Classifications

• • H—ELECTRICITY
  • H01—ELECTRIC ELEMENTS
  • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
  • H01J49/00—Particle spectrometers or separator tubes
  • H01J49/02—Details
  • H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
  • H01J49/0409—Sample holders or containers
  • H01J49/0418—Sample holders or containers for laser desorption, e.g. matrix-assisted laser desorption/ionisation [MALDI] plates or surface enhanced laser desorption/ionisation [SELDI] plates
• • H—ELECTRICITY
  • H01—ELECTRIC ELEMENTS
  • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
  • H01J49/00—Particle spectrometers or separator tubes
  • H01J49/02—Details
  • H01J49/10—Ion sources; Ion guns
  • H01J49/16—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission
  • H01J49/161—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission using photoionisation, e.g. by laser
  • H01J49/164—Laser desorption/ionisation, e.g. matrix-assisted laser desorption/ionisation [MALDI]

Definitions

• the present invention relates to a matrix cartridge for MALDI, a process for making such a matrix cartridge and the use of the cartridge to coat a sample for MALDI.
• MALDI matrix assisted laser desorption/ionization mass spectrometry imaging
• the sample After preparing the matrix coated sample, the sample is targeted with a laser beam. By absorption of energy from the laser beam, analytes are released from the matrix (desorption) and arrive in ionised form in the gas phase (ionisation). In an analyser the mass to charge ratio (m/z) is further determined to identify, quantify and localize the molecules.
• the samples are prepared by spray coating the matrix material onto the surface of a substrate containing the sample material.
• the matrix material is dissolved in a water/organic solvent mixture, sprayed onto the sample material and dried.
• the spray drops collide and crystallize, spatial information is lost.
• the matrix layer is created by spraying several times.
• the challenge is to create a uniform matrix layer with minimal crystal size so that maximal spatial resolution can be achieved, while de-localisation is minimized.
• a sublimator apparatus is put under vacuum. Then the matrix material is heated in a heating unit until sublimation takes place. The gaseous material is then deposited on a cooled sample plate containing the sample to be analysed.
• the matrix material is put as a powder at the base of the sublimator.
• a vapour deposition source which can be a boat type, a basket type, a crucible type and a wire type.
• the drawback of adding the matrix material as a powder is that it is difficult to control the amount of matrix which influences the reproducibility of the measurements. Also, the use of a powder requires strict handling instructions for the operator, making the work more laborious and time consuming.
• the current invention provides a matrix cartridge for MALDI comprising a base layer coated with a film layer of matrix material.
• the film layer has a uniform thickness of from 0.1 ⁇ m to 1000 ⁇ m, more preferably from 0.5 ⁇ m to 500 ⁇ m, even more preferably from 1 ⁇ m to 300 ⁇ m.
• the advantage of using the matrix cartridge according to the invention in preparing MALDI samples by sublimation is that it provides a predetermined amount of matrix so that it results in an accurate and reproducible deposition of the matrix material onto the sample to be analysed.
• uniform thickness of the film layer is meant that the thickness of the film layer does not deviate more than 20% from the average thickness of the film layer.
• the thickness of the film layer anywhere on the surface of the base layer is between 80 and 120 ⁇ m.
• the thickness of the film layer does not deviate more than 10%, more preferably not more than 5% or even not more than 1%, from the average thickness of the film layer over the entire surface of the base layer.
• the film layer thickness can be determined by any method known in the art, for instance by using (electron) microscope technologies.
• a uniform thickness of the film layer on the cartridge has the advantage that it will result in uniform heating of the matrix material during the sublimation process.
• the amount of matrix material on the base layer is generally from 5 to 100 ⁇ g/mm 2 , preferably from 10 to 50 ⁇ g/mm 2 .
• the film layer of matrix material is in direct contact with the base layer over at least 70 % of the surface of the base layer, preferably at least 90%, more preferably at least 99%, or even 100%. This means that there is no further material present between film layer and base layer.
• the base layer is made of a material that it is heat conductive, so that it is particularly suitable for being heated in a uniform way in a sublimation process for preparing a sample for MALDI as will be discussed in more detail later. In particular it should be able to withstand temperatures of from 100 °C to 200 °C at reduced pressures (vacuum).
• Suitable heat conductive materials are metals and glass. Metals are preferred in view of the heat transfer capacity. Examples of metal that can be used are stainless steel, aluminium and copper.
• the base layer is made of metal and is further connected to heating means.
• the heating means can be heating element such as a heat conductive metal coil.
• the heating element has electrodes that can be connected to the sublimator.
• the heating means can be separate or the heating means can be integrated in the base layer.
• the matrix cartridge further comprises an adhesive layer.
• the matrix cartridge further comprises an adhesive layer and a release layer.
• the matrix cartridge is a layered system comprising, one adjacent to the other in the following order: film layer, base layer, adhesive layer and release layer.
• the release layer is removed from the adhesive layer and then the remaining cartridge is adhered to the heating unit in the sublimator.
• the adhesive layer is selected such that it is able to withstand the conditions in the sublimator in terms of high temperature and low pressure and that the cartridge can be easily removed after use. The selection of materials is readily available to the skilled person.
• the base plate has a size of about 10-150 mm in length and 10-150 mm in width.
• the thickness of the base plate will generally be from 0.1 to 10 mm.
• Matrix materials for MALDI are known in the art.
• the matrix materials facilitate the production of intact gas-phase ions from the material in the sample to be analysed.
• a laser beam serves as the desorption and ionization source.
• the preferred matrix material is thus capable of absorbing radiation at a specific wavelength from the laser source (typically ultraviolet or infrared laser source). Further requirements are (amongst others) that it can be soluble in appropriate solvents and that it is stable in vacuum.
• matrix materials are: ⁇ -cyano-4-hydroxycinnamic acid (CHCA), sinapic acid (4-hydroxy-3,5-dimethoxycinnamic acid), 2,5-dihydroxybenzoic acid (DHB), 2-(4-hydroxy phenyl azo) benzoic acid (HABA), succinic acid, 2,6-dihydroxy acetophenone, ferulic acid, caffeic acid (3,4-dihydroxy-cinnamic acid), 2,4,6-trihydroxy acetophenone, 3-hydroxypicolinic acid, 2-aminobenzoic acid, nicotinic acid, trans-3-indoleacrylic acid, isovanillin, dithranol and ⁇ -carboline (Norharmane). Due to ease of use and availability, preferred matrix materials are DHB, sinapic acid and Norharmane.
• the total amount of matrix material in the film layer is generally from 10 to 500 mg, preferably 30 to 300 mg.
• the invention further relates to a sealed container comprising the matrix cartridge for MALDI as described above and an inert gas.
• the sealed container consist of a reservoir suitable for containing the matrix cartridgde and a seal layer, that closes of the reservoir.
• the seal layer is designed such that it hermetically closes of the reservoir and that it can easily be removed from the reservoir when it is needed to take out the matrix cartridge from the container.
• the present invention provides a process for making the matrix cartridge for MALDI as described above.
• the process comprises the steps of:
• the base layer and matrix material are as defined above.
• any solvent suitable for creating a solution of the matrix material that can be sprayed is suitable.
• suitable solvents are: methanol, ethanol, water, chloroform, acetonitrile and mixtures thereof.
• the concentration of the matrix material in the solvent can be determined by the skilled person. In order to achieve a relatively fast process, the concentration should be as high as possible. For example, the concentration can be from 5 to 150 mg/ml.
• the equipment for spraying the solution can be standard equipment available to the skilled person that allows to quickly spray a uniform layer of the matrix solution.
• An example is a HTX TM-sprayer.
• the process comprises the steps of
• the matrix cartridge is manufactured using a sublimation process.
• the base layer and matrix material are as defined above.
• the equipment for sublimation is known in the art.
• One example is a Matrix Vapor Deposition System iMLayer from Shimadzu.
• the source of matrix material for this process can be the matrix material in solid (powder) form. It can be added to the sublimator in a suitable container, for instance a boat type container.
• the matrix material will be heated to a suitable temperature to bring it in a gaseous state, in particular from 80 °C to 250 °C.
• the base layer is cooled to a low temperature so as to condense the matrix material on the base layer. In general the temperature will be from -80°C to 40 °C.
• the reduced pressure in the vacuum chamber is from 0.1 to 1000 Pa, preferably from 1 to 10 Pa.
• the sublimator is brought back to normal temperature and pressure. For example, first the base layer is brought back to room temperature, then the pressure is brought back up to normal, followed by stopping the heating of the source of matrix material.
• the matrix cartridge of the invention can be used to coat a sample to be analysed by MALDI with the matrix material using a sublimation technique.
• the sample to be analysed can be biological material, but can also be a polymer, organic or inorganic coating or a medicinal product (drug).
• the present invention thus also relates to a process for coating a sample for MALDI with a matrix material, comprising the steps of:
• the matrix cartridge is as described above.
• the cartridge will be heated to a suitable temperature to bring it in a gaseous state, in particular from 80 °C to 250 °C.
• the substrate is cooled to a low temperature so as to condense the matrix material on the substrate. In general the temperature will be from -80°C to 40 °C.
• the reduced pressure in the vacuum chamber is from 0.1 to 1000 Pa, preferably from 1 to 10 Pa.
• the time for deposing the matrix material is generally in the order of minutes, e.g. from 0.5 to 30 minutes, preferably 0.5 to 5 minutes.
• the deposition time can be determined experimentally based on the signal obtained from the tissue. It can also be determined based on weight per area or density via light scattering.
• the sublimator is brought back to normal temperature and pressure. For example, first the substrate is brought back to room temperature, then the pressure is brought back up to normal, followed by stopping the heating of the matrix cartridge.
• the matrix cartridge of example 2 was used to coat a sample of mini-pig intestine cryosections on a glass substrate by sublimation, resulting in a uniform coating of the matrix on the sample.
• the matrix cartridge of example 6 was used to coat a glass substrate by sublimation, resulting in a uniform coating of the matrix on the sample.

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• Physics & Mathematics (AREA)
• Optics & Photonics (AREA)
• Chemical & Material Sciences (AREA)
• Analytical Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Plasma & Fusion (AREA)
• Sampling And Sample Adjustment (AREA)

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Cited By (1)

Citations (2)

• 2018
  • 2018-08-31 EP EP18192007.5A patent/EP3618097A1/fr not_active Withdrawn

Patent Citations (2)

Non-Patent Citations (2)

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