An aliquot of the indicated papaverine hydrochloride solution (100 μl) was mixed with aliquots of the reserpine solution (200 μl) and dH ₂ O (400 μl) and DMF (300 μl). A solution of 2,5-dihydroxy benzoic acid (DHB, 10 mg / ml) in 50:50 by volume acetonitrile: water was also prepared. The final calibration solution was prepared by mixing 10 μl of each DHB solution and a mixture of reserpine and papaverine. Aliquots (1 μl) of this solution were used in each experiment, and m / z for the molecular ions of two standards were used to calibrate the system.

Preparation of hydrophobic particles of silicon dioxide with the material introduced into them

This method is an adaptation of the preparation of empty silica-based nanoparticles [12] and involves mixing 30 ml of ethanol, 5 ml of dH ₂ O, 2.5 ml of tetraethoxysilane and 2.5 ml of phenyltriethoxysilane in a centrifuge tube. To the resulting 2 ml of ammonium hydroxide solution (28%) was added to initiate the formation of nanoparticles and the solution was rotated overnight. The resulting suspension (suspension) of the powder is extracted repeatedly with a solution of methylene dichloride / water or ethanol / water (50:50 in both cases). The suspension is centrifuged (for example, 5 minutes at 3000 rpm). The supernatant was removed and 10 ml of dH ₂ O and the same volume of dichloromethane were added. The suspension is rotated an additional 10 minutes and then centrifuged again. The aqueous upper layer of the solution is removed and aliquots of water and dichloromethane are additionally added. This washing and centrifugation process is repeated 4 times until an additional addition of water is required: dichloromethane. After that, the particles are dried from dichloromethane in a thermostat at 40 ° C.

Dry particles are ground in a mortar with a pestle and sieved to the desired size. Hydrophobic particles are sieved manually through a bronze mesh brass test sieve (Endecot Ltd., London UK). The fraction of particles that are used in this test have a size of approximately below 63 microns. To determine the particle size distribution, a Malvern Mastersizer analyzer (Malvern Instruments Ltd., Malvern, UK) is used.

To obtain particles containing titanium dioxide, 25 mg of titanium dioxide is introduced into the centrifuge tube, after which silanization reagents are added. To obtain particles containing carbon black (CB), add 5 ml of a 1: 2-1: 100 solution of a suspension of carbon black in water in a precursor solution instead of TiO ₂ . To obtain magnetic particles, powder magnetite is used in accordance with the published method [13, 14], and then 5 ml of this suspension in water are introduced into the precursor solution instead of dH ₂ O.

Demonstration that contact residues are effectively raised from latent fingerprints

The fingertip was placed in a solution of rhodamine 6G (Rh 6G; 100 μl / ml in EtOH). A finger was moved (waved) to evaporate the excess EtOH before printing on a clean glass slide. Three prints were applied, 2 with Rh 6G and 1 blank control. The prints were examined using a Tecan LS300 scanner (λex 543 nm, λem 590 nm, gain 120). Then the prints were sprinkled with hydrophobic silicon dioxide microparticles, which contain titanium dioxide introduced into them, previously visualized under the same scanning conditions. After that, the prints were lifted using a serial lifting tape (11.5 × 6.5 cm), and then both the remainder on the slide and the lifting tape were scanned.

Experiment to demonstrate MALDI-TOF-MS detection of elevated contact residue (rhodamine 6G)

In this experiment, several grains of Rh powder. 6G was applied directly to the fingertip, previously printing on a glass slide, which was produced similarly to the previously described, but without the addition of a dusting agent (powder). The fingerprint was then raised using double-sided conductive tape and applied to a metal target. A conductive tape suitable for use with MALDI-TOF was kindly provided by Shimadzu Biotech. The target plate was then introduced into the MALDI-TOF-MS system and the imprint was examined for the presence of Rh. 6G. The obtained MSs with m / z detected for Rh 6G of about 444 were compared with a standard (0.1 μl of a solution of 100 μg / ml in ethanol) equivalent to 100 ng of dye deposited on a flat tape.

An experiment to demonstrate the effectiveness of silica particles with carbon black introduced into them as reinforcing agents for SALDI-TOF-MS

The response of the instrument to silicon dioxide particles with carbon black introduced into them was studied in the presence and absence of cocaine, which were introduced into circular cells / regions on the surface of the target metal plate. All results were obtained in triplicate. Cocaine hydrochloride (1 μg as 1 μl in 1 mg / ml ethanol solution)) was injected into 2 sets of recesses on the target. In a positive test, 1 μl of a 10 mg / ml suspension of nanoparticles with carbon black introduced into them (matrix) was introduced into the cell. In another control test, the response of the instrument to cocaine without a matrix was studied. In another control test, the response of the instrument to the matrix in the absence of cocaine was studied using empty cells of a metal target. The cells were dried as previously described before analysis.

Detection of contact residues of cocaine using MALDI-TOF-MS A small amount (several grains) of cocaine hydrochloride were applied to the fingertip when immersed in the drug. After that, the finger was brought into direct contact with a clean metal plate of the target, resulting in an imprint on the surface of the target, covering many cells (recesses) on the surface. Latent prints were then sprinkled with hydrophobic particles of silica with magnetite introduced into them, or sprinkled with serial dusting agents, which were aluminum powder, Magneta Flake ™ and white powder, possibly containing titanium dioxide. A serial magnetic probe was used to apply two magnetic powders and a serial brush was used to apply non-magnetic powders. A serial DHB matrix was used to prove the presence of cocaine on the fingerprint, 10 μl of 10 mg / ml was mixed with 10 μl of a 10 mg / ml cocaine hydrochloride solution, and 1 μl of this mixture was injected into the MALDI target cells. The metal target was then dried in a heater (Shimadzu / Kratos Instruments) and then introduced into the MS system.

An experiment to demonstrate the effectiveness of TiO ₂ / PTEOS particles for detecting cocaine residues in raised fingerprints

Blank and cocaine-containing prints were applied to slides as previously described. Fingerprints were developed using TiO ₂ / PTEOS particles applied with a Zephyr brush. The prints were then lifted using a serial lifting tape, which was turned over and placed upside down on the target plate of the MALDI system. The prints were then analyzed using a mass spectrometer (MS) for the presence or absence of cocaine.

Detection of cholesterol and squalene from fingerprints using the MALDI-TOF-MS system, using hydrophobic particles as an amplification matrix

The target metal plate was cleaned and dried. Then 12 fingerprints of the right index finger were applied to the target metal plate. The imprinted plate was then dried in a thermostat at 37 ° C for about 1.5 hours. Each imprint was then sprinkled with ten different compositions of hydrophobic particles prepared in accordance with those described above. The agent introduced into the particles is indicated below in parentheses. In addition, some of the obtained powders (1, 5, 6, and 8) were then thoroughly mixed with DHB to obtain 1% by weight of the compositions. They were used to determine that the presence of this matrix improves the detection of squalene in the latent imprint after sprinkling, when analyzed in the MALDI-TOF-MS system. Four compositions of black powder were used, which have different proportions of carbon black in them, obtained from the initial synthesis. The numbers in parentheses refer to the ratio of carbon black to dH ₂ O used in the initial synthesis:

1. White powder A (titanium dioxide); 1.0% by weight of DHB; powder)

2. White powder B (titanium dioxide)

3. Purple powder A (violet crystal)

4. Black Powder A (1:10)

5. Black powder B (1:10, 1.0% by weight of DHB; powder)

6. Purple powder B (violet crystal; 1.0% by weight of DHB; powder)

7. Red fluorescent powder A (rhodamine 6G)

8. Red fluorescent powder B (rhodamine 6G; 1.0% by weight of DHB; powder)

9. Black powder C (1: 5)

10. Black powder D (1: 2)

Each of the first ten prints was individually sprinkled with the corresponding powders 1-10 and the obtained prints were photographed using a digital camera (photographs not shown). The fingerprints were then individually analyzed in the MALDI-TOF-MS system, including a fingerprint that was not treated with a dusting agent or matrix (fingerprint 12), and a fingerprint treated with dihydroxy benzoic acid only (fingerprint 11).

Direct detection of cholesterol and squalene using the MALDI-TOF-MS system, using hydrophobic particles as reinforcing matrices

Solutions of cholesterol and squalene (both 2 mg / ml) were prepared in 100% ethanol and 500 μl aliquots of each solution were mixed with each other. 24 drops (each 0.5 μl) of this solution were applied to a clean, dry metal target and left to dry for 2 hours. Then, the depressions were individually sprinkled with each composition of eight hydrophobic powders (3 drops for each composition). The compositions were identical to those described in 2.8:

1. Red fluorescent powder A

2. Black powder A

3. Black powder B

4. Purple powder A

5. Purple powder B

6. Red fluorescent powder B

7. Black powder C

8. Black powder D

Powder sprinkling was carried out in accordance with the previously described here above, directly on the surface of the metal plate, each of the powders. A separate solution was also prepared by mixing 500 μl of a cholesterol / squalene solution with 500 μl of a solution of 10 mg / ml DHB (matrix). Three drops (0.5 μl each) of this solution were also applied to a metal target and left to air dry. Each drop (spot) on a metal target was analyzed using the MALDI-TOF-MS system and peaks of cholesterol (m / z 386) and squalene (m / z 410) and their metal adducts were recorded.

results

Demonstration that contact residues are effectively raised from latent fingerprints

We studied the effectiveness of the method of lifting the print and the details of the protrusion of the obtained prints. Rhodamine 6G was used as a model contact agent due to its high fluorescence. Thus, prints with high fluorescence were obtained after contact (upper and lower prints in Fig. 1a), while the normal print (in the center in Fig. 1a) is not fluorescence in the scanning modes used.

When hydrophobic particles of silicon dioxide were applied to three prints, they became visually clear (not shown), and now all three prints are clearly visible during fluorescence scanning (Fig.1b) due to fluorescence of rhodamine, as well as due to the scattering of light from silicon dioxide particles, printed on the print. After lifting the prints using a lifting tape, a small fluorescence is stored on the surface of the glass slide (figs), which indicates the effectiveness of the lifting method. Scanning the surface of the tape now reveals “fluorescent” prints, which means that the raised prints and dusting means remain intact during this process (Fig. 1d).

The results shown in FIG. 1 confirm that a serial lifting tape can be used to transfer the entire volume of deposited latent imprint material from a glass slide to the tape. This is the first step in detecting any material in a latent fingerprint using the MALDI-TOF-MS system.

MALDI-TOF-MS detection of elevated contact residue (rhodamine 6G)

Successful results from a previous experiment suggest that rhodamine raised from the surface of a microscope slide can be detected using the MALDI-TOF-MS system. The results are shown in figure 2. The upper curve corresponds to the elevated imprint and has a peak at m / z 443 corresponding to Rh 6G. The lower spectrum is obtained directly from the Rh 6G standard applied directly to the target of the MALDI system. Similar peak shapes suggest the presence of the same compound. The reduced mass on the upper curve is probably caused by the slight lifting of the tape used when applying the lifting tape to the target plate, which reduces the transit time of the ionized species.

The conductive tape should be well glued to the plane of the MALDI target, with the sample applied to the upper surface of the tape. This tape is relatively fragile and easily stretched, which can deform the original developed print. This tape can hardly be used to lift the print from a microscope slide and to apply it to the target of the MALDI system in the best way. It is not possible to align the tape on the surface due to its fragility and stickiness.

The results shown in FIG. 2 indicate that contact residues, in powder form, can be lifted from the surface and can be successfully analyzed using the MALDI-TOF-MS system.

Efficiency of using silica particles with carbon black introduced into them as reinforcing agents in the detection of cocaine using the MALDI-TOF-MS system

To demonstrate (verify) that the results were obtained due to the presence of the matrix and cocaine, and not the background signal, additional analyzes were performed using known amounts of cocaine and the matrix. The responses of magnetic particles of silicon dioxide in the presence and absence of cocaine were compared with the responses of an empty target and cocaine without any matrix.

We compared the responses at m / z 304 of silica particles with carbon black introduced into them, in the presence and absence of cocaine, with the responses of an empty target and cocaine without any DHB matrix. The intensities of the MS peaks due to cocaine are shown in FIG. The presence of particles of silicon dioxide leads to a 10-fold increase in the intensity of the peak compared to the peak for cocaine in the absence of these particles. Good reproducibility is observed (average intensity 90.275 and rsd 5.8%, n = 3) compared with the corresponding results in the presence of DHB (average intensity 56.552 and rsd 112%, n = 3).

The potential of the SALDI system using particles with carbon black introduced into them was investigated in comparison with a standard DHB matrix. Cocaine hydrochloride was used as a given analyte, since its identification on prints is of interest to law enforcement agencies, as it shows that the subject who left the printout had contact with cocaine. The mass spectra of cocaine in the presence of a standard chemical matrix (DHB) and a new powder matrix are shown in figure 4, for prints deposited directly on a metal target of the MALDI system. The lower spectrum corresponds to cocaine in the presence of DHB, while the upper spectrum corresponds to cocaine in the presence of magnetic PTEOS nanoparticles deposited with a magnetic probe. Both spectra are similar and correspond to the spectrum of cocaine, but the response is clearly increased in the presence of magnetic particles. This suggests that magnetic PTEOS particles contribute to the process of desorption of cocaine similarly to the standard matrix. Magnetic PTEOS particles also have the potential to be used as a powder for imaging a print, since a good protrusion is visible on the developed prints (not shown).

Similar results were also obtained for cocaine peaks at 304.5 m / z in the case of contact prints deposited on the target metal plate and sprinkled with hydrophobic powders containing titanium dioxide, carbon black (1:10), and also treated with DHB or left untreated (details not shown), and the relative intensities are respectively 1,700, 10,000, 9,000 and 100 mV (fig.4b).

The effectiveness of particles with introduced TiO ₂ in detecting cocaine residues in the raised prints

Successful demonstration of the effectiveness of the lifting process (FIGS. 1 and 2) and the detection of cocaine from latent fingerprints on targets of the MALDI system (FIGS. 3-5) suggest that cocaine can be detected on raised fingerprints obtained directly from a serial lifting tape. The results are shown in figure 5. The lower curve is obtained for a raised fingerprint that was not in contact with cocaine, while the upper curve is obtained for a fingerprint that was not in contact with cocaine. A clear peak is visible only on the upper curve at m / z 304, which corresponds to cocaine. In both cases, the original prints were sprinkled with hydrophobic particles of silicon dioxide containing TiO ₂ . 7 shows the target region of the MALDI-TOF system containing a fingerprint enriched in cocaine. A similar pattern is observed for negative control (not shown). The diameter of the recess is 3.4 mm, with a distinct ridge detail of the print. The spectra in Fig. 6 were obtained by laser scanning of the areas inside the cells, the signals being averaged to obtain the spectra.

Detection of cholesterol and squalene from prints using the MALDI-TOF-MS system, using hydrophobic particles as an amplification matrix

In this experiment, 12 prints were applied directly to the target plate of a stainless steel mass spectrometer (MS). After aging (aging), the prints were sprinkled with one of ten different compositions of hydrophobic powder. The prints were then lifted and analyzed using the MALDI-TOF-MS system.

Both cholesterol and squalene were expected to be detected, but in practice only squalene was detected. It was observed at m / z 433 corresponding to the molecular ion plus sodium adduct. As shown in FIG. 7, the intensity in the absence of any enhancing agent is about 2,000 mV (imprint 12). This intensity rises to 14,000 mV with DHB. Among the powders, those containing the highest proportions of carbon black give the highest responses (prints 9 and 10), and the higher the proportion of carbon black, the higher the peak intensity, and the sample with the highest intensity of about 22,000 mV has a carbon black ratio to diluent 1: 2.

Typically, the presence of DHB in the powder slightly increases the peak intensity (prints 1 and 2 — titanium dioxide, and prints 4 and 5 — the ratio of carbon black to thinner 1:10), but this trend is not preserved for powders that contain crystal violet dyes ( crystal violet) (3 and 6) and rhodamine 6G (7 and 8), in which case they receive low signals equivalent to fingerprint 12 without processing or even lower signals.

Direct detection of cholesterol and squalene using MALDI-TOF-MS, using hydrophobic particles as reinforcing matrices

Since cholesterol was not detected in MS fingerprints, attempts were made to detect this compound using the MALDI-TOF-MS system in the presence of DHB and new dusting agents. To do this, the standards of a mixture of squalene and cholesterol were applied to the surface of the target metal plate and eight sets of these spots in triplicate were sprinkled with eight hydrophobic powder formulations used in 3.6, or were treated with DHB, or were left untreated.

In this case, peaks associated with squalene were again observed at m / z 433 due to the sodium adduct of the compound, with powders 1, 2, 7 and 8 giving intensities (signals) significantly higher than the intensities in the presence of DHB (Fig. 8). The highest intensity was obtained using a red fluorescent powder (rhodamine 6G, powder 1, 49,000 mV), however, powders with carbon black also give good responses (1:10, 6000 mV; 1: 5, 36,000 mV; 1: 2, 4,000 mV). The peak was now observed at m / z 386, however, in all cases, the intensities were much lower than for squalene. The highest intensity was obtained in the presence of a powder that contains a crystal violet dye (1,800 mV, powder 4), and carbon black powders also give relatively good peaks (about 800 mV for powder 3, 1:10 plus DHB, for powder 7, 1: 5, and for the powder 8.1: 2) (Fig. 9).

The actual spectra of these two compounds are shown in FIG. 10a (squalene) and FIG. 10b (cholesterol). The best response for squalene is clearly visible in these experimental conditions.

Direct detection of exogenous metabolites from smokers in strewn latent prints with MALDI-TOF-MS

As a hydrophobic powder, a powder was used with carbon black introduced into it, with an initial carbon black: PTEOS ratio of 1: 2, the synthesis of which is described in the next section of this description.

These particles were uniformly introduced into a mixture of small particles of iron (diameter <60 μM) and stearic acid (1.0% by weight), so that the ratio of hydrophobic particles to iron particles is 2:98 by weight. Latent prints were sprinkled with a powder using a serial magnetic probe and then analyzed at the location (in situ) of the prints on the surface of stainless steel plates (Shimadzu MALDI-TOF-MS target plates), or were lifted using a serial lifting tape. The raised prints were attached to the target plates, print side up, using serial adhesive tape. MALDI-TOF-MS analysis was performed using a Kratos Axima CFR setup plus MALDI-TOF-MS (Shimadzu Biotech, Manchester, UK) operating in the reflectron mode of the positive ion. As a serial matrix, 2,5-dihydroxy benzoic acid (DHB) was used (10 mg / ml in a solution of 50:50 acetonitrile: deionized water [dH ₂ O]).

The results shown in FIG. 11 demonstrate the absence of a detected peak of nicotine (FW 162) or cotinine (FM 176) in the absence of a matrix of a reinforcing agent, for a latent print that was enriched with a mixture of nicotine and cotinine (10 ng each, FIG. 11 a) . When a conventional matrix enhancer in the form of DHB is added to the fingerprints enriched with the indicated compounds, then peaks are observed at 163 and 177 (Fig. 11c). When a hydrophobic powder is added to the enriched latent prints, then peaks are again observed at 163 and 177, which suggests that this powder acts as a reinforcing agent. The additional peak now observed at m / z 199 is probably caused by the sodium adduct of cotinine. The peak at m / z 157 is observed in the spectrum of the non-enriched imprint sprinkled with hydrophobic powder, together with the peak at m / z 163 having approximately the same intensity. No peaks were observed at 177 and 199 in this spectrum (Fig. 11d). It should be borne in mind that in Fig. 11b, the peak at 163 has a significantly higher intensity than the peak at 157, possibly due to the presence of nicotine.

11 clearly shows that nicotine and cotinine (both at a content of 10 ng per fingerprint), when introduced into latent fingerprints, cannot be detected using TOF-MS only by adding such an amplification matrix as DHB (FIG. 11a comparison with figs). Figure 11 also shows that the hydrophobic powder used to develop a latent imprint also acts as a reinforcing agent to detect these compounds (Figure 11b). The main peaks were found at m / z values of 163.3 (weight of the formula for nicotine 162.23) and 177.3 (weight of the formula for cotinine 176.22) and 199.2. This last peak is possibly caused by the sodium adduct of cotinine (C ₁₀ H ₁₂ N ₂ ONa, weight of formula 199.21). These peaks are absent in a pre-sprinkled latent imprint that was not enriched in nicotine and cotinine (Fig. 11d).

The corresponding spectra for a former smoker who has not smoked for the past 24 hours are shown in FIG. The main peaks not associated with the powder matrix are visible at m / z 163.1 (nicotine) and 199.1 (cotinine sodium adduct), with a smaller peak at 177.1 (cotinine) being also visible.

It is interesting to note that when the spectra are examined at higher mass numbers, then they see peaks at 307.4 and 433.4 for prints directly deposited on stainless steel plates. These peaks are possibly caused by the sodium adduct of stearic acid (FW 307.47) and the sodium adduct of squalene (FW 433.71), respectively. It should be borne in mind that stearic acid is used in the preparation of a dusting agent, and natural squalene is released as an endogenous component of the print.

These peaks can be used to calibrate the peaks obtained from the raised prints, as they are present on the surface of the lifting tape, which itself is attached to the surface of the stainless steel target plate. This raises the surface, so that the flight time of the ionized species now becomes shorter than in the case of a steel surface, and the visible m / z peak values become smaller. Thus, for the spectra of prints from smokers on raised ribbons, peaks for sodium adduct are visible at 305.1 and 431.2, along with peaks at 174.6 (main) and 197.4. Lower peak masses can be adjusted by 2.3 m / z units, giving peaks at 176.9 (cotinine) and 199.7 (sodium adduct cotinine).

Peaks at 161, 167 and 168 are always visible in MS only for smokers. It is known that, despite the fact that nicotine forms about 95% of the alkaloids in the tobacco leaf, it also contains various other alkaloids, among which the most abundant are nornicotine (RMM 148) and anatabine (RMM 160) [22]. The peak at m / z 161 can be caused by anatabine, since the protonated forms of nicotine and cotinine are observed in the spectra, however, additional work is necessary to confirm this.

Preparation of carbon black containing agent

The main method for preparing empty nanoparticles involves mixing 30 ml of ethanol, 5 ml of dH ₂ O, 2.5 ml of tetraethoxylan (TEOS) and 2.5 ml of phenyltriethoxylan (PTEOS) in a centrifuge. To this mixture was added 2 ml of ammonium hydroxide solution and the solution was rotated overnight. After this, the suspension is centrifuged (3 minutes at 3.000 rpm).

The product is isolated in the following series of centrifugation and washing operations using 10:90 by volume of ethanol in water, and then kept in a suspension of 3:97 by volume of ethanol in water. After that, an analysis of the particle size distribution, as well as SEM and TEM microscopy is carried out.

To obtain particles with carbon black, 5 ml of a carbon black solution is added to a precursor solution at a dilution of 1: 100 with water. To obtain magnetic particles coated with TEOS: PTEOS, magnetite powder is prepared in accordance with the published procedure, and 5 ml of a suspension in water is added to the precursor solution.

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Claims (21)

1. A method for determining the presence of a residue in a print by mass spectrometry using a matrix, which includes the following operations:
i) applying a powder to a fingerprint that is capable of acting as a matrix in mass spectrometry using a matrix; and facilitate the detection and / or visualization of the imprint in order to form an imprint with particles deposited thereon; and
ii) mass spectrometry of the imprinted material selected from MALDI-TOF-MS or SALDI-TOF-MS, the powder used contains hydrophobic particles of silicon dioxide, as well as metal, metal nitride, metal oxide or carbon to detect the presence or absence of a residue. 2. The method according to claim 1, wherein the metal oxide is selected from the group consisting of titanium dioxide, iron oxide, hematite, and combinations thereof. 3. The method according to claim 1, in which carbon is selected from the group consisting of carbon black, fullerene compound, carbon nanotubes, graphite and its analogue, as well as combinations thereof. 4. The method according to claim 1, wherein the metal is selected from the group consisting of aluminum, iron, and combinations thereof. 5. The method according to claim 1, in which the particles of metal, metal nitride, metal oxide or carbon are introduced into hydrophobic particles of silicon dioxide. 6. The method according to claim 5, in which the average particle diameter is ≤100 microns, possibly from 10 to 90 microns, preferably from 45 to 65 microns, and more preferably from 65 to 90 microns. 7. The method according to claim 5, in which the average particle diameter is ≤1 μm, possibly from 200 to 900 nm, preferably from 300 to 600 nm, and more preferably from 400 to 500 nm. 8. The method according to claim 1, in which the powder further comprises a dye molecule. 9. The method of claim 8, in which the dye molecule is fluorescent or colored. 10. The method of claim 8 or 9, in which the dye molecule and / or colored molecule is introduced into a hydrophobic particle of silicon dioxide. 11. The method according to claim 1, in which the powder is magnetic or paramagnetic. 12. The method according to claim 1, in which the print is lifted from its place of application using a lifting tape and brought into contact with the substrate of the sample mass spectrometer, after which the print is applied to the powder. 13. The method according to claim 1, in which mass spectrometry using a matrix selected from the group consisting of MALDI-TOF-MS-MS or SALDI-TOF-MS-MS and combinations thereof. 14. The method according to claim 1, which further provides for the visualization and / or imaging of the fingerprint. 15. The method according to claim 1, in which the residue is an endogenous residue, in particular an endogenous metabolite and / or exogenous metabolite. 16. The method according to clause 15, in which the endogenous metabolite is squalene. 17. The method according to clause 15, in which the exogenous metabolite is a metabolite of nicotine, such as cotinine. 18. The method according to claim 1, in which the residue is a contact residue. 19. The method according to p, in which the contact residue is a drug. 20. The method according to claim 19, in which the drug is cocaine. 21. The method according to claim 1, in which the print together contains at least one endogenous residue and at least one contact residue.

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