DE19923761C1 - Processing of sample molecules of liquids, involves making the sample droplets stand or suspend from lyophilic or lyophobic anchors on flat support surfaces - Google Patents

Abstract

Processing of sample molecules of liquids involves making the droplets (1) stand or suspend from lyophilic or lyophobic anchors (4,3) on a flat support (2). An Independent claim is also included for support with lyophilic anchors in a lyophobic environment. The lyophilic anchors are coated with layers which reversibly or non-reversibly bind sample molecules and immobilize them temporarily.

Description

The invention relates to sample carriers for the mass spectrometric analysis of large org African molecules, method for producing the sample carrier and method for loading the sample carrier with samples of the molecules from predominantly aqueous solutions together with Matrix substance for the ionization of the substances by matrix-assisted laser desorption (MALDI).

The invention consists in the intended application areas for the solution droplets fixed on an otherwise hydrophobic sample carrier surface with defined amounts of Io to document exchange materials. The ion exchange materials can be polymerized be fixed or glued on; they are naturally hydrophilic and serve the sample droplets as an anchor for sitting on the sample carrier and for crystallization during the Drying process. They remove all of the drying solution for the MALDI process harmful metal ions and can be removed by washing in defined puf Regenerate fer solutions for reuse.

State of the art

For the analysis of large organic molecules, such as those of plastics or biopolymers, mass spectrometry has been supported with ionization through matrix Laser desorption and ionization (MALDI = Matrix-Assisted Laser Desorption and Ionization) established as a standard procedure. Usually time-of-flight mass spectrometers (TOF-MS = Time-of-flight mass spectrometer) is used, but ion cyclotron Resonance spectrometer (FT-ICR = Fourier Transform Ion Cyclotron Resonance) or high frequency quadrupole ion trap mass spectrometer (short: ion traps) are used. In the following, the large molecules to be examined are simply "Analytmo leküle ". The analyte molecules are usually very dilute in aqueous Solution, pure or mixed with organic solvents.

The "biopolymers" in particular include the oligonucleotides (ie the genetic material in various forms such as DNA or RNA), polysaccharides and proteins (i.e. the essential building blocks of the living world), including their special ones Analogs and conjugates such as glycoproteins or lipoproteins.

The choice of matrix substance for MALDI depends on the type of analyte molecules; there are well over a hundred different matrix substances have now become known. The in high The matrix substances used in excess have the task, among other things, of the ana Separate lyt molecules as possible and attach them to the sample holder during the laser shot by forming a vapor cloud without destroying the analyte molecules and  if possible without addition of the matrix molecules or other molecules into the gas phase transferred, and eventually ionize under protonation or deprotonation. For this It has proven to be advantageous to individually insert the analyte molecules in any manner into the Crystals of the matrix substances during their crystallization or at least finely divided into the Incorporate interface areas between the crystals. It seems essential be to separate the analyte molecules from each other, i.e. no clusters of analyte molecules in allow the applied sample.

A number of different methods are known for applying analyte and matrix become. The simplest of these is to pipette up a solution with analyte and matrix a cleaned, metallic sample holder. The drop of solution forms on the metal surface surface, the size of which is several times larger on purely hydrophilic surfaces corresponds to a drop diameter and the hydrophilicity of the metal surface and the Properties of the droplet, especially the solvent, depends. It forms after drying the solution a sample spot made of small matrix crystals in size this wetting area, but usually there is no uniform coverage of the benet surface shows. The crystals of the matrix usually begin in aqueous solutions on Edge of wetting the metal plate with the sample droplets to grow. You grow to Inside the wetting surface. They often form radiation-like crystals, such as for 5-dihydroxybenzoic acid or 3-hydroxypicolinic acid, which extends towards the interior of the stain often lift off the carrier plate. The center of the stain is often empty or fine Crystals covered, but often because of their high concentration of alkali salts for the MALDI ionization is useful. The loading with analyte molecules is very uneven moderate. This type of assignment therefore requires a visual assessment during MALDI ionization examination of the sample carrier surface through a video microscope that is commercially available at all mass spectrometers produced for this type of analysis can be found. Ion yield and mass resolution fluctuate in the sample spot from place to place. It is often a tedious task a favorable spot of the sample spot with good analyte ion yield and good masses Finding a solution, and only experience and trying things out helps so far.

With other application methods, the matrix substance is already before the application of the solder droplets of medium, which now only contain the analyte molecules, are present on the carrier plate the.

The applicant has now developed a method that uses very small hydrophi len anchor areas of about 200 to 400 microns in diameter in an otherwise hydro phobic surface leads to very defined crystallization areas (DE 197 54 978 A1). The drops are caught by the hydrophilic anchors, and exactly on them Hydrophilic anchors form relatively dense and closed crystal conglomerates. It could be shown that there is proof according to the reduced surface ratio limit for analyte molecules improved. It can therefore be used with smaller sample preparation  Quantities of analyte and diluted solutions are worked, an advantage that translates into better ongoing biochemical preparation steps and at lower costs leads to chemical consumption.

Under a "hydrophobic" surface it is supposed to be a liquid that is hostile to wetting repellent surface for the sample liquid used, even if it is it should not be an aqueous sample solution. In the case of an oily sample solution solution should therefore be a lipophobic surface. Usually solve however, the biomolecules are best in water, sometimes with the addition of organic, water-soluble solvents such as alcohols or acetonitrile.

Accordingly, a "hydrophilic" surface should be a wetting-friendly surface for the Type of the sample liquid used is meant, even if it is not an aq solution should act.

In principle, the hydrophobicity can be determined from the angle of attack that the liquid speed under standardized conditions at the edge of the wetting surface with the solid surface trains. For droplets on a highly hydrophobic surface, there is the case that forms no wetting surface at all and therefore there is no angle of attack like it can be found, for example, for mercury droplets on a glass or wooden plate.

The crystal conglomerates that form on the hydrophilic anchor surfaces show a fine crystalline structure that is favorable for the MALDI process. The structure gets finer the more the drying process is faster.

However, there are also disadvantages to this method. The MALDI process is through the presence of metal ions, especially alkali ions, significantly disturbed, and alkali Ions also frequently form adducts of varying sizes with the analyte molecules, the one prevent accurate mass determination. The concentration of alkali ions in the sample solution is very low due to previous cleaning steps, but still annoying. Despite the purification steps, the number of alkali is with dilute analyte concentrations to the analyte molecules generally unfavorably higher than that of concentrated solutions. The previous, relatively concentrated sample solutions were able to which the alkali ions through the crystallization process from the crystals of matrix substance and analyte molecules are kept out, they were often placed in the middle of the spot stored and by focusing the laser beam on the edge of the spot outside the MALDI happening left.

With the new application method with diluted solutions on very small hydrophilic Anchoring brings the problem with alkali ions to the fore. Sometimes it succeeds Slow drying of the sample solution droplets of the alkali ions through late crystallization preferentially to deposit on the surface of the crystal conglomerate, whereby the alkali have them removed by a few previous laser shots. Slow drying leads  however on the other hand again to a coarser-grained crystal conglomerate, which for the MALDI ionization of heavy analyte molecules is not that cheap.

The complete removal of all alkali ions from the sample solution is fundamentally extraordinary really difficult. Since the alkali ions are ubiquitous, they easily get into every pipet step, with every sample application, even if the sample solutions are left in the Solutions, either from the ambient air (especially via tiny floating particles, as they are contained in cigarette smoke, for example) or by diffusion from the vessel walls. Glass vessels have been banned from the laboratories concerned, but plastic containers also contain a considerable amount of alkalis. One of the sources for Alkali ions is the metallic sample holder itself, which despite extremely good washing in again diffuse alkali ions from the inside of the carrier to the surface.

Unfortunately, the alkali ions are more disruptive the further you go to the detection limit for the Analyte approach.

It has become known from various sources that the addition of some crumbs or Beads of ion exchange material to the droplet on the sample carrier a Ver improvement in MALDI results. This encore is difficult, it is hardly possible to apply defined quantities. The resulting irregular shape of the surface interferes with the acceleration of the ions, which a homogeneous electric field requires. It is also an attempt was made to reattach the beads after the sample solution dried scrape off. All of these measures, however, have in common that they do a lot of skillful manual work require and stand in the way of automation.

Also the use of Nafion® (Du Pont), a membrane polymer based on Po ly (perfluoroalkylene) sulfonic acid, which emerges from a solution as a membrane on surfaces can bring is known for the purification of MALDI samples on the sample carrier ge been.

Ion exchangers are manufactured industrially on a larger scale, and in the form of parts Chen powders or gravel of selected sizes and shapes on the market. Han Typical names are, for example, Permutit®, Dowex®, Wofatit® or Sephadex®.

Cation exchangers for the binding of metallic ions contain many negative-ionic groups, such as sulfonic acid groups SO ₃ ^- on the inner or outer surfaces, which are able to bind positive ions. They are usually used in protonated form, which means that the negative ion groups are saturated by H ⁺ ions at the beginning of the exchange activity. These protons are replaced by positive metal ions, which have a higher affinity for the negative ions of the exchanger. The hydrogen ions (protons) are released into the surrounding liquid, which therefore becomes increasingly acidic as the proton concentration increases. However, strongly acidic solutions prevent drying and good crystallization. It has therefore been attempted successfully not to use protonated ion exchangers, but rather at least partially exchangers occupied by ammonium ions (NH ₄ ⁺ ). At least the alkali ions, which are particularly harmful to the MALDI process, have a higher affinity for the exchanger than ammonium ions and therefore displace them from their places.

Object of the invention

It is the object of the invention to remove the interfering metal ions, in particular the alkali ions defined and automatable way to remove as completely as possible from the sample solution nen. Since some of the alkali ions from the sample holder itself get into the sample solution, the Removal take place while the drop is on the surface of the support and dries up.

Invention idea

It is the basic idea of the invention to provide the surface of an otherwise hydrophobic sample carrier with small hydrophilic covering areas as an anchor for the sample droplets, as already methodically developed by the applicant, but now to form the hydrophilic anchor areas in a cation-exchanging manner. This can be done by surface conditioning, which binds negatively charged molecular groups, for example sulfonic acid groups (SO ₃ ^- ) or carboxylic acid groups (COO ^- ), to the surface, or by applying an ion-exchange layer from a solution, or by polymerizing or sticking one Layer of ion exchange material. The layers can be applied as foils, as membranes or as coatings with fine-grained powder from regularly or irregularly shaped particles. You can either sit very thinly on the surface, or you can fill in small depressions in the surface to get a sample holder that is as flat as possible.

Ion exchange material is naturally very hydrophilic and can therefore be an excellent addition are used, hydrophilic anchor areas for the sample droplets on an otherwise hyd to form a rophobic surface.

Zeolites, for example, can be used as ion exchange material. These aluminum silicate frameworks have the advantage of not shrinking when drying. On the other hand, they release water or other solvents very slowly, so they are not particularly suitable for maintaining the high vacuum in the MALDI ion source. More suitable are polymeric ion exchangers such as the above-mentioned commercially available products, mostly based on sulfonated styrene-divinylbenol or styrene-acrylic acid copolymers, which are neutralized by a large number of H ⁺ ions after regeneration with light acids, but with The presence of metal ions immediately absorb them and exchange them for the H ⁺ ions. These porous, polymeric ion exchangers are practically insoluble, they come in the form of small spheres or irregularly shaped particles of sieved sizes.

The acidification of the sample solution by the additional H ⁺ ions can be avoided by first replacing the H ⁺ ions in an ammonium salt solution with ammonium ions (NH ₄ ⁺ ). The ammonium ions accumulating in the sample solution when the sample is applied, which are found in the crystal conglomerates after drying, do not interfere with the MALDI process; on the contrary, they appear to have a favorable influence on the MALDI process.

Because some ion exchange materials swell in liquid and shrink on drying, it can easily happen that crystals lying on the surface peel off due to the shrinking of the base be blown up. It is therefore a further development of the inventive concept in the layer Ion exchange material to store non-shrinking materials on which the Can sit on crystal conglomerates. Metal particles are particularly suitable for this purpose can also create a uniform electrical potential on the surface.

If you observe a drying droplet under the microscope, you can see in the droplet an extremely strong turbulence caused by the irregular processes of heating thrusts and evaporation are generated. The droplet remains constant Reduction continues to be fluid, only at last an almost sudden crystallization takes place instead of. The evaporation is so irregular because it is caused by saturation effects and Changes in the density of the outside air also creates external air vortices, which in turn create an incorrect evaporation distributed over the droplet surface. This strong Swirling in the droplet is particularly suitable, the existing in the droplet To transport alkali ions to the underlying ion exchanger, through which not the heat replenishment also takes place. Such a strong swirl is with not to observe a droplet applied flat on a larger hydrophilic surface, the effect of the ion-exchanging anchor surface is therefore surprisingly small Anchor surface particularly effective.

Description of the pictures

Fig. 1 shows a sequence a, b and c of schematic representations for applying the sample droplets ( 3 ) on the sample holder ( 1 ) with the hydrophilic, ion-exchanging areas ( 2 ) from the pipette tips ( 4 ) of a multiple pipette ( 5 ) subsequent drying.

• a) The pipettes have expressed the solution droplets (3) from its tip (4), the Tröpf surfaces (3) are flattened between pipette tips (4) and sample carrier (1). As a result, the droplets reach their hydrophilic, ion-exchanging anchor areas ( 2 ), even if the pipette tips ( 4 ) are not exactly above the anchor area ( 2 ), and wet the sample carrier ( 1 ) there.
• b) The pipette tips ( 4 ) are lifted off, the droplets ( 3 ) have taken the form of spheres and are located exactly above their hydrophilic, ion-exchanging anchor areas ( 2 ). The drying of the sample droplets ( 3 ) leads to a strong swirling of the liquid in the droplet, which leads the alkali ions to the ion exchanger, where they are retained and are thus removed from the liquid droplet.
• c) The sample droplets have dried up and leave small, monolithic blocks ( 6 ) of precise localization with a microcrystalline structure on the hydrophilic, ion-exchanging areas ( 2 ) of the sample carrier ( 1 ). The alkali ions are extracted from the crystals, ideal for the subsequent MALDI process.

Particularly favorable embodiments

The surfaces of metallic ones normally used for MALDI samples As a rule, sample carriers are naturally slightly hydrophilic compared to the aqueous Pro Solutions, a droplet of sample usually flows slightly apart. The hydrophilicity is generated by the hydroxyl groups, which are exposed to moist air form any metal (even on precious metals).

For reasons of simple production, it is very useful for sample carriers according to this Invention of metal or metallized plastic to remain, however, the surface hydro to make phob. This can be achieved, for example, with a wafer-thin, hydrophobic paint happen, or by sticking a thin, hydrophobic film, for example made of Tef lon®. It can also change the metal surface through a monomolecular, chemical change can be made hydrophobic, for example from fluorinated C18 chains, via sulfur broth be chemically bonded, as a certain electrical conductivity is then retained. Very thin layers of fluorinated ceramic are also known for hydrophobization become. However, there are many other equivalent methods of hydrophobizing to Example using silicones, alkylchlorosilanes or organotin compounds gene.

The hydrophilic anchor areas for the sample droplets can be created in a variety of ways become. An example is the covering of the desired anchor areas before the hydrophobicity with a washable or hydrophilic varnish. To get enough small points can do the topcoat in the form of tiny droplets with a piezo-operated dropper pipette be blown open according to the type of inkjet printer. It is an extraordinary one good local precision of the paint points can be reached. After the hydrophobization, the varnish points are simply washed off. The hydrophilic anchor areas can also be very easily generated by destroying the hydrophobic layer. That can be done by Imprint (for example, again in the manner of the inkjet printer) from chemically changed Changing or enzymatically degrading substance solutions happen by destroying with glü focal tips, but also by ablation of surface material, for example by Spark erosion or laser bombardment.  

The hydrophilic anchor surfaces expediently have diameters between 100 and 500 Micrometers, those of 200, 300 and 400 micrometers have different types Applications proved to be cheap.

If hydrophilic anchor areas have been created, the ion-exchanging areas are applied Layers not too difficult. For example, Nafion® can be applied in solution the. The solution forms small droplets on the hydrophilic anchors after evaporation leave one Nafion film each. But there can also be an adhesive in Solution can be applied with a powder of ions after drying almost complete exchange material is dusted abundantly. If a powder with particles of about 5 to 20 microns rometer diameter (mesh 1000) is used, it results after firm pressing, dry and vigorous washing a very even occupancy that is highly absorbent for alkali ions.

It is also possible to polymerize the materials directly on the hydrophilic surface. Here too, the hydrophilicity makes it easier to apply evenly.

The carrier plates with the ion-exchanging anchor areas are expedient conditioned with sample droplets before loading. To do this, they are first lightly cleaned Rem water (for example HCl) washed thoroughly to remove all metal ions and to be replaced by protons. Then the protons are placed in a solution of Ammonium chloride replaced as completely as possible by ammonium ions. The type of acid (here HCl) depends on the metal base, which of course should not be attacked become.

The sample droplets are normally applied to the sample carrier with pipettes, as shown schematically in FIG. 1. For the simultaneous application of many sample droplets from microtiter plates, multiple pipettes are used, which are moved by pipette robots in automatic pipette machines. It is therefore favorable to use sample carriers the size of microtiter plates and to adapt the grid of the hydrophilic anchor areas to the grid of the microtiter plates. It is also advantageous if the sample carriers also have the shape of microtiter plates, since they can then be processed by the commercially available pipetting robots. Since a much higher sample density can be achieved on the sample holder than is possible in microtiter plates, the grid on the sample holder can be much finer than it corresponds to the grid on the microtiter plate. It can be obtained, for example, by dividing the grid of the microtiter plates in whole numbers. The samples from several microtiter plates can then be applied to a sample carrier. The basic grid of the original microtiter plate consists of 96 small vessels with a grid of 9 millimeters in an arrangement of 8 rows by 12 columns. The microtiter plates have been further developed without changing their size, modern designs show 384 or even 1536 microvessels in a grid of 4.5 and 2.25 millimeters. These (and still fine right) grid dimensions can also be set up for the ion-exchanging anchor areas on the sample carriers; for example, the grid size of 1.5 millimeters results in 3456 anchor areas on one carrier.

The horizontal positional accuracy for the positioning of the multiple pipettes above the horizon Valley sample carrier is limited to about 200 microns. The vertical precise location speed can easily be achieved through the lateral support surfaces of the multiple pipettes and stop bolts about 50 microns can be improved.

The droplets are expediently carried out when the multiple pipette is in the position from 300 to 500 micrometers above the sample carrier. About 500 to 1500 nanoliters of the sample solution are pipetted onto the sample carrier from each pipette tip of the multiple pipette, as shown schematically in FIG. 1; the diameter of the droplets is then about 1 to 1.4 millimeters. The amount of sample solution in the pipette tip is usually closed off by a gas bubble, so there is no longer any solution in the channel of the pipette tip and the contact forces with the hydrophobic pipette tip are very low.

The droplets, which form balls with the above-mentioned diameters in the relaxed state, are now compressed between the pipette tip and the sample carrier, as can be seen from FIG. 1a. Even if the pipette tips are misaligned horizontally, the droplets can reach their respective hydrophilic anchor area and become lodged there. When the multiple pipette is lifted off, the droplets remain on the sample carrier because they have found their anchors there. They stand exactly over the anchor areas and assume their ideal round shape, as shown in Fig. 1b.

The droplets that dry form vortex-like strong liquid flows inside that bring with it that practically all metal ions at some point to the ion exchange end anchor surfaces where they are eagerly held.

On drying, the droplets leave the crystal conglomerates with the sample molecules cool exactly on the hydrophilic anchor areas, as can be seen schematically in FIG. 1c. The chunk-shaped MALDI preparations therefore have exact positioning at previously known locations as desired, their size corresponds to the focal areas of the laser beams. They also offer a high yield of analyte ions and are therefore ideally prepared for an automatic analysis.

These monolithic chunks surprisingly show a very good one and of chunks too Chunks of reproducible ionization of the introduced biomolecules, at least equally good like the cheapest places in previous preparations that were hard to find. The adduct bil Discharge with alkali ions is significantly lower, mostly no longer recognizable. Probably are the analyte molecules in a very favorable for the desorption and ionization process Location embedded at the grain boundaries of the microcrystalline structure.  

From a droplet with a volume of 500 nanoliters and a diameter of one milli meters, a small, 200 micrometer diameter hydrophilic anchor flat block also formed with a diameter of 200 micrometers. This diameter ent speaks about that of the commonly used focus areas of the laser light beam. The turned set sample amount can therefore be greatly reduced without loss of signal. A classic The preparation on hydrophilic surfaces would have a spot diameter of about two Millimeters.

Of course, the droplets can also be applied manually, as are many There are possible uses for the sample carriers shown here, like any subject man will be obvious in this area after these explanations.

It follows from the nature and purpose of the drying process that certain compositions avoid sample solution. So is an admixture of surfactants or detergents genenz harmful because it can wet the hydrophobic surface. Also an admixture of such organic solvents that cause wetting should be avoided. Here, too, it is understandable to any person skilled in the art how he has to carry out the procedure of sample preparation and pipetting in order to obtain a to avoid incorrect sample application.

Both hydrophobic and hydrophilic surfaces can be stored in reverse for a long time air their wetting properties by covering the surfaces with impurities change from the air. It is therefore advisable to vacuum the well-prepared sample carrier or store under clean protective gas.

Claims (7)

1. sample carrier for the mass spectrometric analysis of large organic molecules, which are applied in droplets of a solvent to the sample carrier and dried there together with matrix material for ionization by matrix-assisted laser desorption, characterized in that it is located on an otherwise hydrophobic, flat surface of the sample carrier small ion-exchanging anchor areas for the sample droplets to be applied. 2. Sample holder according to claim 1, characterized in that the ion-exchanging Anchor areas have a diameter between 100 and 500 micrometers. 3. Sample carrier according to one of claims 1 or 2, characterized in that it approximately has the size and shape of a microtiter plate and that the ion-exchanging An core areas form a grid that corresponds to the square basic grid of 9 millimeters for the individual tubes of a microtiter plate or one of them by integer division corresponds to the finer grid. 4. Sample holder according to one of claims 1 to 3, characterized in that in the io A supporting structure made of metal powder is incorporated into the interchangeable anchor areas. 5. A method for producing a sample carrier according to one of claims 1 to 4, characterized characterized in that first a sample carrier with a hydrophobic, flat surface and hydrophilic anchors is made, and that the hydrophilic anchors by application from ion exchange material to ion exchange surface areas be changed. 6. The method according to claim 5, characterized in that on the hydrophilic Ankerbe rich layers of ion exchange particles or membranes are glued on. 7. The method according to claim 6, characterized in that in the layers of ions from exchange material metal particles are stored.