A METHOD FOR THE DETECTION AND QUANTIFICATION OF NANO OR MICRO PLASTIC PARTICLES

Abstract

The present invention relates to a method for the detection of at least one nano or micro plastic particle comprised in a heterogeneous matrix material comprising the following steps: applying of at least one part of a heterogeneous matrix material comprising at least one nano or micro plastic particle onto at least a portion of a surface of a conductive support thereby forming a first layer onto said surface, irradiating of at least a portion of said first layer with at least one ion beam, thereby forming an irradiated layer, detecting of the at least one nano or micro plastic particle comprised in said irradiated layer by a detection method chosen from the group of Raman nanoscopic techniques, or infrared nanoscopic techniques, or charge dependent detection methods or combination thereof. The present invention allowed good detection of micro and nano plastic particles with high resolution and sensitivity.

Claims

1. A method for the detection of at least one nano or micro plastic particle comprised in a heterogeneous matrix material comprising the following steps: a) applying of at least one part of a heterogeneous matrix material comprising the at least one nano or micro plastic particle onto at least a portion of a surface of a focused ion beam irradiable electrically conductive support thereby forming a first layer onto said surface, said first layer having an average thickness equal to or lower than 10 μm, the heterogeneous matrix being a medium in which the at least one nano or micro plastic particle is embedded together with a substance or a mixture of substances, the medium being selected from the group consisting of a liquid, a solution, a suspension, a paste, a cream, a tissue and wherein the heterogeneous matrix is a naturally occurring substance selected from the group consisting of cell cultures grown on a surface, creams obtained from seafood, and cultures obtained by spreading-plating methods; b) irradiating of at least a portion of said first layer with at least one ion beam, thereby forming an irradiated layer, the at least a portion of said first layer being irradiated by using a Ga.sup.+ focused ion beam with a radiation dose which varies from 1*10.sup.13 to below 1*10.sup.17 ions/cm.sup.2 or with a radiation dose which varies from above 1*10.sup.17 to 1*10.sup.20 ions/cm.sup.2, c) detecting of the at least one nano or micro plastic particle comprised in said irradiated layer as formed in step b) by a detection method chosen from the group of a mass spectroscopic technique, or a Raman nanoscopic technique, or an infrared nanoscopic technique, or a charge dependent detection method, or a combination thereof, wherein the charge dependent detection method is chosen from the group of SEM, EDX and combinations thereof.

2. (canceled)

3. The method according to claim 1, wherein said conductive support has a top surface and a bottom surface and at least part of the top and bottom surfaces of said conductive support are substantially flat and parallel to one another or the top surface of the conductive support comprises at least a plurality of recesses having a width W and a depth D.

4-6. (canceled)

7. The method according to claim 1, wherein the at least one portion of said first layer possesses an area comprised between 2000 μm.sup.2 and 500 μm.sup.2.

8. The method according to claim 1, wherein the at least one portion of said first layer possesses an area comprised between 1500 μm.sup.2 and 800 μm.sup.2.

9. (canceled)

10. The method according to claim 1, wherein the conductive support is placed substantially parallel to a first horizontal plane when the heterogeneous matrix material is applied in step a) onto the at least portion of the surface of the conductive support.

11. The method according to claim 1, wherein a drying step is performed prior to the irradiation step b) thereby forming a dried layer.

12. The method according to claim 11, wherein prior or during the drying step, the conductive support is positioned from being substantially parallel to a first horizontal plane to an angle ranging from +60° to −60°, relative to a second vertical plane, even more preferably substantially parallel to a second vertical plane.

13. The method according to claim 11, wherein prior or during the drying step, the conductive support is positioned from being substantially parallel to a first horizontal plane to an angle ranging from +45° to −45°, relative to a second vertical plane, even more preferably substantially parallel to a second vertical plane.

14. The method according to claim 11, wherein prior or during the drying step, the conductive support is positioned from being substantially parallel to a first horizontal plane to an angle ranging from +30° to −30°, relative to a second vertical plane, even more preferably substantially parallel to a second vertical plane.

15. The method according to claim 11, wherein prior or during the drying step, the conductive support is positioned from being substantially parallel to a first horizontal plane to an angle ranging from +15° to −15° relative to a second vertical plane, even more preferably substantially parallel to a second vertical plane.

16-18. (canceled)

19. The method according to claim 11, wherein the drying step is carried out at a temperature between 30° C. and 50° C.

20. (canceled)

21. The method according to claim 1, wherein, the first layer has an average thickness of equal to or lower than 6 μm.

22-23. (canceled)

24. The method according to claim 1, wherein, the first layer has an average thickness of equal to or lower than 1 μm.

25. (canceled)

26. The method according to claim 1, wherein the at least portion of the first layer is irradiated with a radiation dose which varies from 1*10.sup.15 to below 1*10.sup.17 ions/cm.sup.2.

27. (canceled)

28. The method according to claim 1, wherein the at least portion of the first layer is irradiated with a radiation dose which varies from above 1*10.sup.18 to below 5*10.sup.19 ions/cm.sup.2.

29. The method according to claim 1, wherein the at least portion of the first layer is irradiated with a radiation dose which varies from above 5*10.sup.18 to below 5*10.sup.19 ions/cm.sup.2.

30. (canceled)

31. The method according to claim 1, wherein the method further comprises a step (d) of quantifying the at least one nano or micro plastic particle comprised in the irradiated layer as formed in step b) by using, a mass spectroscopic technique, or a Raman nanoscopic technique, or an infrared nanoscopic technique, or a charge dependent detection method or a combination thereof.

32. (canceled)

33. The method according to claim 1, wherein the Raman based nanoscopic technique is chosen from the group consisting of Confocal Raman spectroscopy, Tip Enhanced Raman Spectroscopy, and a combination thereof, or the infrared based nanoscopic technique is nano Infrared Absorption spectroscopy or the mass spectroscopic technique is TOF-SIMS.

34. The method according to claim 31, wherein the detection step (c) and the quantification step (d) of the micro plastic particles comprised in the irradiated layer as formed in step b) are carried out by using scanning electron microscopy.

35. The method according to claim 1, wherein the micro or nano plastic particle is made of at least a polymer selected from the group consisting of vinyl polymers, polyurethanes, polyesters, polyethers, polyamides, polyureas, polycarbonates, polydiene, conjugated polymers, mixtures and/or copolymers thereof.

Description

DESCRIPTION OF THE FIGURES

[0082] FIG. 1 is a schematic representation of an embodiment of the present invention in which a positioning step of the conductive substrate is carried out.

[0083] After formation of the first layer (L1), a drying step is preferably performed. During or prior the drying step, the conductive support is substantially parallel to a first horizontal plane P1. Then, the conductive support is positioned so as to form an angle θ ranging from +60° to −60°, preferably ranging from +45° to −45°, preferably ranging from +30° to −30°, more preferably ranging from +15° to −15° relative to a second vertical plane P2, even more preferably substantially parallel to the second vertical plane P2.

[0084] FIG. 2 is a representation of the support and said first layer (L1) after the additional steps described in FIG. 1.

[0085] The FIG. 2 shows the effect of the positioning step shown in FIG. 1 on the first layer (L1). Due to the position of the conductive support 1 resulting from said positioning step shown in FIG. 1, bottom part 2 of the support 1 is lower than the top part 2′ of the support 1. Consequently, in the layer (L1), gradients of matrix and nano and micro plastic particles 3 are created because of the combined actions of gravity and capillary forces. The concentration of nano and micro plastic particles relative to the total concentration of the heterogeneous matrix material becomes higher on the top part 2′ of the support than on the bottom part 2 of the support. As a result, the nano and micro plastic particles located on the top part 2′ of the support are more easily detected than the nano and micro plastic particles located on the bottom part 2 of the conductive support 1 during the detection step c) of the method according to the present invention.

[0086] The invention will be now described in more details with reference to the following examples, whose purpose is merely illustrative and not intended to limit the scope of the invention.

EXAMPLE 1

[0087] This example is designed as a proof of concept in which polystyrene nanoparticles were deliberately added to a commercial juice in order to prove that the method according the present invention allows the detection and quantification of the added polystyrene nanoparticles.

1 ml of commercial tomato juice was mixed with 1 microliter of polystyrene nanoparticles having a concentration of 4% in water whereby said polystyrene nanoparticles have an average particle size of 200 nm. The obtained mixture was then diluted for 50% by adding ethanol. 20 microliters of the obtained diluted mixture was then applied by spin-coating on a 1 cm.sup.2 silicon chip, previously made hydrophilic by plasma activation, thereby forming a first layer (L1) having an average thickness of 10 μm on the silicon ship. The spin coating was performed with a spin coating device at room temperature, at the speed of 2000 spins per minutes for 1 min under of a flow of air.
Then, the first layer was irradiated by Ga+ ions using FIB with an ion dose of 2*10.sup.19 ions/cm.sup.2, an acceleration voltage of 30 keV for a period of 1 minutes, thereby forming an irradiated layer (L2).

[0088] Then a portion having an area of 50*50 μm.sup.2 of the irradiated layer was visualized by SEM using secondary electron detection, 5 keV acceleration voltage and an aperture of 0.98 pA.

[0089] The results are shown in FIGS. 3 and 4. FIG. 3 shows a SEM image with a zone A that was irradiated with Ga+ ions and a zone B that was not irradiated. As it can be clearly seen, in zone A, the contrast is improved and thus the polystyrene nanoparticles are clearly visible. To the contrary, in zone B, the contrast is not good and it is not easy to distinguish the polystyrene nanoparticles. FIG. 4 shows the grey scale analysis along the lines A1 et B1. The line A1 being in the zone A and the line B1 being in zone B. As we can see the grey scale varies more in the case of line A1 than in the case of line B1 when comparing the polystyrene nanoparticles to their surroundings, which confirms that the contrast is improved in zone A which was irradiated in comparison to zone B which was not irradiated.

[0090] This is due to a combination effects resulting from the FIB Ga+ irradiation. Firstly, in zone A the polystyrene nanoparticles appear more bright than in zone B and secondly, the heterogeneous matrix surrounded the polystyrene nanoparticles is greatly reduced in zone A in comparison to zone B. This confirms that FIB Ga+ irradiation improves nano and micro plastic particles detection via a combination of at least two effects: digestion of the heterogeneous matrix and implantation of Ga+ in the nano and micro plastic particles.

EXAMPLE 2

[0091] 1 ml of a sample containing a heterogeneous matrix of animal mussels in the form of a cream was mixed with 1 microliter of polystyrene nanoparticles having a concentration of 4% in water whereby said polystyrene nanoparticles have an average particle size of 200 nm.

The heterogeneous matrix is diluted in an equal volume of ultrapure water to obtain a diluted heterogeneous matrix. A portion of 20 microliters of the diluted heterogeneous matrix is then dropped on a 1 cm.sup.2 silicon chip (previously made hydrophilic by plasma activation) so that it covers all the chip and forms a thin wet first layer presenting an average thickness of 10 μm.

[0092] The coated silicon ship was then placed in a vertical position and dried at 40° C. for 5 minutes.

Irradiation with Low Ion Dose

[0093] An area of 50*50 μm of the first layer located at the top of the ship was irradiated by Ga+ ions using FIB with an ion dose of 2*10.sup.16 ions/cm.sup.2, an acceleration voltage of 30 keV, 280 pA for a period of 1 minute, thereby forming an irradiated layer.

[0094] Then that area of 50*50 μm.sup.2 of the irradiated layer was visualized by SEM using secondary electron detection, 5 keV acceleration voltage and an aperture of 0.98 pA.

[0095] The irradiated layer was also analysed by confocal Raman scanning. A good Raman spectrum could be obtained. The obtained spectrum allow the detection, quantification and the determination of the nature of nano and micro plastic particles.

Irradiation with High Ion Dose

[0096] Then, an area of 50*50 μm of the first layer located at the top of the ship was irradiated by Ga+ ions using FIB with an ion dose of 2*10.sup.19 ions/cm.sup.2, an acceleration voltage of 30 keV, 280 pA for a period of 1 minutes, thereby forming an irradiated layer.

[0097] Then that area of 50*50 μm.sup.2 of the irradiated layer was visualized by SEM using secondary electron detection, 5 keV acceleration voltage and an aperture of 0.98 pA.

[0098] FIG. 5 contains three SEM images of the same area at different irradiation time. Image A was taken before irradiation, image B, was taken after 1 minute of irradiation and image C was taken after three minutes of irradiation. It is clear that the contrast increases with increasing irradiation time. The polystyrene nanoparticles become more bright with increasing irradiation time due to Ga+ ion implantation. Furthermore, the heterogeneous matrix surrounding the polystyrene nanoparticles is decreased due to sputtering. This confirms that FIB Ga+ irradiation improves nano and micro plastic particles detection via a combination of at least two effects: digestion of the heterogeneous matrix and implantation of Ga+ in the nano and micro plastic particles.

[0099] The irradiated layer was also analysed by confocal Raman scanning. Since only the nano and micro plastic particles and not those of the biological materials are converted to amorphous carbon, the Raman spectrum becomes the one of an amorphous carbon thereby allowing a direct way to discriminate between the micro and nano plastic particles and those related to biological materials.

EXAMPLE 3: DETECTION OF POLYMER NANOPARTICLES IN CELL MONOLAYERS

[0100] A 150-nm thick pAA layer was deposited on silicon wafer by plasma processing. The coated wafer was then cut in 10×10 mm chips, rinsed in ultrapure water and sterilized under UV light for 1 hour. After sterilization the chips were immersed in 1×PBS (Phosphate Buffered Saline) for 1 day in order to check the film stability. A549 cells (i.e. example 3A) or Balb 3T3 cells (i.e. example 3B) were seeded at a concentration of 50 000 cell/ml on 24 well plates containing the sterilized chips on the bottom. After 72 hours, the silicon chips containing A549 cells samples (i.e. example 3A) or Balb 3T3 cells samples (i.e. example 3B) were transferred to new 24 well plates and the cells were exposed to polystyrene beads of 200 nm diameter (PS-NPs, Polybead® Non-functionalized Microspheres (PS)—these particles contain a slight anionic charge from sulphate ester) for 3 days. Then, the A549 cells (i.e. example 3A) or Balb 3T3 cells (i.e. example 3B) were washed one time with PBS, fixed in 4% formaldehyde, washed three times with PBS, then with distilled water, and dehydrated in increasing concentrations (25, 50, 75 and 100%) of ethanol.

[0101] The so obtained A549 cells (i.e. example 3A) were irradiated by Ga+ ions using FIB with an ion dose of 2*10.sup.19 ions/cm.sup.2, an acceleration voltage of 30 keV for a period of 1 minute, thereby forming an irradiated layer (L2).

[0102] Then a portion having an area of 50*50 μm.sup.2 of the irradiated layer was visualized by SEM using secondary electron detection, 5 keV acceleration voltage and an aperture of 0.98 pA.

[0103] FIG. 6 contains three SEM images of the same area at different irradiation time. Image A was taken before irradiation, image B, was taken after 2 minutes of irradiation and image C was taken after five minutes of irradiation. It is clear that the contrast increases with increasing irradiation time. The adherent A549 cells are straightforwardly recognizable on the sample as they have triangular or elongated shape with darker nuclei within the cell contour. The polystyrene nanoparticles become more bright with increasing irradiation time due to Ga+ ion implantation. Furthermore, the great majority of the heterogeneous matrix (cell membranes, nucleic acids, proteins, lipids, sugars, etc.) was digested by the ion beam for a selective sputtering effect as observed in FIG. 6. This confirms that FIB Ga+ irradiation improves nano and micro plastic particles detection via a combination of at least two effects: digestion of the heterogeneous matrix and implantation of Ga+ in the nano and micro plastic particles.

[0104] The so obtained Balb 3T3 cells (i.e. example 3B) were irradiated by Ga+ ions using FIB with an ion dose of 2*10.sup.19 ions/cm.sup.2, an acceleration voltage of 30 keV for a period of 1 minute, thereby forming an irradiated layer (L2).

[0105] Then a portion having an area of 50*50 μm.sup.2 of the irradiated layer was visualized by SEM using secondary electron detection, 5 keV acceleration voltage and an aperture of 0.98 pA.

[0106] The result is shown in FIG. 7. The irradiated areas clearly show polystyrene nanoparticles (NPs) inside the cells by the increased contrast between the nanoplastics and the heterogeneous matrix. Furthermore, the great majority of the heterogeneous matrix (cell membranes, nucleic acids, proteins, lipids, sugars, etc.) was digested by the ion beam for a selective sputtering effect as observed in FIG. 7. This confirms that FIB Ga+ irradiation improves nano and micro plastic particles detection via a combination of at least two effects: digestion of the heterogeneous matrix and implantation of Ga+ in the nano and micro plastic particles.

EXAMPLE 4

[0107] 1 ml of a sample containing a heterogeneous matrix of animal mussels in the form of a cream was mixed with 1 microliter of polystyrene nanoparticles having a concentration of 4% in water whereby said polystyrene nanoparticles have an average particle size of 1 μm.

[0108] The heterogeneous matrix was digested, and separated by centrifugation and filtration. Then it was drop coated onto a silicon conductive support comprising a plurality of recesses, having a cylindrical shape with a width W of 1 μm and depth D of 1 μm, and a distance L between the recesses is 2 μm, thereby forming a first layer (L1) which was dried. Due to capillary forces, some polystyrene nanoparticles having the proper particle size (i.e. equal to or lower than 1 μm) felled inside some recesses. Aggregated polystyrene nanoparticles which were trapped in the heterogeneous matrix did not fall into any of the recesses. This is demonstrated in FIG. 8.

[0109] Then, the first dried layer (L1) was irradiated by Ga+ ions using FIB with an ion dose of 2*10.sup.19 ions/cm.sup.2, an acceleration voltage of 30 keV for a period of 1 minutes, thereby forming an irradiated layer (L2).

[0110] Then a portion having an area of 50*50 μm.sup.2 of the irradiated layer was visualized by SEM using secondary electron detection, 5 keV acceleration voltage and an aperture of 0.98 pA.

[0111] The obtained SEM image is shown in FIG. 9 where particles a, b, d, e, g, h, i, m, n, o, p, q can be seen. As can be seen, for example particles a, b, n, o, p and q appear to have a lower contrast than particles g, h, d, i and m. It can also clearly be seen that particles g, h, d, i and m are not located inside a recess. As a result of the FIB Ga+ irradiation, the polystyrene nanoparticles outside the recesses are visible because they are characterized by a larger contrast compared to the polystyrene nanoparticles inside the recesses.

The irradiated layer (L2) was also analysed by Confocal Raman Scanning Microscopy. The scanning was performed at two different levels, level Z1=−1 μm and Z2=+2.5 μm in height relative to the height of the optical focus as shown in FIG. 8. A Raman spectrum per each pixel was acquired. A Raman spectrum was obtained for each position of the scanning. In this way 4-Dimensional hyperspectral data were created (with coordinates: x,y, wavenumber, Raman intensity). By proper univariate analysis (band integration), the hyperspectral data were reduced to 3 dimensional data (with coordinates x, y, Raman intensity). The obtained 3-D data are shown as black/white images where the bright spots correspond to positions where the intensity of the benzene vibration peak (typical of the Polystyrene) spectrally located at 1005 cm.sup.−1 are larger than the one of the Silicon (2.sup.nd order peak) located at 960 cm.sup.−1.

[0112] It has been demonstrated that it is possible to discriminate between particles inside the recesses and outside. In this way it is possible to filter the particles according to their size and characterize only the particles below a certain size, separating them from bigger particles.

When the scanning is performed at the lower position (z=−1 μm) the intensity of the Raman peak the Benzene is more than double than the intensity of the Raman peak of the Silicon. Following this rule a map of the particles can be drawn and the particles inside the recesses can be evidenced.
A map with the relative intensities when the scanning is performed at the lower position (z=−1 μm), is shown in FIG. 10. The bright spots correspond to the Raman peak of the benzene and thus to the polystyrene nanoparticles. By comparing FIGS. 10 and 11, it is possible to see that the particles inside the recesses such as particles a, b, n, o, p and q are detected by Confocal Raman Scanning Microscopy (FIG. 10). A comparison of the Raman intensities for some of the particles is also shown in FIG. 11. As can be seen, when the position is at z=−1 μm, the intensity of a benzene signal for particles located inside a recess is larger than the intensity of the corresponding Si signal (2.sup.nd order). However, when a particles outside a recess can be detected when the position is at z=−1 μm, the intensity of the benzene signal is lower or substantially equal to the intensity of the Si signal, as evidenced in the case of particle g for example.
On the other hand, if the scanning is performed at a level higher than the level of the optical focus (z=+2.5 μm), more particles are detected, in particular all the particles detected. Indeed, in addition to the particles detected at a lower level such as z=−1 μm, the particles located outside the recesses are also detected. FIG. 12 represents a map with the relative intensities when the scanning is performed at a higher level than the level of the optical focus (z=+2.5 μm). As can be seen, particles a, b, c, d, e, f, g, h, i, I, m, n, o, p, q can be detected. FIG. 13 shows a comparison of the Raman intensities for some of the particles shown in FIG. 12.