Capacitively coupled REIMS technique and optically transparent counter electrode

Abstract

A method of analysis is disclosed comprising providing a sample on an insulating substrate such as a petri dish 4 and contacting e.g. the rear surface of the insulating substrate with a first electrode 9. The method further comprises contacting the sample with a second electrode 2 and applying an AC or RF voltage to the first and second electrodes 9,2 in order to generate an aerosol from the sample.

Claims

1. A method of analysis comprising: locating a sample on a first electrode; passing light or photons through said first electrode in order to illuminate, image or analyse said sample; contacting said sample with a second electrode; and applying an AC or RF voltage to said first and second electrodes in order to generate an aerosol from said sample.

2. A method as claimed in claim 1, wherein the step of providing a sample on said first electrode comprises providing said sample in a container, a petri dish, a vial or a microtitre or microwell plate.

3. A method as claimed in claim 1, wherein said sample comprises a biological, bacterial, fungal or yeast sample or a cell line which has been cultured on to or within a culture or growth medium, wherein said culture or growth medium comprises a solid or liquid culture or growth medium.

4. A method as claimed in claim 1, wherein the step of applying said AC or RF voltage to said first and second electrodes is such that electrical energy is predominantly capacitively coupled into said sample.

5. A method as claimed in claim 1, wherein said first electrode comprises a mesh electrode.

6. A method as claimed in claim 1, wherein said first electrode comprises a substrate which is substantially optically transparent or optically translucent.

7. A method as claimed in claim 6, wherein said substrate further comprises a conductive layer or a conductive coating.

8. A method as claimed in claim 7, wherein said conductive layer or said conductive coating is substantially optically transparent or optically translucent.

9. A method as claimed in claim 1, wherein the step of applying said AC or RF voltage to said first and second electrodes causes electrical energy to be transferred into said sample so as to cause said aerosol to be generated.

10. A method as claimed in claim 1, further comprising directing at least some of the aerosol into a vacuum chamber of a mass spectrometer and ionising at least some the aerosol within a or the vacuum chamber of the mass spectrometer so as to generate a plurality of analyte ions.

11. A method as claimed in claim 10, further comprising mass analysing said analyte ions.

12. A method as claimed in claim 10, further comprising spectroscopically imaging or analysing said sample.

13. A method as claimed in claim 12, wherein the step of spectroscopically imaging or analysing said sample further comprises spectroscopically imaging or analysing said sample at substantially the same time as obtaining mass spectral data corresponding to one or more locations on or in said sample.

14. A method as claimed in claim 12, wherein the step of spectroscopically imaging or analysing said sample comprises subjecting said sample to Raman spectroscopy and/or to infra-red (“IR”) spectroscopy.

15. A method as claimed in claim 13, further comprising using said obtained mass spectral data to identify one or more biological substances, one or more bacterial strains, one or more fungal strains, one or more yeast strains or one or more cell lines located at one or more locations on or in said sample.

16. A method of rapid evaporation ionization mass spectrometry (“REIMS”) comprising a method as claimed in claim 1.

17. An apparatus comprising: a first electrode; a first device arranged for passing light or photons through said first electrode in order to illuminate, image or analyse, in use, a sample located on said first electrode; a second electrode for contacting said sample; and a second device for applying an AC or RF voltage to said first and second electrodes in order to generate an aerosol from said sample.

18. An apparatus as claimed in claim 17, wherein said first electrode comprises a substrate which is substantially optically transparent or optically translucent.

19. An apparatus as claimed in claim 17, wherein said first electrode comprises a mesh electrode.

20. An apparatus as claimed in claim 18, wherein said substrate further comprises a conductive layer or a conductive coating.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Various embodiments will now be described, by way of example only, and with reference to the accompanying drawings in which:

(2) FIG. 1 shows a schematic of an automated microbial sampling system according to an embodiment and shows a REIMS sampling probe located above a sample which is provided in a petri dish;

(3) FIG. 2 shows a schematic of an embodiment wherein a counter electrode is provided comprising a transparent conductive indium-tin oxide layer which is deposited upon a glass support plate;

(4) FIG. 3 shows in greater detail a circular glass support plate which may be coated with a transparent conductive layer in order to form a transparent counter electrode according to an embodiment;

(5) FIG. 4 shows a glass support plate located in a metal holder according to an embodiment;

(6) FIG. 5 shows a petri dish resting on a transparent counter electrode assembly according to an embodiment;

(7) FIG. 6 shows a transparent counter electrode according to an embodiment located below a petri dish which contains a culture or growth medium and a microorganism colony which has been cultured on the culture or growth medium together with a simplified equivalent electrical circuit, wherein the colony may be illuminated from below the petri dish in order to aid microorganism identification and wherein a REIMS probe may be used to sample and analyse the microorganism colony;

(8) FIG. 7 shows a total ion current (“TIC”) obtained by analysing a sample according to an embodiment; and

(9) FIG. 8 shows three mass spectra which were obtained by analysing samples using the disclosed REIMS apparatus according to various embodiments and illustrates the ability of the disclosed REIMS apparatus to be able to differentiate between different types of colonies.

DETAILED DESCRIPTION

(10) Various embodiments will now be described in the context of seeking to aid high throughput microbe identification. However, it should be understood that the various embodiments described herewith are merely illustrative and that the present invention is not intended to be limited to microbe identification.

(11) According to an arrangement it is contemplated that a two-electrode (bipolar) sampling probe might be used to analyse a sample. However, using a two-electrode sampling probe is potentially problematic since a two-electrode sampling probe has an increased physical size at the point of contact with the sample. It is also necessary to avoid electrical breakdown between the two electrodes forming the probe and to take steps to avoid potential problems due to cross contamination effects.

(12) According to other arrangements a single analysis probe (e.g. monopolar electrode) may be used to vaporize or ablate a colony and an additional probe (or counter electrode) may be used to make direct electrical and physical contact with the bulk media or agar. Although this approach has been demonstrated to work (to some extent) it suffers from the problem that it requires an additional probe (or counter electrode) to be provided which needs to contact the bulk media or agar. Furthermore, this approach suffers from the problem of having a restricted available analysis area as the heating effect scales quadratically with the current density. It has been recognised that ideally the sampling probe area should be relatively small whereas the additional probe (or counter electrode) should be relatively large so as to avoid heating at the additional probe (or counter electrode).

(13) Further embodiments which are of particular interest will now be described in more detail below. These embodiments are of particular interest since they eliminate any need for an additional probe (or counter electrode) to be placed in direct physical contact with the bulk material e.g. agar or other culture or growth medium thereby avoiding the problem of cross contamination.

(14) The various embodiments which will now be described in more detail below are of particular interest since they enable a precise region of a colony to be analysed without suffering from heating effects at the counter electrode. Furthermore, these embodiments do not suffer from potential cross contamination effects. A yet further beneficial effect is that the various embodiments described below significantly improve the ability to illuminate a sample in order to identify one or more regions of interest to be analysed.

(15) According to various embodiments a sample to be analysed (such as a microbe colony) may be located or provided on a culture or growth medium such as agar. The sample and the culture or growth medium on which it may have been grown may be located or provided in a petri dish, vial or other container such as a microtitre plate or microwell plate. The microtitre or microwell plate may, for example, comprise 6, 24, 96, 384 or 1536 wells. As will become more apparent below, the sample which has been grown on the culture or growth medium in the petri dish can be analysed in situ in the petri dish. This is of particular interest since conventional approaches require a sample portion to be scraped off the surface of the agar or other culture or growth medium and to be analysed whilst being held by a pair of electrosurgical forceps.

(16) The petri dish or other container in which the sample and culture or growth medium may be provided may be made from glass or plastic. As a result, the petri dish or other container may be essentially electrically non-conductive or may comprise an electrical insulator.

(17) According to various embodiments a first or counter electrode may be located underneath (or otherwise in close proximity to) the lower or bottom surface of a petri dish or other container so that the petri dish or other container (which is an insulator) sits or otherwise rests upon the first or counter electrode.

(18) According to various embodiments the first or counter electrode may be arranged to have a similar surface area to that of the petri dish or other container which contains the sample to be analysed.

(19) According to various embodiments a rapid evaporation ionisation mass spectrometry (“REIMS”) sampling system may be provided which comprises a first (counter) electrode which is placed below or underneath an insulating petri dish (or other container) and a second (sample probe) electrode which is brought into direct physical contact with the sample to be analysed in the petri dish (or other container).

(20) According to an embodiment a REIMS sample probe electrode may be brought into contact (from above) with a microbe colony to be analysed and identified whilst a first or counter electrode is located below the petri dish or other container containing the sample to be analysed.

(21) An AC or RF voltage may be applied between the REIMS sample probe electrode (which is direct contact with the sample to be analysed) and the REIMS counter electrode (which is in contact with the rear or lower surface of the petri dish or other container containing the sample and optional culture or growth medium). It will be apparent, therefore, that the REIMS counter electrode is not brought into direct physical with the sample to be analysed. This enables the REIMS counter electrode to be reused without suffering from any cross contamination problems.

(22) According to various embodiments the application of the AC or RF voltage to the first (counter) electrode and the second (sample probe) electrode causes a current to flow but it will be apparent that since the REIMS counter electrode is in electrical or direct physical contact with an insulator (i.e. the bottom of a petri dish) then the petri dish and the contents of the petri dish (i.e. the culture or growth medium) effectively form a dielectric of a capacitor. Therefore, as a result, electrical energy is predominantly capacitively coupled into the sample.

(23) It will be understood that this approach is therefore quite different from the known approach wherein bipolar electrosurgical forceps are used to remove a sample from a culture or growth medium and wherein both electrodes are brought into direct physical and electrical contact with the sample.

(24) In contrast to the known approach, the first (counter) electrode does not make direct physical contact with the sample to be analysed and energy is capacitively coupled into the sample in order to rapidly heat and vaporise the sample thereby generating an aerosol to be analysed.

(25) The frequency of the applied AC or RF voltage may be maximized so as to ensure that the electrical impedance of the bulk material is essentially minimised thereby resulting in maximum energy dissipation into the sample which is desired to be analysed. As a result, AC or RF energy is capacitively coupled into the microbe colony with the result that a portion of the microbe colony in the immediate vicinity of the REIMS probe electrode is vapourised so as to form an aerosol.

(26) A particular embodiment will now be described in more detail with reference to FIG. 1. FIG. 1 shows an embodiment comprising a commercial robotic system 1 such as, for example, a Tecan EVO® robotic system together with a Picolo® colony identification system. The commercial robotic system 1 has been modified so as to include a REIMS sampling probe electrode 2 and a REIMS counter electrode 9. The REIMS sampling probe electrode 2 and the REIMS counter electrode 9 are connected to an electrosurgical RF generator 6 via a feed electrode 10a and a counter or return electrode 10b respectively.

(27) Colonies grown on a culture or growth medium (such as agar) in a petri dish 4 or other container may be identified using a video camera system 3 which may, for example, be attached to a robotic arm of the robotic system 1. Images of any colonies which have grown on the culture or growth medium may then be processed by digital image processing software. According to an embodiment Picolo® digital imaging processing software may be used to process images of any colonies which have grown on the culture or growth medium.

(28) The robotic arm of the robotic system 1 may be arranged to position the video camera system 3 above the petri dish 4 so that one or more digital pictures of the colony may be recorded. The digital image processing software may then analyse the picture(s) and identify the colonies. Colonies may be selected according to various predetermined criteria or may be manually selected from a video picture.

(29) The REIMS sampling probe (or head) 2 may be moved directly above selected colonies and the position of the surface of a colony relative to e.g. the tip 5 of the REIMS sampling probe 2 may be determined using a built-in capacitive probe. According to various embodiments the tip 5 of the REIMS sampling probe 2 may be moved so that the tip 5 of the sampling probe 2 just contacts and makes physical contact with a selected colony. REIMS sampling may then be performed by briefly energizing the RF generator 6.

(30) One pole of the RF generator 6 may be connected to the tip 5 of the REIMS sampling probe 2 via the feed electrode cable 10a and the other pole of the RF generator 6 may be connected to the counter electrode 9 (located in use underneath the petri dish 4 or other container) by the counter electrode cable 10b.

(31) A RF voltage may be applied to a selected colony by, for example, applying a pulse of a RF voltage via the cables 10a,10b to the tip 5 of the REIMS sampling probe 2 and also to the REIMS counter electrode 9 located underneath the petri dish 4 or other container. As a result of capacitively coupling electrical energy into the sample, surgical fumes or an aerosol of sample material may be generated. The surgical fumes or aerosol of sample material may then be passed via a tube 8 or other conduit (which may be attached to the REIMS sampling probe 2) directly into the housing of a mass spectrometer 7. According to an embodiment the surgical fumes or aerosol may be ionised within a vacuum chamber of the mass spectrometer 7 by an ion source which may be located within the vacuum chamber of the mass spectrometer 7.

(32) In order for the image recognition software to be able to identify a colony as effectively as possible further embodiments which will be discussed in more detail below enable the petri dish 4 or other container to be illuminated from below the petri dish 4 or other container. Illumination of sample in a petri dish 4 or other container from below is beneficial since this enables clear colony boundaries to be observed by, for example, the camera system 3. Furthermore, illuminating the petri dish 4 or other container from below removes or substantially removes reflections (which would otherwise be observed) and also reduces the complexity of the overall optical system including the video camera system 3.

(33) According to various embodiments which are of particular interest and which will be described in more detail below, the counter electrode 9 may comprise a transparent substrate (e.g. glass or plastic) having a transparent conductive coating on e.g. an upper surface so as to form a transparent electrode on the transparent substrate.

(34) Other embodiments are also contemplated wherein the counter electrode 4 may instead comprise an electrically conductive mesh. Such an approach has been demonstrated to work in principle although the mesh electrode can generate shadows which can complicate the automated identification of colonies. For this reason a transparent electrode having a transparent conductive coating or layer is particularly of interest.

(35) According to embodiments which are of particular interest a transparent (or translucent) support plate or substrate may be provided which may be coated with a transparent (or translucent) conductive layer. The support plate or substrate may comprise a transparent or translucent glass plate which may coated with a transparent or translucent conductive layer on an upper surface of the glass support plate. It will be apparent, therefore, that the transparent or translucent conductive coating or layer will then be direct contact with the bottom of a petri dish 4 or other container. According to various embodiments the support plate or substrate may, for example, be coated with an indium-tin oxide (“ITO”) layer. Coating the support plate or substrate with an indium-tin oxide layer is beneficial since the indium-tin oxide layer will have a significant conductivity and will also have sufficient mechanical strength for this application.

(36) FIG. 2 shows a glass support plate 11 according to an embodiment which was fabricated. The glass support plate 11 was of optical grade and the top and bottom surfaces of the glass support plate 11 were polished. An indium-tin oxide layer 12 was then deposited onto the glass support plate 11 using a Physical Vapor Deposition (“PVD”) process so that a layer of optically transparent indium-tin oxide having a thickness of about 1-2 μm was deposited onto the upper surface of the glass support plate 11.

(37) The indium-tin oxide layer 12 is optically transparent and has sufficient transmission so as to enable bacterial colonies which may be present on a culture or growth medium such as agar located within a petri dish 4 or other container to be illuminated. The petri dish 4 may be placed in use on top of the glass support plate 11. The petri dish 4 or other container may be illuminated from below so that light passes in turn through the support plate 11, through the transparent layer 12 and then passes through the petri dish 4 in order to illuminate the culture or growth medium and any colonies growing on the culture or growth medium. Illuminating the petri dish 4 from below is particularly beneficial as it allows colony boundaries to be more easily determined and thus identified.

(38) FIG. 3 shows a glass support plate 11 according to an embodiment wherein the glass support plate 11 is arranged to have a 45° angled or bevelled edge. The angled or bevelled edge facilitates electrical contact at the side with the upper transparent conductive electrode 12 such as a layer of indium-tin oxide 12.

(39) As shown in FIG. 4, a metal plate 13 may be placed onto the glass support plate 11 and may be secured to a light box using springs. The edge of the opening in the metal plate 13 may be arranged so as to connect to the angled edge of the glass support plate 11 thereby ensuring that a reliable electrical contact is made between the metal plate 13 and the conductive electrode layer 12 on the upper surface of the glass support plate 11. The metal plate 13 also enables the glass support plate 11 to be precisely located or positioned.

(40) FIG. 5 shows an embodiment wherein a petri dish 4 or other container is located on a transparent conductive counter electrode comprising a glass support plate 11 having an upper conductive electrode layer 12. The petri dish 4 or other container may be fabricated from glass or plastic and hence is an insulator.

(41) After one or more colonies which have grown on the culture or growth medium in the petri dish 4 or other container have been optically identified by, for example, the camera system 3 then the sampling head or REIMS probe 2 may then be moved so that the tip 5 of the REIMS sampling probe 2 comes into direct contact with the colony mass with the height being determined by, for example, using a capacitive liquid level sensor located in the robotic arm of the robotic system 1.

(42) According to various embodiments the RF generator 6 may be energised for a short period of time (e.g. a pulse of 1 s) with the result that electrical energy is capacitively coupled into the sample in close proximity to tip 5 of the REIMS sampling probe 2. The result of the applied RF voltage is a rapid evaporation of the sample to form an aerosol which may then be aspirated into a vacuum chamber of the mass spectrometer 7 via a tube 8. The aerosol comprises analyte which may be ionised by, for example, an ion source located within a vacuum chamber of the mass spectrometer 7 with the result that analyte ions are then mass analysed by the mass spectrometer 7.

(43) FIG. 6 shows an embodiment showing a glass support plate 11 having an upper conductive layer 12. The glass support plate 11 and the conductive layer 12 may be both transparent (or at least translucent) and enable light from a light source (not shown) to pass through the glass support plate 11, the conductive layer 12 and the base of a petri dish 4 or other container located on the upper surface of the glass support plate 11 (and hence in direct contact with the conductive layer 12).

(44) A REIMS sampling probe 2 comprising a tubular sampling head with a sampling tip 5 is shown. The tip 5 of the REIMS sampling probe 2 is in direct contact with a colony 14 on the surface of a culture or growth medium located in the petri dish 4 or other container. The REIMS sampling probe 2 and the upper conductive layer 12 of the glass support plate 11 are shown to be in electrical contact with an RF electrosurgical generator 6.

(45) FIG. 6 also shows a simplified equivalent electrical circuit of the overall system. Current is arranged to pass through resistive layers (e.g. the colony and the agar culture or growth medium) and also through capacitive layers (e.g. the interface between the agar and the petri dish 4 and the interface between the petri dish 4 and the indium-tin oxide or other conductive layer 12). The result of capacitively coupling electrical energy into the sample is the generation of an aerosol comprising analytes which are subsequently mass analysed by a mass spectrometer 7.

(46) FIG. 7 shows a sample total ion current TIC which was obtained when a yeast colony was mass analysed according to an embodiment as described above wherein a REIMS sampling probe 2 was used to sample a yeast colony on a culture or growth medium. A high signal intensity was obtained and the signal was obtained for the duration of the applied RF voltage pulse.

(47) FIG. 8 shows three mass spectra relating to skin samples, yeast samples and normaflore samples wherein the samples were provided on a culture or growth medium and were obtained using a REIMS sampling probe 2 as discussed above. It is apparent from FIG. 8 that a high signal to noise (“S/N”) ratio was obtained and that the different mass spectra enabled different colonies to be identified. The mass spectral peak at mass to charge ratio of 693.43 corresponds to a lipid which is common to all three samples. The different mass spectral peaks correspond predominantly to the colony being analysed with the agar culture or growth medium having an insignificant impact upon the resulting mass spectra.

(48) It is apparent, therefore, that the REIMS sampling probe 2 according to various embodiments is able to identify various different biological substances on an agar substrate without needing to scrap sample from the agar substrate.

(49) Various further embodiments are also contemplated. According to an embodiment the method may be utilised with micro-REIMS imaging experiments wherein, for example, a sample may be microtome sectioned and then a thin slice may be mounted on to a glass slide.

(50) The method may also be used with simultaneous spectroscopy and rapid evaporation ionisation mass spectrometry imaging.

(51) According to other embodiments the support plate 11 may be fabricated from materials other than glass such as a plastic such as polycarbonate, poly(methyl)methacrylate or Plexiglas®. Alternatively, the support plate 11 may be fabricated from quartz or another transparent insulator material.

(52) It will also be understood that it is not essential that the support plate 11 is totally optically transparent. For example, the support plate 11 may be fabricated from an opaque, translucent or semi-translucent material.

(53) It is also not essential that the conductive layer 12 provided on the support plate 11 comprises indium-tin oxide. For example, other embodiments are contemplated wherein the conductive layer 12 may comprise other transparent conductive oxides such as aluminum-doped zinc oxide (“AZO”), indium-doped cadmium oxide, aluminum-doped zinc oxide (“AZO”), gallium-doped zinc oxide (“GZO”) or indium-doped zinc oxide (“IZO”).

(54) According to other embodiments the conductive layer 12 on the surface of the support plate 11 may comprise a thin translucent or transparent metallic layer.

(55) Alternatively, the translucent or transparent layer 12 on the surface of the support plate 11 may comprise a carbon nanotube conductive coating, a graphene film or silver nanowires covered with graphene.

(56) The transparent or translucent layer 12 on the surface of the support plate 11 may alternatively comprise a conductive polymeric layer such as polyaniline or poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (“PEDOT:PSS”) composite.

(57) It is also contemplated that the counter electrode 9,12 may comprise a conductive plastic foil. The transparent supporting plate 11 and the conductive plastic foil may be assembled using a fixture that holds them together.

(58) Other embodiments are contemplated wherein the culture or growth medium may comprise a liquid medium, a cell growth medium such as but not limited to DME (Dulbecco's Modified Eagle's medium), a modified DME medium (e.g. glucose or glutamine free), RPMI (Roswell Park Memorial Institute medium), MEM (Minimum Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium) or another liquid growth medium.

(59) The sample may be spun down using a centrifuge to form a pellet and the supernate may be discarded or used for subsequent analysis.

(60) The sample or pellet may be smeared onto a glass slide or other insulating surface, or may be anlaysed in situ.

(61) Although the present invention has been described with reference to preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the scope of the invention as set forth in the accompanying claims.