Separation of liquid in droplets and sedimented material enclosed therein

Abstract

The invention relates to methods for drawing-off liquid from individual droplets which are in a predefined arrangement on a flat substrate and have sedimented material enclosed in them. A mask of an absorbent material comprising a pattern of indentations or holes which corresponds at least partially to the regular arrangement of the individual droplets, or a stiff, rigid plate of an absorbent material is positioned above the flat substrate in such a way that the droplets come into contact with the absorbent material peripherally so that liquid is drawn off there-into. The invention also relates to a mask of an absorbent material with a substantially rectangular shape which has a predefined pattern of indentations or holes for the purpose of separating liquid and sedimented material enclosed therein.

Claims

1. A method for drawing-off liquid from individual droplets which are in an arrangement on sample spots of a flat substrate and contain sedimented material, the method comprising: positioning a mask of an absorbent material above the flat substrate, the mask comprising a pattern of indentations or holes that corresponds to the arrangement of the individual droplets in such a way that each sample spot has a respective one of said indentations or holes opposite it; and lowering the mask so that edges of the indentations or holes come into contact with peripheral parts of the individual droplets such that liquid is drawn off into the absorbent material.

2. The method according to claim 1, wherein a metal or ceramic plate is used as the flat substrate.

3. The method according to claim 1, wherein 48, 96, 384 or 1536 of said individual droplets are arranged in a regular pattern on the flat substrate.

4. The method according to claim 1, wherein the sedimented material enclosed in the individual droplets contains microorganisms.

5. The method according to claim 1, wherein the volume of any one of said droplets is approximately between one and twelve microliters.

6. The method according to claim 1, wherein the mask and the flat substrate are moved laterally relative to each other to ensure that all of said individual droplets come into contact with the corresponding indentations or hole edges.

7. The method according to claim 1, wherein each of said sample spots comprises a hydrophilic area on which is located one of said droplets, a size of each hydrophilic area limiting a maximum diameter of a droplet located thereupon to being larger than a diameter of a respective one of said indentations or holes that is opposite it.

8. The method according to claim 1, wherein the individual droplets comprise at least one of nutrient liquid and washing liquid.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention can be better understood by referring to the following illustrations. The elements in the illustrations are not necessarily to scale, but are intended primarily to illustrate the principles of the invention (mainly schematically). In the illustrations, the same reference numbers designate corresponding elements in the different views.

(2) FIG. 1 is a schematic diagram of an example embodiment for a mask of an absorbent material with 96 holes (arranged in 8 rows and 12 columns).

(3) FIGS. 2A-2C give a schematic illustration of an embodiment of the methods.

(4) FIG. 3 is a schematic diagram of an example embodiment for a mask of an absorbent material with indentations instead of holes.

(5) FIGS. 4A and 4B provide a schematic illustration of the use of a mask with corresponding frame or vertical guide for the purpose of alignment and guidance.

(6) FIGS. 5A-5C present another schematic illustration of a further embodiment of the methods.

DETAILED DESCRIPTION

(7) While the invention has been illustrated and explained with reference to a number of different embodiments, those skilled in the art will recognize that various changes in form and detail may be made to it without departing from the scope of the technical teaching as defined in the appended claims.

(8) FIG. 1 depicts a plan view of a mask of rectangular form (10), which has an array of 96 holes (12) arranged in a nine-millimeter grid in an arrangement of eight rows by twelve columns. Smaller (e.g. 48) or larger (e.g. 384) arrays are also conceivable, however. The array can generally correspond to the arrangement of sample spots on a conventional, standardized sample support for ionization by means of matrix assisted laser desorption (MALDI). The dimensions of the mask (10) can be 127.76 mm (length)×85.48 mm (width), corresponding to a microtitration plate, with a thickness of around two to five millimeters.

(9) FIG. 2A shows an arrangement of equal droplets (14) in a row of eight on a flat substrate which can correspond to a MALDI sample support plate (16). A mask (10) of an absorbent material, which has an array of holes (12) arranged opposite all the droplets (14), is positioned above the flat substrate. Each sample spot on the support plate (16) thus has a hole opposite it (12). The mask (10) is moved slowly towards the substrate, whereby the droplets (14) come into contact with the absorbent material at the hole edges once the mask has been lowered to a certain point so that the liquid is removed from the droplets (14) laterally via capillary forces, FIG. 2B.

(10) This lowering movement may end when the mask (10) is lying on the flat substrate, as depicted; it is also possible to keep the mask (10) slightly above the substrate without coming into contact. This can prevent a lateral spread of droplet liquid in the gap between mask (10) and substrate, which could lead to the mutual contamination of the individual droplets (14). To ensure that each droplet (14), even when it is applied slightly asymmetrically or does not cover the whole sample spot, comes into contact with the absorbent material of the mask (10), the mask (10) can be moved laterally to and fro slightly, as indicated by the double-headed arrow (18).

(11) The lateral absorption of the liquid from the droplet, starting from the middle of the droplet, is completed in a very short time, usually a few seconds up to around one minute at the most. Afterwards, the mask (10) can be lifted again and removed, FIG. 2C. The liquid removed is safely held in the capillary matrix of the mask (10), so there is no danger that it will drop out again as it is being lifted and contaminate the flat substrate. On the contrary, it is a very safe and reliable way to remove the liquid. The partially saturated mask (10) is typically disposed of as a consumable, which is advantageous particularly for applications in microbiology. It could also be washable and then re-usable where appropriate, however.

(12) Sedimented material, such as microorganisms, which is enclosed in the droplets (14) is not removed when the liquid is gently drawn off with the aid of capillary forces. It does not come into contact with the edges of the holes (12) (or indentations), but remains in the center of the spot on the surface of the flat substrate on which the droplets (14) were deposited. The sedimented material, now largely free of liquid, is thus available for further processing such as sample preparation for ionization by means of matrix assisted laser desorption or similar process steps.

(13) FIG. 3 shows a schematic side view of a row of eight indentations which have, for example, been pressed into a stiff and rigid mask fabric such as non-woven material. When the indentations are designed appropriately and adapted to the expected droplet shape, the liquid can be made to come into contact not only with the edge (as with the hole version) but also with the bottom of the indentation, or at least parts of the indentation surface, which may further accelerate the removal process. Since the droplets are not visible through the mask in this embodiment, care must be taken that the mask is aligned correctly—particularly when this is done manually—with the array of droplets on the flat substrate.

(14) In a preferred embodiment, the dimensions of the strips between the indentations or holes, compared to the spacing of the indentations or holes themselves, are chosen such that the liquids absorbed from the different individual droplets do not run into each other, thus preventing cross-contamination. Furthermore, the thickness of the mask and the width of the side edge are preferably dimensioned so that the liquid is not drawn right to the top or the edges. If we assume cylindrical droplets with a volume πr.sup.2×h as our model (r=droplet radius; h=droplet height), which is drawn into a cylindrical ring around a hole, which for simplicity has the same volume 2πr×dr×h, then the ratio of ring width dr to droplet radius is given by dr/r=0.5. This means that there is no mutual penetration of liquids of neighboring individual droplets when the width of the strips is given by: s>2×dr=2×0.5×r=r. According to this simple model, the strip width is therefore preferably chosen to be larger than half the hole diameter (or indentation diameter). Similar considerations can be applied to the mask edge and the mask thickness.

(15) To make the mask (10) easier to handle, it can be inserted or clamped into a frame (20). The frame (20) can be dimensioned so as to create a flush fit around a sample support (16) which contains the array of droplets, for example, as depicted in FIG. 4A. It can be designed as a disposable article, which is disposed of together with the saturated mask (10), or can be washable and re-usable. Possible designs encompass a frame (20) with stepped inner contour, on which the mask (10) can be placed with friction locking. If the frame (20) slides down around the outer sample support contour, as depicted, contact with the liquid is established below a certain point. The frame (20) has furthermore the advantage that it provides reliable alignment and guidance of the array of holes relative to the array of droplets. If the inner contour of the frame (20) and the outer contour of the sample support (16) are not dimensioned so as to be completely flush, but have a certain amount of play, a slight lateral movement can be executed to guarantee that the liquid of all droplets comes into contact with the mask.

(16) In an alternative embodiment, the frame can be fixed to the mask. A projecting, custom-cut edge of the absorbent material can be folded and then impregnated with a plastic material, for example, which then sets to ensure stability and rigidity (monolithic version). The frame can, if appropriate, also be attached to the outer circumference of the mask using an injection molded plastic.

(17) In a version sketched in FIG. 4B, a sample support (16), as a flat substrate which supports the array of droplets, can be inserted into a vertical guide (22) which surrounds it on all sides. The mask (10) can then have similar dimensions to the sample support (16) and slide slowly downwards onto the sample support (16) from the top opening of the vertical guide. Grip recesses in the walls of the vertical guide (not shown here) can facilitate the insertion and removal of sample support (16) and mask (10).

(18) In another implementation of the principles set out herein, FIG. 5A shows again an arrangement of equal droplets (14) in a row of eight on a flat substrate which can correspond to a MALDI sample support (16), similar to FIG. 2A. A rigid, stiff plate (24) of an absorbent material, without any profile, is positioned above the flat substrate. The plate (24) is moved slowly towards the substrate, whereby the protruding parts of the droplets (14) come into contact with the absorbent material once the plate (24) has been lowered to come to rest on the spacer ridge (26), which is located laterally at a receptacle (28) accommodating the flat substrate. The distance above the flat substrate surface kept by the spacer ridge (26) can amount to about one third to about half the droplet diameter, for example. The peripheral contact facilitates the removal of liquid from the droplets (14) via capillary forces, FIG. 5B. No special alignment of plate (24) and droplets (14) is necessary in this variant.

(19) Instead of locating the spacer ridge at a receptacle (28), it could also be mounted laterally on the surface of the rigid, stiff plate (24) facing the flat substrate itself, as indicated by the dotted contour. This alternative design affords better adaptability to different droplet sizes, in particular when the plate (24) is designed as a consumable. To accelerate the aspiration of the liquid into the absorbent material of the rigid, stiff plate (24) at the points of contact, the plate (24) can be moved laterally to and fro slightly, as indicated by the double-headed arrow (18).

(20) As expounded before, the absorption of the liquid from the droplets (14) is completed in a very short time, usually a few seconds up to around one minute at the most. Afterwards, the partly saturated plate (24) can be lifted again and removed, FIG. 5C. The liquid removed is safely held in the capillary matrix of the plate (24), so there is no danger that it will drop out again as it is being lifted and contaminate the flat substrate. On the contrary, it is a very safe and reliable way to remove the liquid.

(21) Sedimented material, such as microorganisms, which is enclosed in the droplets (14) is not removed when the liquid is gently drawn off with the aid of capillary forces. By virtue of the spacer ridge (26) which keeps the surface of the rigid, stiff plate (24) at a distance, for example about one third to about half a droplet diameter above the flat substrate, it does not come into contact with the absorbent material of the plate (24) at all, but remains in the center of the spot on the surface of the flat substrate on which the droplets (14) were deposited. The sedimented material, now largely free of liquid, is thus available for further processing such as sample preparation for ionization by means of matrix assisted laser desorption or similar process steps, as has been explained before.

(22) Further embodiments of the invention are conceivable in addition to the designs described by way of example. With knowledge of this disclosure, the person skilled in the art is easily able to design further, advantageous sample processing methods for infrared spectroscopic or mass spectrometric measurement using a desorbing ionization method, which are to be contained in the scope of protection of the claims, including any possible equivalents as the case may be.