Apparatus and method for contactless sampling of solutions and interface to mass spectrometry

Abstract

A method of mass spectrometry is disclosed comprising focusing electromagnetic radiation into a region of a liquid sample 3 below a surface of the liquid sample so as to generate one or more bubbles 4. The one or more bubbles 4 rise to the surface of the liquid whereupon one or more droplets of liquid 6 are emitted from the surface of the liquid sample. The method further comprises directing the one or more emitted droplets 6 towards an inlet of a mass spectrometer 8.

Claims

1. A method of mass spectrometry comprising: focusing electromagnetic radiation into a region of a liquid sample below a surface of the liquid sample so as to generate one or more bubbles which rise to the surface of the liquid whereupon one or more droplets of liquid are emitted from the surface of the liquid sample; and directing the one or more emitted droplets towards an inlet of a mass spectrometer or an ion source.

2. A method as claimed in claim 1, wherein the liquid sample is provided in a sample well of a microtitre plate or multi-well sample plate.

3. A method as claimed in claim 2, wherein the step of focusing electromagnetic radiation comprises directing electromagnetic radiation either: (i) through a base or lower portion of the microtitre plate or multi-well sample plate; (ii) through a sidewall or side portion of the microtitre plate or multi-well sample plate; or (iii) from above an upper surface of the liquid.

4. A method as claimed in claim 1, wherein the step of focusing electromagnetic radiation comprises directing the electromagnetic radiation through one or more focusing lenses; and wherein the method further comprises moving or translating the one or more focusing lenses in order to focus or auto-focus the electromagnetic radiation to a desired depth below an upper surface of the liquid sample.

5. A method as claimed in claim 4, wherein the step of focusing electromagnetic radiation comprises directing the electromagnetic radiation through one or more focusing lenses; and wherein the method further comprises passing a second source of electromagnetic of laser radiation through the one or more focusing lenses in order to determine the location of the upper surface of the liquid sample.

6. A method as claimed in claim 1, further comprising reflecting the electromagnetic radiation using a mirror having one or more apertures.

7. A method as claimed in claim 6, further comprising causing or allowing the one or more emitted droplets to pass through the one or more apertures.

8. A method as claimed in claim 1, wherein the electromagnetic radiation has a wavelength in the wavelength range 750-1500 nm.

9. A method as claimed in claim 1, wherein the step of directing the one or more emitted droplets towards an inlet of a mass spectrometer comprises causing the one or more emitted droplets to pass through an electric field defined by two or more electrodes, wherein the electric field is arranged to cause the one or more emitted droplets to elongate and emit oppositely charged jets towards the electrodes.

10. A method as claimed in claim 9, further comprising directing at least one jet through at least one aperture in at least one of the electrodes towards an inlet of the mass spectrometer.

11. A method as claimed in claim 1, wherein the step of directing the one or more emitted droplets towards an inlet of a mass spectrometer comprises directing the one or more emitted droplets into an open port probe sampling interface.

12. A method as claimed in claim 11, further comprising capturing the one or more emitted droplets and diluting the one or more emitted droplets into a continuous flow of solvent.

13. A method as claimed in claim 12, further comprising aspirating the flow into an electrospray ionisation ion source.

14. A method as claimed in claim 1, further comprising increasing or varying the intensity or pulse energy of electromagnetic radiation focused into the region of the liquid sample until one or more bubbles are observed or detected and/or analyte ions are detected.

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 an embodiment wherein a laser is focused from underneath a liquid sample causing or inducing droplet ejection whereupon the ejected droplets are ionised by Field Induced Droplet Ionisation (“FIDI”) and the resulting ions are then mass analysed by a mass spectrometer for detection of dissolved analytes;

(3) FIG. 2 shows another embodiment wherein the laser is focused from above the liquid sample causing or inducing droplet ejection whereupon the ejected droplets are ionised by Field Induced Droplet Ionisation (“FIDI”) and the resulting ions are then mass analysed by a mass spectrometer for detection of dissolved analytes;

(4) FIG. 3 shows another embodiment wherein the laser is focused from above the liquid sample causing or inducing droplet ejection and wherein a second laser is used to determine the surface of the liquid sample whereupon the position of a focusing lens may be adjusted in order to focus the laser to a desired depth below the surface of the liquid sample and whereupon the ejected droplets are ionised by Field Induced Droplet Ionisation (“FIDI”) and the resulting ions are mass analysed by a mass spectrometer for detection of dissolved analytes;

(5) FIG. 4 shows another embodiment wherein the laser is focused from above the liquid sample causing or inducing droplet ejection whereupon the ejected droplets are ionised by an impact ionisation ion source and the resulting ions are then mass analysed by a mass spectrometer for detection of dissolved analytes; and

(6) FIG. 5 shows a microtitre plate sample well for use in combination with a nitrogen laser or other laser source according to various embodiments and which is particularly suitable for frequent non-destructive sampling of cells or organoids.

DETAILED DESCRIPTION

(7) Various embodiments will now be described in further detail.

(8) FIG. 1 shows an embodiment wherein a laser beam 1 is focused by a microscope objective 2 which is positioned underneath a liquid solution 3 to be sampled and analysed. The liquid solution 3 may be provided in a liquid container 5 such as a sample well of a multi-well sample plate such as a microtitre plate.

(9) A pulsed laser beam 1 of suitable wavelength is focused using the microscope objective 2 (or other optical device) to a location just below the surface of the liquid solution or liquid sample 3. Selecting an appropriate location and laser pulse energy, one or more bubbles 4 of solvent vapor may be formed or otherwise generated just below the liquid surface. For example, one or more bubbles may initially be created <1 mm, 1-2 mm, 2-3 mm, 3-4 mm or >4 mm below the surface of the liquid sample 3. According to an embodiment the laser energy may be focused to a depth of 0.5 mm below the surface of the liquid sample 3. As one or more bubbles are created or otherwise generated, the one or more bubbles rise upwards to the surface of the liquid sample 3 and cause one or more liquid droplets 6 to be ejected from the liquid surface. The one or more liquid droplets 6 may be ejected from the liquid surface in a vertical direction.

(10) As will be described in more detail with reference to FIG. 2, the focusing microscope objective 2 (or other optical device) and the laser beam 1 may be located in different positions or orientations to that shown in FIG. 1. For example, according to an embodiment the focusing microscope objective 2 (or other optical means) may be located above the liquid sample 3. If the microscope objective 2 (or other optical means) is located above the liquid sample 3 then the electromagnetic radiation or laser radiation is focused below the surface of the liquid sample 3 so that one or more bubbles are generated just below the surface of the liquid sample 3.

(11) According to other embodiments the laser beam 1 may be focused through the sidewall of the liquid container 5. The liquid container 5 may be made of a transparent material and may have a general construction which is arranged and adapted to adequately transmit the laser beam 1 or other electromagnetic radiation.

(12) Referring again to FIG. 1, the one or more ejected droplets 6 may be caused to rise in a vertical direction through a pair of parallel plate electrodes 7a,7b. The electrodes may be spaced 1.4 mm apart for example or may be spaced 0.5-1.0, 1.0-1.5, 1.5-2.0, 2.0-2.5, 2.5-3.0, 3.0-3.5, 3.5-4.0, 4.0-4.5, 4.5-5.0 or >5.0 mm apart. The one or more droplets 6 may pass through an electric field defined by the two parallel plate electrodes 7a,7b. A first electrode 7a as shown on the left-hand side of FIG. 1 may be held or maintained at ground or zero volts while the other second electrode 7b on the right-hand side of FIG. 1 may be held at a relatively high voltage such as <500 V, 500-1000V, 1.0-1.5 kV, 1.5-2.0 kV, 2.0-2.5 kV, 2.5-3.0 kV, 3.0-3.5 kV, 3.5-4.0 kV or >4 kV. The pair of electrodes 7a,7b may be arranged to generate an electric field that causes the one or more droplets 6 to elongate in a horizontal direction and symmetrically emit two oppositely charged jets from either end towards both electrodes 7a,7b. The jet as shown on the left-hand side of FIG. 1 may be arranged to pass or be directed through one or more apertures, openings or holes which may be provided in the first electrode 7a. The ions thus formed may then be sampled via an inlet 8 of a mass spectrometer. Ions within the mass spectrometer may pass through a plurality of ion optical devices before being mass analysed by a mass analyser.

(13) The embodiment shown in FIG. 1 depicts only one polarity of ions being detected, but according to other embodiments ions of the opposite polarity may be simultaneously detected via a second inlet to the mass spectrometer. According to such an embodiment, ions may be transmitted through one or more apertures, openings or holes provided in the second electrode 7b and are then directed to a second inlet of the mass spectrometer.

(14) Alternatively, the polarity of the high voltage applied to the second electrode 7b can be reversed in order to achieve the same effect. Reversing the polarity of the second electrode 7b may be synchronized with droplet formation. Alternatively, the reversal of polarity may be performed sufficiently rapidly such that each individual droplet 6 can be analyzed for each polarity.

(15) According to various embodiments one or more individual droplets 6 may be trapped in an atmospheric pressure acoustic trap (not shown) such that a sustained droplet or ion stream may then be extracted. The one or more droplets may also be trapped or otherwise retained in an atmospheric pressure electrodynamic trap if the one or more droplets are charged.

(16) It will be understood by those skilled in the art that there are a variety of other ways of extracting ions from droplets. Such approaches may also be used in conjunction with the method of laser induced droplet ejection according to various embodiments.

(17) It is not necessary to utilise a coherent light source to cause droplet ejection. Other embodiments are also contemplated wherein one or more incoherent light sources may be used to cause droplet ejection from the liquid sample.

(18) FIG. 2 shows another embodiment wherein the laser source 1 is arranged above the liquid sample 3. According to this embodiment, laser induced droplet ejection is arranged from above the liquid sample 3 and may be coupled with Field Induced Droplet Ionisation (“FIDI”) for mass spectrometry detection of dissolved analyte.

(19) According to this embodiment a mirror 9 may be provided which may have one or more apertures, openings or holes provided therein. The mirror 9 may be arranged to reflect the laser beam 1 down onto the liquid sample 3 and in particular to ensure that the focal point of the laser beam 1 is just below the upper surface of the liquid sample 3.

(20) The one or more apertures, openings or holes which may be provided in the mirror 9 may be arranged so as to allow one or more droplets 6 which are emitted from the surface of the liquid sample 3 to pass therethrough. The one or more droplets 6 which pass through the one or more apertures, openings or holes provided in the mirror 9 may then be ionised. The portion of the laser beam 1 which is not reflected by the mirror 9 but which passes through the one or more apertures, openings or holes provided in the mirror 9 is omitted for clarity purposes. According to various embodiments the mirror 9 may be planar, curved or parabolic.

(21) One or more droplets 6 which pass through the one or more apertures, openings or holes provided in the mirror 9 may then be arranged to pass through an electric field defined by two parallel plate electrodes 7a,7b in a similar manner to the embodiment described above with reference to FIG. 1. A first electrode 7a as shown on the left-hand side of FIG. 2 may be held at ground or zero potential while a second electrode 7b as shown on the right-hand side of FIG. 2 may be held at high voltage or potential such as <500 V, 500-1000V, 1.0-1.5 kV, 1.5-2.0 kV, 2.0-2.5 kV, 2.5-3.0 kV, 3.0-3.5 kV, 3.5-4.0 kV or >4 kV.

(22) One or more droplets 6 which are ejected from the liquid sample 3 and which pass between the two electrodes 7a,7b may be caused to elongate in a generally horizontal direction. As a result, two oppositely charged jets may be symmetrically ejected towards the first and second electrodes 7a,7b. The jet directed at or towards the first electrode on the left-hand side of FIG. 2 may be arranged to pass through one or more apertures, opening or holes provided in the first electrode 7a. The ions may then be sampled by a mass spectrometer (“MS”). Ions may pass through an inlet 8 to the mass spectrometer and may then pass through a plurality of ion optical devices before being mass analysed by a mass analyser.

(23) Automation of Laser Focus Position and/or Laser Pulse Power

(24) For many applications multiple samples (or compound libraries) to be dispensed and/or analysed may be contained in, for example, 96 well microtitre plates or other multi-well sample plate format.

(25) The different samples present in different sample wells of a multi-well sample plate may comprise different liquids which may have different physical properties. Furthermore, the liquid levels in each individual well may differ from one another.

(26) According to various embodiments parameters such as laser power and the position of the laser focus may be adjusted for individual wells allowing rapid automated analysis of such multiple samples to be achieved.

(27) According to various embodiments the laser power and focus may be optimized automatically under microprocessor control.

(28) For example, it may be desirable to position the laser focus about 500 μm below the surface of the liquid sample 3. This can be achieved by utilising an optical system which automatically focuses a laser beam 1 either on the liquid surface or just below the liquid surface.

(29) Various different methods of auto-focusing a camera or a microscope are known which may be adapted in order to auto-focus the laser beam 1 so that the laser beam 1 focuses just below the surface of the liquid sample. Reference is made to two commercial products namely a Continuous Reflection Interface Sampling and Positioning (“CRISP”) system by Applied Scientific Instrumentation®, Eugene, Oreg. and a Laser Autofocus System by Prior Scientific®, Rockland Mass. as examples of auto-focusing systems which may be utilised according to various embodiments.

(30) FIG. 3 illustrates in more detail an embodiment wherein the laser beam is auto-focused into the liquid sample. As shown in FIG. 3, a dichroic beam splitter 30 may be placed or provided in the path of a primary laser beam 31. A second laser source 32 may be provided which may be arranged to emit a secondary laser beam. The second laser source 32 may comprise a red laser diode source 32 which may be arranged to emit a laser beam that is reflected by the dichroic beam splitter 30 such that the secondary laser beam is aligned with the primary laser beam 31. The red laser diode source 32 may emit the secondary laser beam with a wavelength of 690 nm.

(31) The primary laser beam 31 is focused by a movable microscope objective 35 and the primary laser beam 31 is reflected by a mirror 9 towards the surface of the liquid sample 3. The mirror 9 may be provided with one or more apertures, opening or holes provided therein. The one or more apertures, openings or holes may be arranged so as to allow one or more droplets 6 which are emitted from the surface of the liquid sample 3 to pass therethrough. The portion of the primary laser beam 31 which is not reflected by the mirror 9 but which passes through the one or more apertures, openings or holes is omitted for clarity purposes. The mirror 9 may be planar, curved or parabolic.

(32) The red laser light emitted from the secondary laser source 32 may be reflected by the upper liquid surface of the liquid sample. The reflected red laser light may be arranged to be imaged by the microscope objective 35 (and any necessary auxiliary optics, not shown) onto the imaging sensor of an electronic camera 34. The red reflected light is reflected by the dichroic beam splitter 30 which is arranged to reflect red light but transmit most other wavelengths. The red light is also reflected by a further beam splitter 33 on to the camera 34 or other detector. The image produced by or on the camera 34 or other detector may be analysed by software in a computer (not shown). The software may be arranged to produce a signal that will cause an actuator to move or translate the microscope objective 35 so that it focuses the red laser beam on to the liquid surface (or to a position such the red laser beam is focused just above the liquid surface or just below the liquid surface). Accordingly, the focal point of the primary laser beam 31 can also be adjusted to a desired or optimum depth.

(33) Alternatively, the location of the liquid surface relative to the microscope objective 35 may be determined by optical or ultrasonic range finding techniques and the position of the laser focus may be adjusted accordingly.

(34) Ultrasonic range finding is used, for example, with a Polaroid® SX-70 camera and such a method may be used according to various embodiments.

(35) Laser range finding techniques are also used for autofocusing in some smartphone cameras and in some surveying applications and such techniques may be used according to various embodiments in order to determine the desired or optimum focal point for the primary laser source 31.

(36) Since the primary laser beam 31 entering the rear of the focusing objective 35 is essentially parallel, then the focal point of the primary laser beam 31 will always be at a known fixed distance with respect to the mechanical front of the objective 35. Thus, it suffices to measure the distance of the objective 35 from the liquid surface.

(37) Once the surface of the liquid sample 3 is located or otherwise determined, the laser focus may then be automatically moved, adjusted or set to an appropriate distance below the surface of the liquid sample 3.

(38) Once the laser focus has been automatically moved, positioned, adjusted or otherwise set, the laser pulse energy may also be adjusted or otherwise optimised. For example, the laser pulse energy may be directly controlled by an electrical signal. According to various embodiments the pulse energy may be increased incrementally starting from a sub-threshold energy level until bubble and/or jet formation is observed by the camera 34. These events may be identified by image analysis software running in real-time in the associated computer.

(39) According to various embodiments, an auxiliary lens may be inserted in between the dichroic beam splitter 30 and the camera 34 so that reflected red laser light can be imaged by the camera 34 at different levels at and above the surface of the liquid without disturbing the focal point of the primary laser beam 31. In the case of mass spectrometric detection, the appearance of an output signal from the mass spectrometer may be utilised to indicate that an appropriate pulse energy level has been reached.

(40) Impact Ionisation Ion Source

(41) FIG. 4 shows a further embodiment wherein laser induced droplet ejection is performed from above the liquid sample 3 but the ejected droplets 6 are ionised by an impact ionisation ion source 40.

(42) A laser beam 1 is focused by a microscope objective 2 and is reflected towards the surface of the liquid sample 3 by a mirror 9.

(43) The mirror 9 may be provided which may have one or more apertures, openings or holes provided therein. The mirror 9 may be arranged to reflect the laser beam 1 down onto the liquid sample 3 and in particular to ensure that the focal point of the laser beam 1 is below the upper surface of the liquid sample 3. The one or more apertures, openings or holes are arranged so as to allow one or more droplets 6 which are emitted from the surface of the liquid sample 3 to pass therethrough. The portion of the laser beam which is not reflected by the mirror 9 but which passes through the one or more apertures or holes is omitted for clarity purposes. The mirror 9 may be planar, curved or parabolic.

(44) A co-aligned beam of low power optical radiation (not shown) may be used to locate the liquid surface. An acoustic signal may alternatively be used to locate the liquid surface.

(45) The one or more droplets 6 which pass through the one or more apertures, openings or holes provided in the mirror 9 may then be ionized by an impact ionization ion source 40. The portion of the laser beam 1 which is not reflected by the mirror 9 but which passes through the one or more apertures, openings or holes provided in the mirror 9 is omitted for clarity purposes. According to various embodiments the mirror 9 may be planar, curved or parabolic.

(46) One or more droplets 6 which pass through the one or more apertures, openings or holes provided in the mirror 9 may be arranged to pass through an inlet 8 of a mass spectrometer and may be directed by gas flow effects to impact upon an impact ionisation surface 40. The impact ionisation surface 40 may comprise an impact ionisation pin or impactor pin 40 which may be held at a relatively high voltage such as 1 kV. Droplets which impact upon the impact ionisation pin or impactor pin 40 may become ionised. The impact ionisation method is used to ionise droplets by an impact ionisation ion source for mass spectrometry detection of dissolved analytes.

(47) Microtitre Plate

(48) FIG. 5 shows a microtitre plate well having a plurality of lips or steps for use with a nitrogen laser or other laser according to various embodiments.

(49) Cells or organoids 50 may be disposed in one or more sample wells of the microtitre plate. The cells or organoids 50 may be suspended in a broth or liquid which provides nutrients to the cells or organoids 50. The cells or organoids 50 may also excrete metabolites and other substances and liquids which contribute to the liquid level in the sample well. The upper liquid level 51 of a sample in a sample well is indicated in FIG. 5. According to various embodiments, the sample well may comprise one or more lips, stepped portions or annular surfaces which surround the upper portion of the sample well. In the particular example shown in FIG. 5, three lips, stepped portions or annular surfaces are shown although it should be understood that according to various embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 lips, stepped portions or annular surfaces may be provided. The upper surface of one or more of the lips, stepped portions or annular surfaces may be coated with a metal or metallic film 52. The metal or metallic film 52 may be deposited on one or more lips, step portions or annular surfaces which may be provided on edge layers of the microtitre plate well.

(50) The one or more lips, step portions or annular surfaces may be used to eliminate any risk of directly irradiating cells or organoids 50 which may be located within the sample well. According to various embodiments a laser source is focused onto or just above the metal or metallic film 52 layer rather than being directed into the main body of the sample well thereby ensuring that the laser radiation does not impinge upon any cells or organoids 50 present in the sample well. Instead, it is assured that the laser radiation only impinges upon the liquid or broth provided in the sample well and/or any liquid excreted by the cells or organoids 50.

(51) The use of a metal or metallic surface, film or layer 52 allows use of optical wavelength lasers such as nitrogen lasers. In particular, water and various solvents do not have significant absorbance at such optical wavelengths. According to an embodiment a nitrogen laser may be used which may be arranged to emit in the ultra-violet wavelength region of the electromagnetic spectrum with a wavelength of, for example, 337.1 nm. It will be understood that nitrogen lasers and other lasers operating in the visible or near ultra-violet wavelengths are relatively inexpensive and are often less expensive than comparable infrared lasers. Furthermore, lasers such as nitrogen lasers and other lasers operating in the visible or near ultra-violet can utilise less complex and less expensive focusing and steering optics which are also relatively robust. For example, glass or fused silica optics may be used which are relatively inexpensive.

(52) It should also be understood that whilst metallic surfaces are often thought of as being primarily reflectors, such surfaces also have a significantly higher absorbance at visible or near ultra-violet wavelengths compared to water or other liquids which may be provided in the sample well. As a result, the provision of a metalised area or surface around one or more lips of the sample well enables droplet ejection to be effective with or from these layers 52. It is assumed that the liquid in the sample well is subjected to sufficient mixing and/or diffusion such that sample liquid which is sampled from the one or more lips, step portions or annular surfaces by focusing electromagnetic radiation onto a region of the lip, step portion or annular surface 52 and below the upper surface 51 of the liquid is indicative or representative of the broth or liquid surrounding the cells or organoids 50 and includes any metabolites, chemicals, substances or liquids which may have been excreted by the cells or organoids 50.

(53) It will be understood that according to various embodiments focusing the laser radiation on to the one or more lips, step portions or annular surface rather than into the main body of the sample well avoids any of the optical radiation impinging upon the cells or organoids 50 within the sample well. It will be understood that focusing optical radiation onto the cells or organoids 50 or focusing optical radiation to a region in close proximity to the cells or organoids 50 may have a deleterious or negative impact upon the cells or organoids 50 particularly if liquid from the sample well is repeatedly sampled from the sample well.

(54) Although embodiments are contemplated wherein just one lip, step portion or annular surface is provided around or surrounding an upper portion of the sample well, according to other embodiments a plurality of lips, step portions or annular surfaces are provided since the provision of multiple lips, step portions or annular surfaces eliminates, reduces or avoids any need to fill the liquid level in the sample well precisely or to maintain a certain amount of liquid within the sample well. In particular, sample liquid initially provided in the sample well may be consumed by the cells or organoids 50 over a period of time so that the upper level of the liquid 51 may change or become lower with time. Accordingly, if multiple lips, step portions or annular surfaces are provided then as the liquid level 51 changes with time or becomes lower with time, then different lips, step portions or annular surfaces may become exposed. Exposed surfaces may no longer be used since the laser radiation should ideally be focused below the upper level of the liquid. However, as the liquid level 51 changes with time, different lips, step portions or annular surfaces may be located at the optimum depth below the upper surface 51 of the liquid level. Therefore, according to various embodiments an outermost lip, step portion or annular surface may initially be utilised and as the liquid level drops with time the laser radiation may be directed onto to inner lips, step portions or annular surfaces.

(55) The provision of multiple lips, step portions or annular surfaces which may be provided at different depths relative to the upper liquid level 51 enables bubble formation to occur at a metalised surface 52 which is as close as possible to an optimum depth below the surface 51 of the liquid.

(56) Various embodiments are contemplated wherein the vertical spacing of the lips, step portions or annular surfaces is set so that a laser can always be focused on to a lip, step portion or annular surface which is a desired depth below the upper surface 51 of the liquid in a manner which is independent of the exact liquid level in the well.

(57) The metallic or metal film 52 may be overcoated with a thin layer of a polymeric material in order to prevent any toxicity effects of the metallic or metal film 52 for long term incubation.

(58) 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.