Sample holder for mass spectrometry analysis in MALDI mode, production and use of the sample holder

Abstract

There are described a new type of sample holder for performing analyses of biological samples with mass spectrometry in MALDI mode, the process for its production and some protocols for the use of the sample holder in said technique. The sample holder, in its simplest embodiment (10), consists of a support (11) on a face (12) of which there is at least one porous deposit (13) consisting of nanoparticles of an oxide of a Group 4 metal.

Claims

1. A sample holder for use in the MALDI technique, comprising: a support, selected from: a) a support made from a non-elastomeric polymer loaded with graphitic carbon (carbon-black filled), having a volume resistivity lower than 10.sup.12Ω×cm and a contact angle in a water wettability measurement at least equal to 90°; or b) a support having at least one face covered with a layer of a non-elastomeric polymer loaded with graphitic carbon (carbon-black filled), having a surface resistivity lower than 10 kΩ×square and a contact angle in a water wettability measurement at least equal to 90°; on a face of the support in case a) or on said covered face in case b), one or more deposits of an oxide of a metal of Group 4 of the periodic table of the elements, having a thickness between 100 and 400 nm and consisting of nanoparticles of said oxide having size between 2 and 50 nm, said one or more deposits entirely surrounded by the polymer of the support in case a) or by the polymer of said layer in case b); wherein said one or more oxide deposits are obtained by ballistic growth from said nanoparticles and have a self-affine structure, which has a porosity hierarchy from one nanometer to one hundred of nanometers; and wherein the surface of the sample holder on which there are said one or more oxide deposits is treated with UV radiation in such a way that the deposits show a contact angle smaller than 5° to a water wettability measurement while the same treatment with UV radiation does not alter the hydrophobicity of the support in case a) or of the polymer of said layer in case b).

2. The sample holder according to claim 1, wherein said deposits have a thickness between 150 and 300 nm, and the particle-size distribution curve of the oxide nanoparticles forming them has a maximum in the range between 5 and 15 nm.

3. The sample holder according to claim 1, wherein said one or more deposits of an oxide of a metal of Group 4 of the periodic table of the elements have, at a measurement with an atomic force microscope (AFM), a minimum rms roughness between 3 and 5 nm.

4. The sample holder according to claim 1, wherein said one or more deposits of an oxide of a metal of Group 4 of the periodic table of the elements have a density of nucleation centers higher than 1×10.sup.10 per square millimeter.

5. The sample holder according to claim 1, wherein said non-elastomeric polymer loaded with graphitic carbon (carbon-black filled) of said support or of said layer is selected among polypropylene, polyethylene, polystyrene, poly(methyl methacrylate) and polycarbonate.

6. The sample holder according to claim 1, wherein there is a plurality of deposits of oxide of a metal of Group 4 in an ordered geometric arrangement, and preferably centered at the nodes of a square lattice with a spacing corresponding to the standard adopted in multiwell plates.

7. The sample holder according to claim 1, wherein on the same face there are deposits of oxide of a Group 4 metal and deposits, generally made with the same oxide but having different size, for the positioning on the sample holder of internal calibration standards of MALDI analysis.

8. The sample holder according to claim 1, having lateral dimensions of 25×75 mm and a thickness of 1 mm.

9. The sample holder according to claim 1, wherein said oxide of a metal of Group 4 of the periodic table of the elements is titanium oxide, TiO.sub.2.

10. A process for the production of a sample holder of claim 1, comprising the following steps: obtaining a support consisting of a non-elastomeric polymer loaded with graphitic carbon (carbon-black filled), having a volume resistivity lower than 10.sup.12Ω×cm and a contact angle in a water wettability measurement at least equal to 90°; or obtaining a support having at least one face covered with a layer of a non-elastomeric polymer loaded with graphitic carbon (carbon-black filled), having a surface resistivity lower than 10 kΩ×square and a contact angle in a water wettability measurement at least equal to 90°; positioning in the vicinity of the support, between this and the source of the material to be deposited on the same, a physical mask having one or more openings having geometry corresponding to one or more deposits of an oxide of a Group 4 metal which are intended to be formed on the support, turning towards said source the face covered with said layer in the case of support with a covered face; depositing with the Supersonic Cluster Beam Deposition (SCBD) technique, on a face of the support in case a) or on said covered face in case b), one or more deposits of an oxide of a metal of Group 4 of the periodic table of the elements, consisting of nanoparticles of said oxide of dimensions ranging between 2 and 50 nm and having a thickness between 100 and 400 nm, entirely surrounded by the polymer of the support in case a) or by the polymer of said layer in case b), forming a self-affine porous structure having a hierarchy of porosity from one nanometer to one hundred of nanometers; treating the surface of the sample holder on which there are said one or more deposits by UV radiation, to impart to said deposits a contact angle of less than 5° at a wettability measurement while preserving the hydrophobicity features of the surrounding support.

11. The process according to claim 10, wherein the Supersonic Cluster Beam Deposition technique adopted is based on a Pulsed Microplasma Cluster Source (PMCS).

12. The process according to claim 10, wherein the Group 4 metal oxide deposit is made hydrophilic by irradiation for more than half an hour with a 30 W power UV lamp, kept at a distance of about 40 cm from said one or more deposits.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIGS. 1a and 1b show two possible alternative embodiments of sample holders of the invention;

(2) FIG. 2 shows a schematic representation of the microscopic structure of a metallic oxide deposit of a sample holder of the invention;

(3) FIG. 3 shows a photograph of a sample holder of the invention in a preferred embodiment;

(4) FIG. 4 shows a photograph of a sample holder of the invention in which, on the deposits of metallic oxide, drops of aqueous solution were deposited;

(5) FIGS. 5a and 5b show two microphotographs in different magnifications taken with the scanning electron microscope of the surface of a deposit of metallic oxide present on the sample holder of the invention;

(6) FIG. 6 shows another possible embodiment of a sample holder of the invention;

(7) FIGS. 7 and 8 show two mass spectra made with the MALDI technique using a sample holder of the invention, before and after in situ treatment of the sample, respectively.

DETAILED DESCRIPTION OF THE INVENTION

(8) The invention is described hereinafter in detail with reference to the Figures.

(9) In the figures, the same number corresponds to a same element; also, for clarity of representation, the elements shown are not necessarily to scale.

(10) The sample holder of the invention consists of a support formed by, or having at least one face coated with, a non-metallic antistatic and hydrophobic material, where the term hydrophobic means the characteristic of a surface on which a contact angle measurement made with water provides a value equal to or greater than 90°.

(11) The first possibility (case a, support consisting of non-metallic material with a volume resistivity lower than 10.sup.12Ω×cm) is represented in FIG. 1a, in which the sample holder, 10, consists of a support, 11, made entirely with said non-metallic antistatic and hydrophobic material. On one face, 12, of support 11, there is a deposit, 13, of a Group 4 metal oxide consisting of nanoparticles.

(12) The second possibility (case b, support with a face covered with a layer of a non-metallic material with a surface resistivity lower than 10 kΩ×square) is represented in FIG. 1b; in this case, the sample holder of the invention, 10′, consists of a support, 11′, having a face covered with a layer 14 of non-metallic antistatic and hydrophobic material. On the exposed face 12 of layer 14, here is the deposit 13 of oxide nanoparticles.

(13) In the sample holder 10, the support 11 may be made, for example, with a polymeric material (such as polypropylene, polyethylene, polystyrene, poly(methyl methacrylate) or polycarbonate) filled with conductive material powders, such as graphitic carbon; alternatively, support 11 may be made of doped silicon.

(14) In the case of the sample holder 10′, support 11′ may be made with any material that has mechanical properties suitable for the purpose, and which allows the adhesion of layer 14 (for example, it may be made of glass, plastic or metal); layer 14 may be made with one of the materials mentioned above for the production of support 11, or with a conductive oxide, such as for example the mixed indium and tin oxide (ITO).

(15) The preferred materials for making support 10′ are polymers (for example, polypropylene, polyethylene, polystyrene, poly(methyl methacrylate), polycarbonate) charged with graphite powder, or glass slides with a face covered with ITO.

(16) The dimensions of the support may vary within wide limits, but they are suitably similar to those of the slides already employed in routine analyses, either automated or not, in the medical and biological field, to allow the management (handling, storage) of the sample holder of the invention in standard operating modes, including the use of automated means and instrumentation already employed in the sector also for analyses other than MALDI; for example, a typical sample holder of the invention may have lateral dimensions of 25×75 mm and a thickness of 1 mm, standard values of common glass slides.

(17) On one of the main faces of support 11, or on the exposed face of layer 14, there is at least one porous deposit 13 of an oxide of a Group 4 metal, preferably titanium and zirconium, entirely surrounded by the material of support 11 or of layer 14.

(18) The deposit (the production of which is described below) is formed by particles of said oxide having a size of between about 2 nm and 50 nm, with a maximum of the particle-size distribution curve of the same in the range between 5 and 15 nm. Moreover, the deposit has a thickness of between about 100 and 400 nm, and preferably between about 150 and 300 nm. The porosity of the deposit is a consequence of a growth mode of the deposit itself due to the “ballistic” model, in which the flying nanoparticles stop at the exact point at which they impact on the substrate or on the nanoparticles already deposited, without diffusion or rearrangements. Due to the ballistic growth mode and to the thickness values indicated above, the deposit has within certain limits a self-affine or more generically fractal structure. Self-affinity consists in that the three-dimensional geometry (or a two-dimensional section thereof) of the oxide deposit always appears similar to itself even when observed at different magnifications, in which details of different sizes are displayed at the same apparent size for some orders of magnitude; for a confirmation of the self-affine nature of these deposits, see for example articles “Self-affine properties of cluster-assembled carbon thin films”, R. Buzio et al., Surface Science 444 (2000), and “Nanomanufacturing of titania interfaces with controlled structural and functional properties by supersonic cluster beam deposition”, A. Podesta et al., Journal of Applied Physics 118 (2015). In the particular case of the oxide deposits of the present invention, there is a self-affine porous structure characterized by a porosity hierarchy, of a size from one nanometer to one hundred of nanometers. This situation is schematized in FIG. 2, which shows a series of particles 20 of different sizes, assembled in ballistic mode to form a deposit on a support (not shown in the figure); reference numeral 21 shows by way of example, represented by dashed circles, some empty spaces of different sizes representing in a schematic manner the hierarchical structure of porosity observed in the deposit and due to the ballistic growth mode of deposit 13. In particular, the subject porous structure is characterized in that conditions for the nucleation of proteins and biomolecules in general are determined inside the pores and this allows a particularly effective capture of the biomolecules themselves in amounts that exceed what is expected by the geometric increase of the specific surface area, as described in articles “The effect of surface nanometrescale morphology on protein adsorption”, P. E. Scopelliti et al., PLoS One 5(7), e11862 (2010); and “Nanoscale roughness affects the activity of enzymes adsorbed on cluster-assembled titania films”, L. Gailite et al., Langmuir 30(20), 5973-5981 (2014).

(19) Due to the size distribution of the nanoparticles, of between about 2 and 50 nm, and centered between 5 and 15 nm, the deposit surface has a minimum peculiar roughness, related to this distribution, and independent from the level of development of the hierarchical structure of the porosity.

(20) The preferred material for making deposit 13 is titanium oxide, TiO.sub.2.

(21) This oxide has the peculiar feature of being able to be made super-hydrophilic as a result of UV irradiation, as initially described in article “Light-induced amphiphilic surfaces”, R. Wang et al., Nature 388,431 (1997). The term super-hydrophilic, in the present invention, refers to the feature of a surface on which a contact angle measurement made with water provides a value equal to or smaller than 5°. A possible explanation of this phenomenon is given in article “TiO.sub.2 photocatalysis: A Historical Overview and Future Prospects”, K. Hashimoto et al., Japanese Journal of Applied Physics 44(12), 8269-8285 (2005); the feature described in the article by Hashimoto et al. for TiO.sub.2 is also found in zirconia, ZrO.sub.2, as shown in article “Light-Controlled ZrO.sub.2 Surface Hydrophilicity”, Rudakova A. V. et al., Scientific Reports 6, 34285 (2016).

(22) Unlike other processes for the treatment of surfaces such as, for example, exposure to an oxygen plasma (as described for example in paragraph [0060] of patent application US 2011/0281267 A1, assigned to the present Applicant), which has the known effect of inducing hydrophilicity indistinctly on different materials exposed to it, including polymeric materials, UV irradiation was observed to have no effect on the original hydrophobicity of the support.

(23) If on the one hand the original hydrophobicity of the support is not altered by UV radiation, it is known however that UV radiation can also cause photo-desorption of volatile compounds from polymeric materials. These compounds can re-settle on the surfaces arranged in the vicinity of the irradiated polymeric material, causing a substantial modification of the wettability thereof: in particular, originally hydrophilic surfaces, arranged in the vicinity of polymeric materials, can be made hydrophobic through this mechanism. The phenomenon is clearly described, for example, in the article Nagai H. et al. “Flexible manipulation of microfluids using optically regulated adsorption/desorption of hydrophobic materials”, Biosensors and Bioelectronics 22, 1968-1973 (2007), where it is used to make a hydrophilic TiO.sub.2 surface hydrophobic.

(24) Surprisingly, the inventors have observed that such a photo-desorption process of volatile compounds and consequent induction of hydrophobicity of the surfaces in the vicinity of the irradiated polymer does not occur in the case of polymers such as polypropylene, poly(methyl methacrylate), polycarbonate and other similar non-elastomeric polymers. In particular, the inventors have unexpectedly observed that areas of TiO.sub.2 very limited in size (1 mm diameter dots) deposited on the polymers mentioned above and completely surrounded by them, when subjected to UV irradiation do not acquire any feature of hydrophobicity as a result of photo-desorption of volatile compounds from the surrounding polymer, and conversely become super-hydrophilic.

(25) Therefore, the UV radiation can be advantageously exploited to make the porous deposits of oxides of Group 4 metals super-hydrophilic and at the same time not affect the hydrophobic character of the support. This leads to the remarkable result of generating a strong hydrophilic-hydrophobic discontinuity in the properties of wettability of the sample holder surface, and in particular at the edge of the porous oxide deposit. The hydrophilic-hydrophobic barrier therefore acts as a containment structure for drops of liquid and allows the in situ treatment of biological samples without the aid of external physical containment structures.

(26) The UV treatment of the sample holder can conveniently and easily be carried out by operator of the MALDI analysis, before using the sample holder, by irradiation for at least half an hour by means of a 30 W power UV lamp, kept at a distance of about 40 cm from the sample holder of the invention. It is noted that a radiation such as that described herein is easily obtainable within a chemical hood aspirated and equipped with a UV lamp sterilizer, commonly available in any biology laboratory. In the case of aspirated hoods with UV lamps of different power, it is always possible to suitably rescale distance and time to easily obtain the described irradiation.

(27) While the sample holder of the invention has been described thus far as having a single deposit 13, it will be apparent to those skilled in the art that in the preferred embodiment thereof, this will have a plurality of type 13 deposits; this configuration allows multiple treatments and parallel analysis of similar samples to be carried out, or to repeat treatments and analyses on multiple fractions of the same sample in order to improve the reliability of the results.

(28) The deposit (or deposits) 13 are preferably circular in shape, and have a diameter between 1 and 3 mm.

(29) When on the sample there are multiple type 13 deposits, these are normally in an ordered geometric arrangement, and preferably centered at the nodes of a square lattice, whose spacing corresponds to the standard adopted in the sample holders used in other analyses in the biomedical field, such as “multiwell” plates, and facilitates the integration of MALDI analysis in the typical operational sequences of the field. FIG. 3 shows a photograph of a sample holder with multiple deposits 13 according to this preferred embodiment; the sample holder in the photograph was produced from a plastic support loaded with graphite, but of course sample holders with multiple type 13 deposits may be produced both with supports with the simple structure shown in FIG. 1a, and with supports with a coated face of the type shown in FIG. 1b.

(30) The sample holder may also have, on the same face on which there are deposits 13, different deposits, for example, for the positioning on the sample holder of internal calibration standards of the analysis. These different deposits may be located on nodes of the above lattice (for example square), at points where type 13 deposits have not been produced.

(31) In a second aspect thereof, the invention relates to the method for producing the sample holders described above.

(32) The supports for the production of the sample holder are commercially available or easy to produce; in particular, of common commercial availability are supports made of plastic material loaded with graphite or doped silicon, as well as slides having a uniform ITO coating on one or both faces. The latter may possibly be made in a simple manner starting from a mixed solution of indium and tin precursors (for example, in an alcohol or hydro-alcohol solvent) with well-known techniques, such as sol-gel or spray-drying.

(33) The formation of deposits 13 can be achieved using various techniques, in particular those known as Supersonic Cluster Beam Deposition (SCBD) and, among these, in particular the one based on a Pulsed Microplasma Cluster Source (PMCS).

(34) These techniques are known in the production of thin films on a substrate. The PMCS-based SCBD technique is described in various publications, such as the article “Cluster beam deposition: a tool for nanoscale science and technology”, K. Wegner et al., Journal of Physics D: Applied Physics 39(22): 439-459, 2006; patent application WO 2011/121017 A1; and chapter “Pulsed microplasma cluster source technique for synthesis of nanostructured carbon films”, P. Milani et al., pages 561-564 of book “New trends in intercalation compounds for energy storage”, NATO Science Series, vol. 61, 2002.

(35) With the SCBD technique a beam of particles of the material of interest is produced, and the beam is directed, within a reduced pressure chamber, on a support generally arranged orthogonally to the axis of the beam itself.

(36) The shape and size of the deposit (or deposits) 13 is determined with the interposition of a physical mask (typically metal) along the beam and in the proximity of the support; during the deposition, the mask is positioned parallel to the support, generally at a distance of less than 1 mm therefrom. In order to carry out the deposition of the desired material over relatively large areas, such as for example in the case of sample holders with multiple type 13 deposits, or in the case of simultaneous deposition on multiple sample holders, it is possible to carry out a scan of the deposit area, for example by laterally moving the support (and mask) in the plane perpendicular to the beam axis. A same mask may have openings having multiple geometries and/or dimensions, in order deposit sample holders having different configurations in a single deposition session.

(37) After the depositions with the above techniques, deposit 13 is generally hydrophobic.

(38) The hydrophilicity required for the intended applications is imparted to the deposit via UV radiation in air, as described above. By operating according to this preferred mode, deposit 13 can conveniently be made super-hydrophilic, such as by radiation for at least half an hour by means of a 30 W power UV lamp, kept at a distance of about 40 cm from the sample holder of the invention, without having any effect on the original hydrophobicity of the support or affecting the super-hydrophilic nature of the deposits by photo-desorption of volatile compounds from the support. The UV treatment of the sample holder can conveniently and easily be carried out by the operator of the MALDI analysis prior to use the sample holder, using for example an aspirated chemical hood equipped with UV lamp sterilizer, commonly available in any biology laboratory.

(39) The sample holder of the invention has a series of advantages and features that make it particularly suitable and versatile in various MALDI analysis modes.

(40) The inventors have first noted that deposit 13 is able to bind very different biological materials, in particular with respect to their size: from single isolated biomolecules (e.g., peptides and proteins in solution), up to more complex and larger biological entities, such as exosomes, microvesicles, bacteria, or cells. In particular, the inventors have unexpectedly observed that, in the case of exosomes or microvesicles, whose size is in the range of 10-100 nm, the hierarchical structure of the porosity, a consequence of the ballistic growth dynamic of the deposit, has cavities of a size suitable to their capture. In general, the inventors believe that the versatility of the subject sample holder with respect to the capture of very different biological materials comes from the combined effect of nanoporosity at the scales of the size of interest and of the bioaffinity between titanium oxide, and generally of the oxides of Group 4 metals, and the biomolecules present on the membranes of these biological entities (e.g. membrane proteins). In addition, both the oxide and the materials of face 12 or 12′ are chemically inert, and allow the in situ treatment of the biological sample, for example with acids or bases. The deposits of oxides of Group 4 metals at the thicknesses at which they are used are substantially transparent, allowing, when deposited on glass slides, optionally coated with ITO, optical microscope analysis before or after the MALDI analysis; these materials are also not self-fluorescent, so as not to overlap a spurious signal to that of the sample in fluorescence-based analyses. Finally, in the common case in which the sample holder has suitably large type 13 deposits, these allow the adhesion of biological tissue sections; this avoids the problem, which occurs with some sample holders of the prior art (and which irremediably leads to the impossibility of using the sample in the MALDI Imaging analysis), of the movement or folding on itself of the histological section as a consequence of washing or treatments with special reagents carried out in situ prior to the analysis (such as, for example, treatment with chloroform for the delipidation of a tissue), or even subsequent to this (for example, treatment with methanol solutions for the removal of the matrix). Surprisingly, the inventors have observed that the adhesion of the tissue to the deposit offers an unexpected advantage, particularly relevant with respect to the problem, commonly known in the MALDI practice on histological samples (MALDI Imaging), of the variation in the planar dimensions of the histological sections as a result of in situ treatments. This problem consists in a slight expansion or contraction of the planar dimensions of the tissue, which makes it difficult to superimpose MALDI images with optical images of the tissue, obtained for example by an optical scanner, if between the ones and the others there have been, as is common, treatments of the sample. Experts of the field currently seek to overcome this drawback through the use of software algorithms applied to the images, which act by altering the dimensions thereof. Due to the improved adhesion of the tissue, the use of the sample holder of the present invention prevents the variation of the planar dimensions of the histological sections following in situ treatments and makes image processing operations superfluous, with advantages in terms of results analysis time, as well as with regard to the risk of introducing artifacts due to processing of the images.

(41) The improvement of the adhesion of the histological sections also assumes particular importance in the use of the MALDI technique for pharmacokinetic studies, whose purpose is the identification of the spatial distribution of a drug (or its active ingredient or one or more of its metabolites, or one of its components) within a tissue. In this application of the MALDI technique, unlike what is illustrated above, no chemical treatment of the tissue can be made: any treatment would in fact cause the elution of the test compounds and the consequent loss of information on their spatial distribution in the tissue. As the use of special reagents (such as chloroform) can cause the detachment of the histological section and the consequent uselessness of the sample, also the total absence of treatments, such as imposed in the MALDI experiments of pharmacokinetics, makes the sample particularly fragile: any assays subsequent to the MALDI analysis performed on the same sample (for example, immunohistochemistry staining) can easily lead to the detachment of the histological section and consequent loss of the sample. This problem is instead solved by the sample holder of the present invention, in which the adhesion of the histological section is substantially improved by the presence of the porous oxide.

(42) With further reference to the use of the sample holder of the invention, the hydrophilicity of deposits 13 and the hydrophobicity of the surrounding surface of face 12 or 12′ allows precisely and uniformly attracting and locating on deposits 13 the drops of aqueous solution containing the species to be analyzed, even when the dispensing of the liquid is not very precise. Also, thanks to the hydrophilicity of deposits 13 and the hydrophobicity of the surrounding area, the drops of solution will concentrate on the deposits also in case of relatively high amounts of liquid, without the need for any raised areas or other containment structures around the deposits themselves; this effect is shown in FIG. 4, in which drops of water-based solution are shown, whose imprint on the sample holder remains confined within deposits 13 despite the large volume of the drop; the same volume, without the confinement given by the hydrophilic-hydrophobic discontinuity, would occupy a much larger area. Likewise, any drops of reagent solution are also confined to the area of deposits 13 for the in situ treatment of biological samples previously deposited on the same areas. Unexpectedly, it has been observed that the confinement given by the hydrophobic barrier around the hydrophilic surface is effective to the point of allowing mechanical actions on the drop itself, such as handling, by manual pipettor or automated liquid-handling systems, of a suitable volume of water back and forth, tens of times, in order to carry out the washing of the sample previously absorbed on the hydrophilic porous oxide (for example, for the removal of salt residues, a consequence of protein samples preparation).

(43) Finally, the inventors have found an unexpected advantage provided by the sample holder due to the peculiar minimum roughness of the porous oxide deposit and correlated to the size distribution of nanoparticles used to produce the deposit: this roughness provides a very high density (per unit area of the deposit) of the nucleation centers on which the crystallization of the matrix used in the MALDI technique can be initiated, when the solution containing the matrix itself, present on the deposit, begins to evaporate. This allows obtaining an optimal distribution of MALDI matrix crystals, i.e. a high surface density of small crystals, uniformly distributed over the entire area of deposit 13, which eliminates the problem of the (manual or automatic) search for the most suitable crystal to be irradiated by the laser pulse (in jargon called “hot spot”). The matrix materials may for example be α-cyano-4-hydroxycinnamic acid (generally indicated in the field with the abbreviation CHCA) or sinapic acid (usually abbreviated as SA), in acetonitrile and/or trifluoroacetic acid solutions in water. It is possible to quantify the minimum peculiar roughness of the porous oxide deposit correlated to the size distribution of the nanoparticles used to produce the deposit by means of atomic force microscopy (AFM) measurements on deposits consisting of a single monolayer of nanoparticles. The method is described in section II.B of the article by Podestà et al. mentioned above. At a series of measurements carried out by the inventors on deposits consisting of a single monolayer of nanoparticles, the minimum rms roughness of the oxide deposits formed on the supports was found to be about 3-5 nm. It is also possible to estimate the number of nucleation centers per unit area, imagining a monolayer of spherical particles of 10 nm diameter (therefore, between 5 and 15 nm) and assuming that each nanoparticle constitutes a nucleation center. In these assumptions, it is found that the number of nucleation centers is greater than 1×10.sup.10 per square millimeter. The three-dimensional development of the nano-porous film for thicknesses higher than the monolayer further increases the number of nucleation centers for geometric unit area.

(44) After being activated by exposure to UV in the simple manner described above, the sample holder of the invention can be used in the preparation for MALDI analysis according to various possible protocols, such as for example those exemplified below; all the drops of sample solutions or of calibration standards have a volume in the order of magnitude of microliters.

Protocol 1—Basic Procedure

(45) 1.a) Pipetting a drop of the biological sample to be analyzed on a deposit 13 of the sample holder of the invention;

(46) 1.b) waiting for the drop to dry;

(47) 1.c) pipetting a drop of a MALDI matrix solution, such as SA or CHCA, on the same area of point 1.a;

(48) 1.d) waiting for the drying of the drop;

(49) 1.e) pipetting a drop of solution of a calibration standard in a dedicated type 13 deposit of the sample holder;

(50) 1.f) waiting for the drying of the drop;

(51) 1.g) if the calibration standard solution of point 1.e not is not already provided with the MALDI matrix, pipetting a drop of a MALDI matrix solution, such as SA or CHCA and waiting for the drying of the same.

Protocol 2—Procedure with Sample Washing

(52) 2.a) Pipetting a drop of the biological sample to be analyzed on a deposit 13 of the sample holder of the invention;

(53) 2.b) waiting for the drying of the drop;

(54) 2.c) washing the sample (for example for removal of the salt) by pipetting a drop of water on the same area of point 2.a, and moving the plunger of the pipette forward and backward; then, eliminating the drop of water;

(55) 2.d) repeating step 2.c at least once;

(56) 2.e) pipetting a drop of a MALDI matrix solution, such as SA or CHCA, on the same area of point 2.a;

(57) 2.f) waiting for the drying of the drop;

(58) 2.g) pipetting a drop of solution of a calibration standard in a dedicated type 13 deposit of the sample holder;

(59) 2.h) waiting for the drying of the drop;

(60) 2.i) if the calibration standard solution of point 2.g not is not already provided with the MALDI matrix, pipetting a drop of a MALDI matrix solution, such as SA or CHCA and waiting for the drying of the same.

Protocol 3—Procedure with Chemical Treatment of the Sample

(61) 3.a) Pipetting a drop of the biological sample to be analyzed on a deposit 13 of the sample holder of the invention;

(62) 3.b) waiting for the drying of the drop;

(63) 3.c) chemically treating the sample by pipetting a drop of an aqueous solution of a suitable reagent (e.g. ethanol for the dehydration of the sample) on the same area of point 3.a, and moving the plunger of the pipette forward and backward; then, discarding the drop of aqueous solution;

(64) 3.d) repeating step 3.c at least once;

(65) 3.e) pipetting a drop of a MALDI matrix solution, such as SA or CHCA, on the same area of point 3.a;

(66) 3.f) waiting for the drying of the drop;

(67) 3.g) pipetting a drop of solution of a calibration standard in a dedicated type 13 deposit of the sample holder;

(68) 3.h) waiting for the drying of the drop;

(69) 3.i) if the calibration standard solution of point 3.g not is not already provided with the MALDI matrix, pipetting a drop of a MALDI matrix solution, such as SA or CHCA and waiting for the drying of the same.

Protocol 4—Procedure with Enzymatic Treatment of the Sample

(70) 4.a) Pipetting a drop of the biological sample to be analyzed on a deposit 13 of the sample holder of the invention;

(71) 4.b) waiting for the drying of the drop;

(72) 4.c) performing the enzymatic digestion of the sample by pipetting a drop of the enzyme in the digestion buffer (e.g. trypsin in ammonium bicarbonate) over the same area of point 4.a; then, incubating at a suitable temperature and for a suitable time (such as 50° C. for 30 minutes), preferably in a closed volume in order to limit the evaporation of the buffer;

(73) 4.d) waiting for the drying of the drop;

(74) 4.e) pipetting a drop of a MALDI matrix solution for peptides, such as CHCA, on the same area of point 4.a;

(75) 4.f) waiting for the drying of the drop;

(76) 4.g) pipetting a drop of solution of a calibration standard in a dedicated type 13 deposit of the sample holder;

(77) 4.h) waiting for the drying of the drop;

(78) 4.i) if the calibration standard solution of point 4.g not is not already provided with the MALDI matrix, pipetting a drop of a MALDI matrix solution, such as SA or CHCA and waiting for the drying of the same.

Protocol 5—Procedure for Pre- and Post-Enzymatic Treatment Measurements

(79) 5.a) After carrying out any one of protocols 1 to 3 and after collecting the MALDI analysis data, removing the MALDI matrix by pipetting a drop of an aqueous solution of methanol on the same area being analyzed, moving the plunger of the pipette forward and backward; then, discarding the drop of aqueous solution;

(80) 5.b) performing the enzymatic digestion of the sample by pipetting a drop of the enzyme in the digestion buffer (e.g. trypsin in ammonium bicarbonate) over the same area of point 5.a; then, incubating at a suitable temperature and for a suitable time (such as 50° C. for 30 minutes), preferably in a closed volume in order to limit the evaporation of the buffer;

(81) 5.c) waiting for the drying of the drop;

(82) 5.d) pipetting a drop of a MALDI matrix for peptides, such as CHCA, on the same area of point 5.a;

(83) 5.e) waiting for the drying of the drop.

Protocol 6—Procedure for Preparing the Sample for Molecular Histology Measurements

(MALDI Imaging) for Pharmaco-Kinetics

(84) 6.a) Cutting a cryo-preserved tissue section using a cryomicrotome and placing it on a sample holder of the invention, at at least one type 13 deposit of appropriate size;

(85) 6.b) mounting the tissue through thawing (for example by the heat of a finger placed on the back of the sample holder) and allowing it to adhere on the surface of the sample holder;

(86) 6.c) pipetting a drop of solution of a calibration standard not including the MALDI matrix in a dedicated type 13 deposit of the sample holder;

(87) 6.d) waiting for the drying of the drop;

(88) 6.e) with the aid of dedicated instrumentation (such as a Bruker Immagine Prep instrument), atomizing a MALDI matrix solution for proteins/peptides, such as SA or CHCA, at least in the areas defined in points 6.a and 6.c;

(89) 6.f) waiting for the drying of the matrix.

Protocol 7—Procedure for the In Situ Preparation and Treatment of the Sample for Molecular Histology Measurements (MALDI Imaging) for Proteomics

(90) 7.a) Cutting a cryo-preserved tissue section using a cryomicrotome and placing it on a sample holder of the invention, at at least one type 13 deposit of appropriate size;

(91) 7.b) mounting the tissue through thawing (for example by the heat of a finger placed on the back of the sample holder) and allowing it to adhere on the surface of the sample holder;

(92) 7.c) treating the tissue section with suitable chemicals, such as ethanol for dehydration, acetone for fixation or chloroform for delipidation;

(93) 7.d) pipetting a drop of solution of a calibration standard not including the MALDI matrix onto a dedicated type 13 deposit of the sample holder;

(94) 7.e) waiting for the drying of the drop;

(95) 7.f) with the aid of dedicated instrumentation (such as a Bruker Immagine Prep instrument), atomizing a MALDI matrix solution for proteins/peptides, such as SA or CHCA, at least in the areas defined in points 7.a and 7.d;

(96) 7.g) waiting for the drying of the matrix.

Protocol 8—Procedure for Sequential Analysis MALDI and Immunohistochemistry

(97) 8.a) After carrying out any one of protocols 6 or 7, and after collecting the MALDI analysis data, removing the MALDI matrix, such as by means of weak sonication in an aqueous solution of methanol;

(98) 8.b) waiting for the drying of the sample thus removed;

(99) 8.c) performing an immunohistochemistry (IHC), or any generic staining assay of the sample (for example, hematoxylin).

(100) As will be apparent to those skilled in the art, other protocols are also possible, for example derived from combinations of the eight protocols described above; the MALDI sample holders of the invention are suitable for performing all of these protocols in automated form, with a robotic platform for the handling of liquid samples and treatment solutions, allowing the analysis of a large number of samples per unit of time to be performed. In order to employ automated procedures, the sample holder of the invention may be mounted on a suitable adapter allowing the stable insertion thereof into the sample chamber of a mass spectrometer.

(101) The invention will be further described by the following experimental section.

EXAMPLE 1

(102) This example relates to the production and characterization of a first sample holder for MALDI analysis according to the invention.

(103) A polypropylene plate loaded with graphite was used as a support, having a volume resistivity of the order of 3×10.sup.2Ω×cm and dimensions of 75×25×1 (mm).

(104) The support was introduced into the deposition chamber of an apparatus for SCBD depositions. In front of the support, at a distance of 1 mm from this, a physical mask (perforated metal plate) was positioned, having two series of circular apertures of diameter of 1 mm and 2 mm, each having a spacing of 4.5 mm between the centers of the respective circular openings, and mutually offset by 2.25 mm in vertical and horizontal direction.

(105) Through the SCBD technique, at the openings of the mask, nanoporous deposits of TiO.sub.2 were obtained on the support, having a thickness of about 200 nm, produced by deposition of nanoparticles generated by PMCS. The main process parameters used were: Argon gas (process gas) line pressure=40 bar, discharge voltage=900 V, discharge duration=60 μs, number of pulses per unit time=4 (repetition rate=4 Hz), nominal opening time of pulsed valve=220 μs, average pressure in the expansion chamber during the process=2.4×10.sup.−3 mbar, source-support distance=50 cm.

(106) The result of the deposition test is shown in the photograph reproduced in FIG. 3. FIGS. 5a and 5b show photographs of the surface of a TiO.sub.2 deposit obtained by scanning electron microscopy (SEM) at 70,000 and 200,000 magnification, respectively. These photomicrographs show the nanoparticles that make up the deposit and highlight both the nanoporous structure and the porosity hierarchy thereof.

EXAMPLE 2

(107) The procedure of Example 1 was repeated but using in this case, as a support, a glass plate of dimensions of 75×25×1 (mm) having a surface coating of ITO, which imparts a surface resistance to the support of the order of 100Ω×square, and a suitable mask with three square openings of 15 mm side and two circular openings of 2 mm diameter. A sample holder like that shown in FIG. 6 is obtained. The TiO.sub.2 deposits, analyzed by SEM, show the same morphological characteristics (particle size of the TiO.sub.2 nanoparticles and distribution of porosity) as those in Example 1.

EXAMPLE 3

(108) The sample holder produced as described in Example 1 was used to perform MALDI analyses according to Protocol 5 (procedure for pre- and post-enzymatic treatment measurements), operating on one of the 2 mm diameter deposits present on the sample holder.

(109) A volume of 2 μL BSA (Bovine Serum Albumine) in aqueous solution was pipetted on one of the deposits of the sample holder, waiting for the evaporation of the solvent (Milli-Q water).

(110) Once the evaporation was completed, on the same deposit was pipetted a volume of 1 μl of a solution with saturated concentration of a MALDI matrix precursor for proteins (sinapic acid) in acetonitrile and water in a proportion of 1:2 (volume). It was waited for the evaporation of the solvent and the formation of matrix-sample crystals.

(111) The sample holder thus prepared was introduced into the MALDI mass spectrometer (Bruker UltrafleXtreme) and the signal produced by the BSA sample was acquired; the test result is shown in FIG. 7.

(112) The sample holder was then extracted from the spectrometer and the removal of the crystallized MALDI matrix was performed by in-situ washing with 2 μl of a methanol solution at 70% by volume in water.

(113) The enzymatic digestion of the sample was then carried out by pipetting a volume of 5 μl of an aqueous solution of ammonium bicarbonate 40 mM (buffer) to which was added a volume of 1 μl of aqueous solution of Trypsin at a concentration of 0.05 μg/μl. The sample holder thus treated was maintained for about 30 minutes at a temperature of 45-50° C. in a closed volume (to limit the evaporation of the buffer).

(114) After incubation, it was waited for the complete evaporation of the residual volume of ammonium bicarbonate buffer in water.

(115) After complete evaporation, on the same deposit was pipetted a volume of 1 μl of a saturated concentration solution of MALDI matrix precursor for peptides (CHCA) in acetonitrile and water in proportions of 1:2 (by volume). It was waited for the evaporation of the solvent and the formation of matrix-sample crystals.

(116) The sample holder thus prepared was again introduced into the MALDI mass spectrometer and the signal produced by the peptides generated by the enzymatic digestion of the BSA sample was acquired. The test result is shown in the spectrum in FIG. 8.