WO2014108323A1

WO2014108323A1 - Method and apparatus for depositing droplets onto a substrate

Info

Publication number: WO2014108323A1
Application number: PCT/EP2014/000005
Authority: WO
Inventors: Petra Dittrich; Martin Pabst; Simon KUESTER
Original assignee: Eidgenoessische Technische Hochschule Zurich ETHZ
Current assignee: Eidgenoessische Technische Hochschule Zurich ETHZ
Priority date: 2013-01-10
Filing date: 2014-01-06
Publication date: 2014-07-17
Anticipated expiration: 2015-07-10

Abstract

A method for depositing droplets (24) separated by a first fluid from a channel (20) onto a substrate (10) is disclosed. To prevent evaporation of the droplets and to enable combination of droplets, the substrate is covered with a second fluid, and the outlet end (26) of the channel is immersed in the second fluid. The method can be used as an interface between a separation technique, e.g., liquid chromatography, and an analytical technique such as MALDI-MS. A further chemical dimension can be added by splitting a single peak of the separation technique over a plurality of sample spots and subjecting only selected sample spots to a chemical reaction. Applications include the analysis of post-translational modifications of proteins and peptides.

Description

I

TITLE

Method and apparatus for depositing droplets onto a substrate

TECHNICAL FIELD The present invention relates to a method for depositing single droplets from a channel, in particular from a microchannel such as a capillary, onto a substrate. The present invention further relates to a corresponding apparatus.

PRIOR ART

A fundamental problem when handling very small liquid samples (in the nanoliter to picoliter range) is usually their high surface-to-volume ratio, which results in their ultrafast evaporation. Evaporation alters the content of each sample (e.g. the concentration of analytes) and ultimately leads to the drying up of the sample. For liquid samples with volumes in the nanoliter to picoliter range, this drying up usually occurs within a few seconds or even faster. This severely limits the time period in which the molecules of interest are mobile in a liquid environment and can perform reactions or be observed in a mobile state. As a consequence, the miniaturization of sample volumes restricts the time available to incubate the sample, to run a chemical reaction or to perform a biological assay. Furthermore, evaporation impedes the addition and mixing of two small liquid samples. This prevents the realization of liquid-liquid reactions and the addition of further reagents or auxiliaries such as nutrients in a cell culture medium.

EP 2 436 444 Al addresses some of these problems. Droplets exiting from the outlet of a capillary fall under the action of gravity into wells of a microliter plate. The wells are pre- filled with oil prior to droplet deposition. Since the density of the droplets is larger than the density of the oil, the droplets fall into the well, settle under the oil phase, and remain isolated from air and from each other. Thereby the droplets are protected from contamination and evaporation.

This technique necessarily requires the use of an oil that has a density lower than the density of the sample droplets. However, such oils often tend to dissolve lipophilic constituents of the droplets, and many such oils are not chemically inert. Furthermore, since the technique requires the presence of relatively deep wells in the microtiter plate, the lateral distance between sample spots cannot be decreased arbitrarily, limiting the sample spot density on the microtiter plate. In addition, due to the specific topographic structure of a microtiter plate, it is not readily possible to use the microtiter plate as a sample plate for further analytical investigations of the deposited samples with various analytical methods, such as mass spectrometry.

M. Hartmann, J. Sj6dahl, M. Stjernstrom, J. Redeby, T. Joos, and J. Roeraade, "Non- contact protein microarray fabrication using a procedure based on liquid bridge formation", Anal. Bioanal. Chem. 2009, vol. 393, 591-598 discloses a method of spotting aqueous solutions to a hydrophilic microscope glass slide under an inert liquid fluorocarbon having a density of 1.77 g/ml, which is significantly higher than the density of aqueous or organic solutions to be spotted. The glass slide was placed in the bottom of a glass cuvette filled with fluorocarbon liquid, next to a silicon chip with shallow recesses acting as anchors for large drops of different solutions. A capillary filled with fluorocarbon was employed to aspirate the solutions from the large drops on the chip. Aliquots of this solution were deposited onto the glass slide in an array. The aspiration and dispensing of fluids was accomplished by regulating the pressure in the capillary. Prior to each deposition round, the content of the capillary was emptied into a wastewater reservoir. The capillary was then flushed twice with pure water, and subsequently the desired solution was aspirated from a recess on the silicon chip. Using pressure pulses, aliquots of the aspirated solution were deposited as droplets onto the glass slide. During deposition, the vertical distance between the capillary and the glass slide was varied with the aid of a linear translation stage so as to detach droplets from the liquid column in the capillary. The distance was initially 10 μηι and was increased to 150 μηι once the solution had made contact with the glass slide. Thereby, the liquid bridge between the slide surface and the capillary was cut in two and a droplet remained on the surface. A similar deposition technique is also disclosed in the following publication: W. Villanueva, J. Sjodahl, M. Stjernstrom, J. Roeraade, and G. Amberg, "Microdroplet Deposition under a Liquid Medium", Langmuir 2007, vol. 23, 1171-1 177. These deposition techniques are rather complicated. They require that all solutions which are to be deposited are already present in the cuvette before the start of the deposition procedure. A relatively large amount of solution must be taken up into the capillary, and aliquotation by droplet formation takes place only during the deposition procedure itself. This requires a rather complicated up-and-down movement of the capillary relative to the glass slide in order to detach droplets from the liquid column in the capillary. In order to avoid cross-contamination, extensive flushing is required before another solution can be deposited. This makes it virtually impossible to prepare a sample plate having a different sample in each spot. Furthermore, the required up-and-down movements and the extensive flushing steps limit the throughput of these deposition techniques.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method for depositing droplets onto a substrate that can be carried out rapidly, that is suitable for depositing droplets which differ between each sample spot, and that is capable of achieving a high sample density while preventing evaporation and undesired interactions of the droplets with their environment as well as cross- contamination between sample spots.

This object is achieved by a method according to claim 1. Further embodiments of the invention are laid down in the dependent claims.

The present invention provides a method for depositing single droplets from a channel (in particular, from a microchannel) onto a substrate. The droplets are separated by a first fluid, which is essentially immiscible with the droplets. The method comprises, not necessarily in the following order:

(a) placing an outlet end of the channel in proximity to the substrate;

(b) delivering a stream of the first fluid containing the droplets through the channel towards the outlet end thereof; (c) depositing at least one (preferably exactly one) individual droplet from said stream to a sample spot on the substrate;

(d) changing the relative position of the substrate and the channel to place the outlet end of the channel in proximity to another sample spot; and

(e) repeating steps (c) and (d).

According to the present invention, the substrate is covered by a second fluid, and the outlet end of the channel is immersed in said second fluid during deposition of the droplets, the second fluid being essentially immiscible with the droplets.

In this method, single droplets, separated by the first fluid, are pre-formed and transferred through the channel towards the substrate where they are deposited, rather than being created only during deposition. This enables very rapid deposition of the droplets to the sample spots ("spotting"). By carrying out the spotting process under a bath of the second fluid, evaporation of the droplets is effectively prevented. The bath of the second fluid in combination with the segmentation of the droplets by the first fluid prevents the formation of "bridges" of the droplet fluid between sample spots and thus effectively prevents sample carry-over and cross-contamination. The technique readily enables the addition of further fluids to each sample spot without cross-contamination, e.g., the addition of auxiliaries or the addition of reagents for inducing chemical reactions within the droplets, including biological assays.

The droplets can consist of any kind of liquid, including pure liquids like water or organic liquids and mixtures, in particular solutions, suspensions or emulsions based on any liquid. The droplets can contain suspended matter, in particular, cells. The composition of the droplets can vary between droplets. The droplets can be created in any suitable manner. In particular, the droplets can be created by combining continuous streams of immiscible phases in a network of microfluidic channels, e.g. in a microfluidic T-junction. It is however also possible to create the droplets by a "droplet-on-demand" system as described, e.g., in J. Wu, M. Zhang, X. Li, and W. Wen, "Multiple and High-Throughput Droplet Reactions via Combination of Microsampling Technique and Microfluidic Chip", Analytical Chemistry 2012, 84, 9689-9693. The present invention is not restricted to any particular method of droplet creation. Optionally, further components can be admixed to the fluid that forms the droplets before or during combination of the immiscible phases, e.g., a chemical reagent or MALDI matrix.

The first fluid can be a liquid or a gas. The second fluid is preferably a liquid. Preferably the first fluid and the second fluid are miscible. However, it is also possible that the first fluid and the second fluid are immiscible. In this case, it is preferred that the first fluid has a lower density than the second fluid, so as to cause the first fluid to rise to the surface of the second fluid after it has left the channel. Preferably the first fluid and the second fluid are liquids of the same type (e.g., both liquids can be oils or aqueous liquids) and more preferably essentially identical liquids. In particular, both the first and the second fluid are preferably a low-viscosity oil. It is preferred that the oil is a perfluorinated oil, in particular, a perfluorocarbon such as the family of perfluorocarbons offered by 3M Company under the trade name Fluorinert™. Examples of suitable perfluorocarbons are perfluorohexane, available under the trade name Fluorinert™ FC-72; a mixture of perfluorotri-n-butylamine with perfluoro-n-dibutylmethylamine, available under the trade name Fluorinert™ FC-40; perfluoro(2-butyl-terrahydrofurane), available under the trade name Fluorinert™ FC-75; and perfluorodecalin (octadecafluoronaphthalene). However, it is also possible to use other types of liquids. For instance, the first and/or second fluid can be aqueous liquids if the droplets consist of an oil phase or any other liquid that is immiscible with said aqueous liquids such as many pure liquids, solutions or suspensions containing organic solvents.

The method of the present invention is applicable even if the first fluid, which separates the droplets, and/or the second fluid, which covers the substrate, have a higher density than the droplets themselves. This is possible because the droplets can be caused to adhere to the surface of the substrate by wetting. If the separation force that is required for separating a droplet from the substrate surface exceeds its buoyancy, the droplet will remain adhered to the substrate surface despite its buoyancy. In particular, the density of the second and/or third fluid can be larger than 1.0 gram per milliliter (i.e., larger than the density of water at normal conditions). Most perfluorinated oils have such a high density.

In order to ensure good adhesion of the droplets to the substrate, at least portions of the substrate surface (in particular, the sample spots) are preferably wettable by the droplets while having a lower affinity for the first and second fluid, and consequently, the sample spots are wetted by the droplets during their deposition in step (c). The substrate can be structured to have surface portions with different wettability for the droplets and for the first and/or second fluid. In particular, the sample spots can be wettable by the droplets while being surrounded by surface portions having a lower wettability for the droplets. . This helps to "draw" the droplets to the correct position, even if the outlet end of the channel and the sample spots are not perfectly aligned with each other. For instance, if the droplets consist of an aqueous liquid, the sample spots are preferably more hydrophilic than the surface portion that surrounds each sample spot. The substrate can be microstructured. In particular, the sample spots can have a next-neighbor center-to-center distance of less than 1 mm while being fully surrounded by regions having a lower wettability for the droplets than the sample spots. Examples of suitably microstructured substrates are provided in WO 201 1/144743 Al . The contents of this publication are incorporated herein by reference in their entirety for teaching suitable substrates for use in conjunction with the present invention. However, it is also possible to provide an unstructured substrate. The term "sample spot" is therefore to be understood broadly as referring to a selected location or site on the substrate, without implying any particular structure or chemical composition of the substrate surface at this location. In the simplest case, the substrate can be, e.g., a microscopy glass slide or a simple stainless-steel plate. At least portions of the substrate can be translucent or transparent. This facilitates subsequent investigations of the deposited samples by optical methods.

The substrate can form the bottom of a vessel for holding the second fluid. To this end, the substrate can be held in a frame that sealingly engages the substrate. The frame and the substrate can be made in one piece. If at least portions of the substrate are transparent (in particular, the part where the droplets are deposited), observation from below is facilitated. The method of the present invention can further comprise observing the substrate from below before, during and/or after the deposition of droplets to the substrate. The channel is preferably a microchannel having inner lateral dimensions below 1 mm and preferably below 500 micrometers, more preferably below 300 micrometers. The channel can be, e.g., a capillary made from glass or from a suitable plastic, e.g., from PTFE, FEP or PFA. In other embodiments, the channel can be an integral part of a microfluidic device that has been manufactured, e.g., in a MEMS process.

The distance between the outlet end of the channel and the substrate during deposition should preferably be sufficiently small to ensure that the droplets adhere to the substrate by wetting the substrate before they detach from the channel, or that the droplets readily combine with a droplet that had previously been deposited to the sample spot. To this end, the distance to the substrate or to the surface of a previously deposited droplet should be smaller than the diameter of a sphere having the droplet volume. Expressed as a mathematical formula, the distance d should be chosen as follows:

where V is the droplet volume. This is especially advisable if the first and/or second fluid have/has a density that is larger than the density of the droplets. Since the droplets are preformed before being transported through the channel, it is not necessary to carry out any complicated up-and-down movements of the outlet end of the channel relative to the substrate during deposition of the droplets. Droplets are detached automatically by the "plug" of the first fluid that follows each droplet. By choosing a suitable material for the channel walls, detachment can be further facilitated. In particular, the material can be chosen not to be wettable by the droplets. The method of the present invention allows to maintain an essentially constant distance between the outlet end of the channel and the substrate during the entire deposition of droplets to the sample spot in step (c), and optionally also during the entire change of relative position in step (d).

Preferably the stream of the first fluid containing the droplets is delivered continuously (i.e., without interrupting the flow between the deposition of consecutive droplets) during steps (c)-(e). The change in relative horizontal position in step (d) preferably takes place only during time intervals in which the first fluid that separates the droplets exits the outlet end of the channel, whereas the outlet end preferably has a fixed horizontal position relative to the substrate (i.e. it does not move in the plane defined by the substrate surface) while droplets exit the outlet end, or the horizontal moving speed of the outlet end relative to the substrate is reduced during time intervals in which the droplets exit the outlet end. This prevents splitting of the droplet across several sample spots or the application of a droplet to a larger substrate area. The stream of the first fluid containing the droplets need not necessarily have a constant flow rate, and droplets can be present in irregular intervals, i.e., they need not have a constant distance from another.

The droplets deposited on the substrate can act as micro- or nanoreactors that are chemically isolated from the environment by the second fluid (to be precise, by the resulting mixture of the first and the second fluid, if these fluids are miscible). The method of the present invention is therefore particularly well-suited for carrying out (bio-) chemical reactions or other (bio-) chemical processes (including biological assays) in the droplets or on the droplet surface and studying the products, or for studying cells in the droplets. The method can thus further comprise:

(f) allowing a (bio-) chemical reaction (including, e.g., enzymatic reactions) or a (bio-) chemical process (e.g., a crystallization process) to take place in the droplets or at their surface after deposition onto the substrate; and

(g) observing products from said reaction or process.

Observation of the products (including the outcome of any biological assay) can be carried out by any known analytical method, including microscopy and/or spectroscopy (using for example fluorescence and/or chemoluminescence for detection, e.g. using protein/DNA microarray readout systems), mass spectrometry etc.

The droplets can be modified after deposition on the substrate by adding one or more further droplets. In other words, the method allows for combining two or more droplets on the substrate. To this end, the method can further comprise:

(h) while the substrate remains covered by the second fluid (to be precise, by the resulting mixture of the first and the second fluid, if these fluids are miscible), depositing at least one (preferably exactly one) further droplet to each of a set of selected sample spots, the further droplet preferably having a different composition than the droplet(s) deposited in step (c), so as to create a combined droplet comprising a mixture.

If the substrate is intended to be used as a MALDI sample plate, the droplet(s) deposited in step (c) or the further droplet(s) deposited in step (h) can comprise a MALDI matrix. However, it is also possible to pre-load the sample spots with MALDI matrix (see below).

Instead or additionally, the further droplet(s) deposited in step (h) can comprise a reagent for inducing a (bio-) chemical reaction or process in the droplets. In particular, the droplet(s) deposited in step (c) can comprise at least one peptide or protein, and the further droplet(s) deposited in step (h) can comprise an enzyme, or vice versa.

It is possible to modify droplets only in selected sample spots, i.e., to deposit further droplets only to selected sample spots. In this manner, e.g., reactions can be induced in selected droplets only, and a direct comparison can be made between modified (e.g., "reacted") droplets containing the reaction products, side-by-side with unmodified (e.g., "unreacted") droplets, on different sample spots on one and the same substrate. This adds a further dimension (a "chemical dimension") to the analytical arsenal available for investigating fluids. Especially if the droplet composition varies slowly from one sample spot to the next, as it can be the case when the droplets comprise an eluate from a separation technique (see below), it may be advantageous to interleave or interdigitate modified and unmodified droplets. In this context, the term "interleaved" is to be understood as meaning that each sample spot to which a further droplet is applied is no more than Five, preferably no more than three sample spots, most preferably exactly one sample spot away from a sample spot to which no further droplets have been deposited. In this manner it can be ensured that for each modified droplet there is a nearby unmodified droplet having a similar composition as the modified droplet before modification. This enables a direct comparison between droplets of similar chemical composition before and after modification.

It is also possible to pre-load the sample spots on the substrate with one or more substances and to dry the pre-loaded sample spots before covering the substrate with the second fluid. Pre-loading can be carried out by any known method, including coating techniques, pipetting (e.g. by the use of pipetting robots) or by droplet-based deposition techniques, including the method of the present invention, with or without the presence of a bath of second fluid. The substances that are pre-loaded to the sample spots can include, e.g., auxiliary substances such as a MALDI matrix, they can include one or more reagents for chemical reaction with the droplets to be subsequently deposited on the sample spots, or they can constitute a sample of interest for modification by the droplets and subsequent investigation. If the pre-loaded substance comprises a sample of interest, the droplets that are subsequently deposited in step (c) or step (h) can contain a chemical reagent for reaction with the pre-loaded samples. In particular, the pre-loaded samples can comprise at least one protein or peptide, and the chemical reagent deposited in step (c) or step (h) can comprise at least one enzyme, or vice versa. Again, it is possible to deposit droplets in step (c) or step (h) to selected sample spots only, these spots being optionally interleaved with sample spots to which no droplets are applied, to achieve the above-mentioned additional "chemical dimension".

The method of the present invention can be employed as an interface between two different analytical techniques, in particular, between a separation technique such as liquid chromatography (LC) or capillary electrophoresis (CE) and a second analytical technique such as mass spectrometry (MS) and/or optical analysis techniques such as microscopy and/or spectroscopy. The separation technique can be a high-performance nano- or microflow technique. In particular, the droplets that are deposited either during the preloading step or during step (c) or step (h) may comprise the eluate of a CE or LC apparatus, in particular, of a so-called nano-LC apparatus (i.e. an LC apparatus having a flow rate in the range below 1000 nanoliters per minute). In practice, this can be achieved by eluting the fluid that will later form the droplets from a CE apparatus or from an LC column and combining a continuous stream of this eluate with a continuous stream of the first fluid before step (b). Optionally, further components can be admixed before or during combination of the immiscible phases, e.g., a chemical reagent or MALDI matrix.

The method can further comprise removing the substrate from the bath of the second fluid (and the first fluid that has possibly entered the bath during step (c)) after step (e), or removing the first fluid and the second fluid from the vessel in which the substrate is received or whose bottom is formed by the substrate, and evaporating any remaining first fluid and second fluid from the substrate and drying the droplets on the sample spots, followed by obtaining optical images, optical spectra and/or mass spectra of the dried droplets on the sample spots.

The addition of a further "chemical dimension", as explained above, is particularly useful if the method of the present invention is used as an interface between a separation technique such as LC or CE and another analytical technique such as MS. To explain just one example in more detail, post-translational modifications (e.g., glycosylation and phosphorylation as well as methylations, acetylation or any kind of esterifications) of peptides and proteins often make it difficult to identify these in mass spectrometry due to the resulting increase of mass and of heterogeneity. The method of the present invention can be used to fractionate the eluate into droplets at such a high frequency that an eluted peak is typically fractionated into multiple droplets. This is a distinct advantage of droplet- based methods. By subjecting the peptide or protein contained in this peak to an enzymatic reaction (e.g., by addition of a glycopeptidase (e.g. PNGAseA and/or PNGAseF), addition of an O-glycosidase (e.g. O-glycanase), addition of an endoglycosidase (e.g. ENDO F2), addition of an exoglycosidase (e.g. neuraminidases), addition of an alkaline phosphatase or acid phosphatase or addition of an esterase (e.g. methyl- or acetyl-esterases)) after the separation by LC but before analysis by MS, the modifications can be selectively removed. Since this is done only after deposition of the droplets to the substrate, and, e.g., only to selected sample spots, it becomes possible to detect the unmodified peptide or protein side- by-side with the modified peptide or protein. In the same manner, reactions could be carried out using transferases (e.g. sialyltransferases) or synthetases/synthases (e.g. core O- glycan synthases). This facilitates screening for potential substrate (acceptor) substances in an LC separated and fractionated run of lowest amounts of analytes. Furthermore, the method of the present invention allows a "results determined analysis workflow" of the fractionated peaks (analytes). Results of a pre-scan of one part of the fractionated peak can be used to design the next chemical experiment performed on the same part of the fractionated peak or on any other part of the fractionated peak. Since more than five fractions per peak are expected, a higher number of consecutive experiments in unrestricted time frames can be arranged.

The method of the present invention can not only be employed for applying the eluate of the separation technique, but instead or in addition for applying a further reagent to sample spots that have been pre-loaded with eluate. In particular, the present invention provides a method for interfacing a separation technique with an analytical technique, the method comprising:

pre-loading sample spots on a substrate with samples by depositing an eluate of the separation technique onto the sample spots in such a manner that a fraction of the eluate that corresponds to a single analyte peak is fractionated over a plurality of sample spots; and

depositing droplets of a reagent from a channel onto a sample spot, the droplets being separated by a first fluid, the first fluid being essentially immiscible with the droplets, the reagent being a chemical or biochemical reagent for reaction with the preloaded samples on the sample spots,

wherein depositing the droplets comprises:

(a) placing an outlet end of the channel in proximity to a sample spot on the substrate;

(b) delivering a stream of the first fluid containing the droplets through the channel towards the outlet end thereof;

(c) depositing at least one individual droplet from said stream to a sample spot on the substrate;

(e) repeating steps (c) and (d). Preferably, the droplets are deposited in step (c) only to selected sample spots. The selected sample spots are preferably interleaved with sample spots to which no droplets are applied in step (c). A different reagent can be pre-loaded or subsequently applied to those sample spots to which no droplets are applied in step (c). The substrate is preferably covered by a second fluid, and the outlet end of the channel is immersed in said second fluid during deposition of the droplets, the second fluid being essentially immiscible with the droplets.

The separation technique can be, e.g., liquid chromatography or capillary electrophoresis, in particular, a high performance (micro- and nanoflow) separation technique such as nano-LC. The eluate is preferably deposited onto the substrate by a deposition technique based on fluid-separated droplets. In particular, a method with steps (a)-(e) as defined above may not only be employed to deposit the reagent droplets, but also to deposit the eluate droplets onto the substrate to pre-load the sample spots. The same fluid as for separating the reagent droplets or a different fluid may be used for separating the eluate droplets. Depositing the eluate droplets may be carried out under air or while the substrate is covered with a fluid, the outlet end of the channel that is used for depositing the eluate droplets being immersed in said fluid during deposition of the eluate droplets. If the substrate is covered with a fluid, this fluid can be the same as for covering the substrate during deposition of the reagent droplets, or it can be a different fluid. Preferably a solvent of the separation technique is evaporated after pre-loading the sample spots and before depositing the reagent droplets. This is particularly preferred if the separation technique is liquid chromatography, where often solvents or solvent additives are used that are incompatible with the subsequent reaction system. Evaporating the solvent or the solvent mixture and all volatile additives within this solvent or solvent mixture is particularly preferred if one of the analytical method comprises mass spectrometry as various solvents and additives can disrupt the analysis.

The order in which the eluate and the reagent are applied can also be reversed. In other words, the present invention also relates to a method for interfacing a separation technique with an analytical technique, the method comprising:

pre-loading selected sample spots on a substrate with a chemical or biochemical reagent; and

depositing droplets of an eluate of a separation technique onto the sample spots, the droplets being deposited in such a manner that a fraction of the eluate that corresponds to a single analyte peak is fractionated over a plurality of sample spots, the droplets being separated by a first fluid, the first fluid being essentially immiscible with the droplets, wherein depositing the droplets comprises:

(e) repeating steps (c) and (d).

The selected pre-loaded sample spots can be interleaved with^" sample spots that are not preloaded with said chemical or biochemical reagent. A different reagent can be pre-loaded or subsequently applied to those sample spots that are not pre-loaded with the chemical or biochemical reagent.

In some embodiments, the eluate can comprise at least one protein or peptide, and the chemical or biochemical reagent can comprise at least one enzyme. In some embodiments, the enzyme can act to remove a post-translational modification from the peptide or protein. In particular, the enzyme can be selected from the group consisting of glycopeptidases, O- glycosidases, endoglycosidases, exoglycosidases, alkaline phosphatases, acid phosphatases, sulfatases and esterases. In other embodiments, the enzyme can be selected from the group consisting of transferases, synthetases, and synthases.

In another aspect, the present invention provides an apparatus that is configured to carry out the method of the present invention. More specifically, the present invention provides an apparatus for depositing single droplets onto a substrate, the droplets being separated by a first fluid, the first fluid being essentially immiscible with the droplets, the apparatus comprising:

a vessel configured to receive a second fluid, the vessel having a bottom forming a substrate, so as to cover the substrate with the second fluid, or a substrate holder configured to receive the substrate and to receive a second fluid so as to cover the substrate with the second fluid;

a channel having an outlet end;

a holding structure configured to hold said channel in such a manner that the outlet end of the channel is immersible in said second fluid (to be precise, in the resulting mixture of the first and the second fluid, if these fluids are miscible) in proximity to the substrate; a transporting device for delivering a stream of the first fluid containing the droplets through the channel towards the outlet end thereof;

a detection system for determining time intervals in which first fluid that separates the droplets exits the outlet end of the channel;

a positioning device configured to change the relative position of the substrate and the channel while the outlet end of the channel is immersed in said second fluid (to be precise, in the resulting mixture of the first and the second fluid, if these fluids are miscible); and

a control device configured to operate said positioning device in such a manner that a change in relative position takes place during time intervals in which first fluid that separates the droplets exits the outlet end of the channel (but not necessarily exclusively during such time intervals).

Preferably, the relative horizontal position of the substrate and the channel is kept essentially constant during the deposition of the droplets to the substrate (i.e., no movement occurs in the horizontal plane defined by the substrate surface), or the horizontal moving speed of the outlet end relative to the substrate is reduced during time intervals in which the droplets exit the outlet end. The positioning device, the control device and the holding structure can be configured to maintain an essentially constant distance between the outlet end of the channel and the substrate during deposition of droplets to the substrate, and optionally also during any change of relative position of the substrate and the channel. The holding structure can be formed by part of the detection system, in particular, by one or more optical fibers of the detection system. The substrate holder can itself take the form of a vessel in which the substrate is received. The vessel may then act to form a bath of the second fluid, and the substrate may be fully submersible in the bath. In alternative embodiments, the substrate holder may take the form of a frame that sealingly engages and preferably surrounds the substrate, the substrate thus forming the bottom of a vessel that is laterally delimited by the frame. In such cases the second fluid will be filled into the thus-formed vessel, covering only the upper surface of the substrate. The frame may be removable (in particular, applied to the substrate without the use of adhesives) to facilitate further handling. The frame may comprise one or more sealing elements, in particular, annular sealing rings of an arbitrary shape, in particular, of a shape that is adapted to the shape of the substrate. In other embodiments, it is possible for the substrate holder or frame and the substrate to be formed in a single piece.

If at least portions of the substrate are transparent (in particular, the part where the droplets are deposited), optical surveillance of the spotting process from below is facilitated.

The same considerations apply, mutatis mutandis, for the apparatus of the present invention as for the method according to the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings:

Fig. 1 shows, in a highly schematic manner, the setup of a droplet-spotting apparatus according to the present invention;

Fig. 2 shows, in a highly schematic manner, the manner in which an apparatus according to the present invention can be coupled to a nano-LC apparatus; Fig. 3 shows, in a perspective sectional view, an embodiment in which the sample plate is held in a frame, forming the bottom of a vessel for receiving an oil bath;

Fig. 4 shows the embodiment of Fig. 3 in an exploded view (non-sectional);

Fig. 5 illustrates sample spots on a sample plate, droplets comprising a reactant having been deposited to selected sample spots only;

Fig. 6 shows results of a phosphorylation analysis of tryptic peptides; and

Fig. 7 shows results of a N-glycosylation analysis of the human IgG Fc region.

DESCRIPTION OF PREFERRED EMBODIMENTS

Figure 1 illustrates, in a highly schematic manner, an apparatus according to the present invention and the corresponding method.

As illustrated in Fig. 1, a substrate in the form of a sample plate 10 contains a large number of hydrophilic sample spots 11, separated by hydrophobic regions. The sample plate has been manufactured as described in the publication WO 201 1/144743 Al, the contents of which publication are incorporated herein by reference in their entirety for teaching sample support plates that are suitable for use in conjunction with the present invention. The sample spots are arranged in a plurality of rows, each row consisting of a plurality of sample spots whose centers are regularly spaced along the x direction. The rows themselves are regularly spaced along the y direction, which is perpendicular to the x direction, the centerline distance of the rows corresponding to half of the center distance of the sample spots within each row. Adjacent rows are shifted with respect to each other along the x direction by half of the center distance between adjacent sample spots within each row. This results in a "checkerboard"-type of arrangement of the sample spots. In this manner, a sample plate is obtained that has a high density of well-separated sample spots. The sample plate 10 is placed in a vessel 70 in the form of a flat, open basin. The vessel 70 acts to receive and hold the sample plate 10 and in this sense acts as a substrate holder. The vessel 70 is filled with a volatile perfluorinated oil, such as perfluorodecaline. In this manner, an oil bath 71 is formed. The sample plate 10 is fully immersed in the oil bath 71. The vessel 70 is mounted for horizontal movement on an x-y-stage 50 (shown only in a highly schematic manner) driven by a controller 40. Above the sample plate 10, a channel in the form of a capillary 20 is mounted, as will be described in more detail below. The outlet end 26 of the capillary 20 is also immersed in the oil bath 71.

An aqueous phase 21 containing the sample of interest and an oil phase 22 that is immiscible with the aqueous phase 21 are fed to a T-junction 23, where the aqueous stream and the oil stream are combined in such a manner that aqueous droplets 24 separated by oil plugs 25 are formed. The manner in which such droplet formation can be achieved is well known in the art. In the present case, the aqueous phase 21 is pushed towards the T- junction 23 by means of a first syringe 61 driven by a first motor 62, while the oil phase 22 is pushed towards the T-junction 23 by means of a second syringe 63 driven by a second motor 64. Both the first and the second motor are controlled by the controller 40. The two syringe pumps consisting of the syringes 61, 63 and the motors 62, 64 together form a transporting device 60 for transporting a continuous stream of oil-separated droplets 24 through the capillary 20 towards the outlet end 26 thereof. In the present example, the oil phase 22 consists of the same perfluorinated oil as the oil bath 71.

A detection system 30 determines the time intervals during which the oil plugs 25 that separate the droplets 24 exit the outlet end 26 of the capillary 20. The detection system comprises a light emitting diode 31, whose light is guided to the capillary 20 by means of an optical fiber 32 having a transverse hole through which the capillary 20 is threaded. The light transmitted through the capillary 20 is received by the second part of the optical fiber 32 and detected by a photodetector 33. Signals from the photodetector 33 are digitized and fed to the controller 40. Discrimination between droplets 24 and oil plugs 25 is achieved by performing a threshold analysis. The detection system 30 facilitates reliable deposition of the droplets even if their spacing varies significantly, or if the droplet stream is temporarily stopped. In the present embodiment, the detection system 30 also acts to hold the capillary 20 in a defined position above the sample plate 10, the optical fiber 32 forming a holding structure for the capillary 20. The entire holding structure is movable in the z direction, perpendicular to the sample plate 10, by means of a z stage 34 (shown only in a highly schematic manner). The z stage 34 is controlled by the controller 40 so as to adjust and maintain a predetermined distance between the outlet end 26 of the capillary 20 and the sample plate 10 even in case of misalignment between the upper surface of the sample plate 10 and the x-y plane defined by the x-y stage 50.

Droplets are deposited to the sample spots 11 on the sample plate 10 in the following manner. Initially, the outlet end 26 of the capillary 20 is placed in close proximity to a first sample spot 11 on the sample plate 10. A continuous stream of oil containing the droplets 24 is transported through the capillary 20 by operating the transporting device 60. Once a droplet 24 exits the outlet end 26, it wets the hydrophilic sample spot 1 1 and thus adheres to it. When the detection system 30 detects that an oil plug is about to exit the capillary, after a certain delay time, which depends on the distance between the detection system and the outlet end 26 and on the flow rate of the first fluid together with the droplets, the controller 40 commands the x-y-stage 50 and the z-stage 34 to move the sample plate 10 to a position in which the next sample spot 1 1 is placed immediately below the outlet 26 of the capillary 20. The movement is either carried out only while an oil plug exits the capillary 20, or the moving speed of the outlet end relative to the substrate is reduced during time intervals in which the droplets exit the outlet end. In this manner droplet splitting is avoided. This sequence is repeated until droplets have been deposited to all desired sample spots 11. The droplets adhere to the sample spots despite the fact that the density of the perfluorinated oil in the oil bath 71 is higher than the density of the droplets. This is a result of the hydrophilic anchor-like character of the sample spots. As the perfluorinated oil is almost completely inert and practically immiscible with the aqueous phase making up the droplets, the deposited droplets 12 remain stable at least on the time scale of a few hours.

An apparatus as illustrated in Figure 1 was constructed and tested, as described in the following.

The sample plate 10 consisted of a micro-array plate based on a standard stainless steel ALDI-MS plate (123 x 81 x 1.2 mm³), which was coated with a 10 μπι thick hydrophobic PTFE layer (EpoFlon 375/6504, Eposint AG, Pfyn, Switzerland). Subsequently, the hydrophobic' layer was structured by picosecond laser ablation to form an array of 26'444 circular hydrophilic sample spots of 300 um diameter each. A Nd:YAG laser (SuperRapid, Lumera Laser, Kaiserslautern, Germany) delivering 10 ps pulses was employed, focused by a telecentric lens to a spot size of approximately 10 μηι at a working distance of 102 mm. Scanning was carried out by a galvanoscanner (hurrySCAN 10 from Scanlab, Puchheim, Germany) with the following parameters: wavelength 355 nm, repetition rate 50 kHz, average power 100 mW, hatch 3 μπι, scan speed 150 mm/s; three loops of two orthogonal passes each per sample spot. The galvanoscanner in combination with the telecentric lens enabled a scan field of 5x5 cm². As the dimensions of the total array exceeded the scan field, individual segments of the array were ablated separately and stitched together by repositioning the coated substrate using a precision x-y-stage consisting of two linear translation stages (XML210 and XML350, Newport, Darmstadt, Germany). The size of the individually ablated segments was chosen to be much smaller than the available scan field to reduce inaccuracies of the beam position at large deflection angles by the galvanoscanner. Thereby, a precise spacing of the hydrophilic spots was achieved with high resolution across the whole array.

As the oil phase, perfluorodecalin (ABCR, Karlsruhe, Germany) was used. Aqueous droplets having a volume of approximately two to three nanoliters were created in the microfluidic T-junction 23 (PEEK, through-hole diameter 152 μπι). Both the aqueous droplet phase and the oil phase were driven by neMESYS syringe pumps (cetoni GmbH, Korbussen, Germany) equipped with 250 μΐ glass syringes (Agilent Technologies, Basel, Switzerland). FEP capillaries with 250 μιη I.D. (BGB Analytik, Boeckten, Switzerland) were used to connect the syringes to the T-junction 23. The T-junction 23 was connected to a perfluoroalkoxyalkane (PFA) capillary (360 μηι O.D., 100 μτη I.D.; Upchurch), which formed the capillary 20, using a microsleeve (F-242x, Upchurch). An Olympus IX inverted microscope equipped with a UK1 1 17 CCD camera (EHD imaging GmbH, Damme, Germany) was used to monitor droplet transport inside the capillary 20. The vessel 70 was mounted on an x-y-stage consisting of two linear translation stages (Physik Instrumente, Karlsruhe, Germany) with a travel range of 150 mm in the x direction and 100 mm in the y direction. The capillary and the fibers of the droplet detection system were mounted on another linear translation stage in the z direction to adjust the spacing between the outlet end 26 of the capillary 20 and the sample plate 10. The spotting process was monitored with a DinoLite handheld USB microscope (AM31 1-RO, AnMo Electronics Corporation, Hsinchu, Taiwan). Alignment marks on the sample plate were used to compensate for possible alignment errors of the sample plate 10 relative to the x-y-stage, so as to ensure that the outlet end 26 of the capillary 20 moved exactly along the rows and columns of the sample spots.

Droplet detection was carried out with a detection system 30 consisting of a red LED (DF- E97, Industrial Fiber^" Optics, Tempe, AZ, USA), a polymeric fiber (Distrelec, Nanikon, Switzerland), and a phototransistor (IF-D92, Industrial Fiber Optics, Tempe, AZ, USA). The optical fiber had been stretched out to reduce the diameter of the fiber core (PMMA) to approximately match the outer diameter of the capillary. A hole of 360 μπι had been drilled into the stretched-out section and the capillary was threaded through the hole. The light from the LED was coupled into the optical fiber, and the photodetector attached to the other side of the optical fiber was used to detect changes in the transmittance between the aqueous and the oil phase inside the capillary. The analog signal from the photodetector was evaluated using a real-time data processing system (ADwin-Gold II, Jager Computergesteuerte Messtechnik GmbH, Lorsch, Germany). A custom-made evaluation software smoothed the raw data and performed a threshold analysis to differentiate droplets from oil plugs. For each droplet, the evaluation software triggered a LabVDEW script (National Instruments, Austin, Texas, USA) which controlled the x-y stage 50 and the z- stage 34 to perform a movement to the next empty hydrophilic sample spot. This ensured that only a single droplet was deposited per spot. Furthermore, an implemented delay time between the detection of the droplet and the movement ensured that the movement is only performed while an oil plug was exiting the capillary. This prevented splitting of a single droplet across multiple hydrophilic spots and hence precluded cross-contamination.

Droplets were deposited to the sample spots as described above, every spot acting as a recipient for one individual droplet.

MALDI-MS measurements were performed using a 4800 MALDI TOF TOF Analyzer (AB Sciex, Concord, ON, Canada). A spotset for the novel spot geometry was created using a custom- written MATLAB script (The MathWorks, Natick, MA, USA) and imported into the Data Explorer software (AB Sciex) of the mass spectrometer. All spectra were acquired using a Nd:YAG laser (355 nm, 200 Hz repetition rate, 30 μηι circular spot size) and a mass detector in reflector operation mode. The laser intensity and number of shots per spot were optimized (base on the signal-to-noise ratio) for each experiment individually using test sample spots. Subsequently, all spectra were acquired with the automatic acquisition control (batch mode) using the same settings.

Matrix droplets were created and deposited in the same way as the analyte solution. The MALDI matrix solution consisted of 2,5-dihydroxybenzoic acid (DHB) dissolved at 10 mg/mL in a mixture of DI water, acetonitrile, and phosphoric acid 49.5:49.5:1 v/v/v. Fig. 2 illustrates how the method of the present invention can be employed as an interface between a high performance separation technique (here using a LC system as an example) and a further analytical technique. Parts having a similar functionality as in Figure 1 carry the same reference numbers as in Figure 1. A solution of a sample of interest and an eluent is pumped through a pre-column 81 and an analytical column 82 of an LC apparatus 80. A T-junction 83 enables the eluate stream from the pre-column to be diverted to a waste channel 84. In a T-junction 23, the eluate stream from the analytical column 82 is combined with an oil stream 22 to form a stream of eluate droplets 24 separated by oil plugs 25. Figs. 3 and 4 illustrate an embodiment wherein the sample plate 10 is surrounded by a frame 90, the sample plate 10 directly forming the bottom of a vessel for receiving the oil bath. In this embodiment, only the upper surface of the sample plate is covered with oil, while the lower surface remains dry. The sample plate 10 is illustrated in a highly schematic fashion only; in particular, the sample spots are not shown in Figures 3 and 4.

The frame 90 comprises an annular lower frame portion 91, an annular central frame portion 92, and an annular upper frame portion 93. The sample plate 10 is held between the lower frame portion 91 and the central frame portion 92. The lower frame portion 91 circumferentially surrounds the perimeter of the sample plate, a peripheral region of the underside of the sample plate 10 resting on an inwardly extending flange of the lower frame portion 91. The central frame portion 92 has a region that presses downwardly onto a peripheral region of the upper side of the sample plate. To ensure leak tightness, two sealing rings 95 are provided, the first of the sealing rings being arranged between the underside of the sample plate 10 and the inwardly extending flange of the lower frame portion 91, and the second of the sealing rings being arranged between the central frame portion 92 and the upper side of the sample plate 10. Bores 96, 97 can receive screws for fastening and pressing the lower frame portion 91 and the central frame portion 92 to one another. The central frame portion 92, the upper sealing ring 95 and the sample plate 10 together delimit a vessel for receiving the oil bath, the sample plate forming the vessel bottom, and the central frame portion together with the upper sealing ring forming the circumferential vessel side wall. A transparent plate 94 is held in a similar manner between the central frame portion 92 and the upper frame portion 93, again with the aid of two annular sealing rings 95. The plate 94 forms a lid for the vessel that can be used after deposition of the droplets to prevent evaporation of the oil, and to prevent spillage during transport. The upper frame portion 93 can be fastened to the central frame portion 92 by screws received in bores 98, 99.

The frame portions 91, 92, and 93 may consist, e.g., of stainless steel. The sealing rings 95 may be made, e.g., of NBR (Nitrile Butadiene Rubber). The plate 94 may consist, e.g., of mineral glass, PC or PM A. No adhesive is required to hold the sample plate 10 in the frame 90. After deposition of the droplets and removal of the oil bath, the sample plate 10 can be easily separated from the frame 90 without any residues and can be subjected to further analysis, such as MALDI- MS. The embodiment with the frame 90 can be used in a similar manner as the embodiment with a pre-formed vessel as described in conjunction with Figs. 1 and 2 once the frame 90 has been mounted on the x-y stage. Of course, many modifications can be made to the frame construction.

The sample plate 10 can be at least partially transparent, allowing the sample spots to be observed from below, e.g., by an inverted microscope setup. The fact that the sample plate itself forms the transparent bottom of the vessel for the oil bath facilitates such observation, in particular when high magnification is desired, since high magnification usually requires a low distance between the microscope objective and the region of interest. Furthermore, problems with autofocus systems, which might otherwise result from multiple reflections, are avoided. Fig. 5 illustrates how an additional "chemical dimension" can be created on the sample plate by depositing a reactant to selected sample spots after a sample has been applied to the sample spots. The sample is applied as described above. In the present example, the sample is applied in horizontal rows in a serpentine pattern (part (a); see sequence of sample spots numbered as 1, 2, 3, 9). The sample may be the eluate from a separation column, e.g. from an LC or CE column, and the composition of the sample may therefore vary between sample spots. In the present example, each fraction from the separation column is distributed over a plurality of sample spots (shaded sample spots indicating sample spots loaded with a fraction from the separation column). Depending on the separation technique, the sample may be applied under air, allowing the carrier to evaporate, or the sample may be applied under oil. If the sample is applied under oil, the sample plate may be removed from the bath and dried before the following procedure. In particular, if the separation technique is LC, the eluent often comprises an organic solvent such as acetonitrile, and it might be necessary to allow for evaporation of the solvent before the next steps are carried out because some of the next steps might be incompatible with the solvent or solvent mixture used as eluent. On the other hand, if the separation technique is CE, evaporation of the carrier might not be necessary. After application of the samples, and optionally after evaporation of the solvent as described above, the sample plate is submerged in the oil bath (or it remains therein if it has never been removed), and reactant droplets are applied to selected sample spots in vertical columns 13 (part (b)) in the above-described manner. In the present example, the columns have a width of two sample spots each, leaving out a distance of two sample spots each between adjacent vertical columns. In this manner, due to the checkerboard-like arrangement of the sample spots on the substrate, a pattern of modified sample spots to which the reactant has been applied, interleaved with unmodified sample spots to which no reactant has been applied, is created. It is of course possible to create different interleaved patterns, e.g. by spotting reactant droplets in a horizontal serpentine pattern as in part (a), but leaving out every other sample spot in the spotting process. The reactant droplets are allowed to react with the samples on the respective sample spots. Subsequently, MALDI matrix is deposited to all sample spots in the above-described manner. The sample plate is then removed from the bath and dried. Subsequently, MALDI mass spectra are recorded for all sample spots or for a subset of sample spots. In particular, the samples can contain a peptide or protein, and the reactant can be an enzyme.

Example

In the following, an example will be described with reference to Figures 6 and 7, illustrating the versatility of a droplet-based interface between nano-LC and MALDI-MS for the reliable identification of post-translational protein modifications. A tryptic protein digest containing phosphorylated and glycosylated peptides was separated by a standard nano-LC run, the eluate was compartmentalized into microdroplets and spotted in a high- frequency manner onto a sample target plate. A single eluting peak was split over multiple sample spots. Specific enzymes were applied to every second sample spot to remove the modification, which resulted in a mass shift for originally modified peptides. Due to this, a comparison between two consecutive MS spectra allowed a robust identification of peptides carrying post-translational modifications.

The analysis of post-translational modifications (PTMs) can be crucial in the context of protein and biomarker analysis to understand, characterize and monitor molecular and cellular function or regulation. PTMs alter or add selective protein properties and functions and hence, affect a wide range of biological processes including catalytic activity, cellular signal cascades and cell-to-cell signaling. Due to the high complexity of PTMs, their localization and identification in complex mixtures of proteins at low concentrations poses a big challenge for conventional analytical methods and requires refined sample preparations, sensitive detection methods as well as the combination of several analytical methods. Even by using advanced nano-LC-MS/MS based methods it is not always possible to reliably answer questions about the presence or the nature of a modification.

To address this challenge, a droplet-based interface was employed to interface standard nanoflow-liquid chromatography (nano-LC) with matrix-assisted laser desorption ionization mass spectrometry (MALDI- S). The interface enabled enhanced aliquotation of eluted peaks at yet unmatched temporal resolution. Moreover, the system enabled the integration of a further analytical dimension into the standard LC-MS workflow. This facilitated reliable detection and analysis of modifications as well as a convenient and simple analysis of the modified peptide itself. To show the potential of the novel interface, the workflow for the most popular PTMs will be illustrated: phosphorylation and glycosylation.

A tryptic protein digest was performed on standard proteins carrying either phosphorylations and or N-glycosylations (human polyclonal IgG, alpha casein, beta casein; each around 25 ng/pL). The resulting mixture of phosphopeptides, glycopeptides and unmodified peptide molecules was separated using a nano-LC system (Bruker easy- nLC II) equipped with a standard reversed phase column (75 pm inner diameter, 10 cm length, 3 μηι particle size). The separation was performed using a 20-minute water- acetonitrile gradient at a total flow rate of 500 nL/min. Using a microtee junction and a stream of perfluorinated oil (perfluorodecalin), the eluate was directly compartmentalized into nanoliter droplets. The oil segments between the aqueous eluate droplets prevented peak broadening during droplet transport and enabled the deposition of lowest nL volumes even at very low flow rates.

As most peaks from the nano-LC eluted over at least 10 seconds or more, a single peak could be spread over at least 10 hydrophilic spots due to the high sampling frequency of the droplet interface at this low flow rate regime (typically 1 Hz spotting frequency). The repetitive information could thus be utilized for further chemical analyses, which introduced a further analytical dimension. To this end, a second nanoliter droplet was deposited to every other spot, which contained either a solution of alkaline phosphatase or PNGaseF. These enzymes selectively removed the phosphorylation or N-glycosylation, respectively. Deposition of the second droplets was carried out under a film of perfluorinated oil covering the sample target plate to prevent droplet evaporation during this step and to facilitate long-term incubation of the nanoscale reaction. The oil film was subsequently removed and remaining oil residues were evaporated together with the aqueous droplets. Finally, droplets containing MALDI matrix were deposited on all spots. Using MALDI-MS, it was possible to reliably detect peptide modifications such as phosphorylation (Fig. 6) or glycosylation (Fig. 7), just by performing a pairwise comparison of mass spectra obtained from neighboring spots.

Figure 6 shows results for the phosphorylation analysis: a) Overview spectrum of a digest spot (center) and the two adjacent control spots. Five phosphorylated peptides (marked with stars) can be found by comparison, b) Scale up showing that the phosphorylation of peptides can be determined from a peak shift of n x 80 Da in the digest spectrum (n=l for a single phosphorylation site) while non-phosphorylated peptides remain unmodified.

Figure 7 shows results for the glycosylation analysis, illustrating a N-glycosylation found in one eluted peak from human IgG. The control spectra show five different mass peaks (the three most abundant are highlighted). The corresponding peaks are missing in the digest spectrum (center) marking them clearly as glycopeptides. The digest spectrum shows the deglycosylated peptide chain and the three most abundant glycans (G0F, GIF and G2F), which are all missing in both control spectra. Many modifications are possible without leaving the scope of the present invention. For example, it is possible to pre-load the sample spots with different reactants and to spot droplets of the same sample substance to such pre-loaded sample spots to test for chemical reactions of the sample substance with the reactants. The pre-loading in this scenario can also be performed using an interleaved pattern as described above.

Claims

1. A method for depositing single droplets (24) from a channel (20) onto a substrate (10), the droplets (24) being separated by a first fluid, the first fluid being essentially immiscible with the droplets, the method comprising:

(a) placing an outlet end (26) of the channel (20) in proximity to the substrate (10);

(b) delivering a stream of the first fluid containing the droplets (24) through the channel (20) towards the outlet end (26) thereof;

(c) depositing at least one individual droplet (12) from said stream to a sample spot (11) on the substrate;

(d) changing the relative position of the substrate (10) and the channel (20) to place the outlet end (26) of the channel (20) in proximity to another sample spot; and

(e) repeating steps (c) and (d),

characterized in that the substrate (10) is covered by a second fluid and the outlet end (26) of the channel (20) is immersed in said second fluid during deposition of the droplets (24), the second fluid being essentially immiscible with the droplets.

2. The method of claim 1,

wherein, before depositing the droplets, the sample spots are pre-loaded with samples by depositing an eluate of a separation technique onto the sample spots in such a manner that a fraction of the eluate that corresponds to a single analyte peak is fractionated over a plurality of sample spots;

wherein the droplets (24) that are deposited in step (c) are droplets containing a chemical or biochemical reagent for reaction with the pre-loaded samples on the sample spots, and

wherein the droplets are deposited in step (c) only to selected sample spots.

3. The method of claim 1, wherein the selected sample spots are interleaved with sample spots to which no droplets (24) are applied in step (c).

4. The method of claim 2 or 3, wherein a different reagent is pre-loaded or subsequently applied to those sample spots to which no droplets (24) are applied in step (c).

5. The method of any one of claims 2-4, wherein the eluate is deposited onto the substrate by a deposition technique based on fluid-separated droplets.

6. The method of any one of claims 2-5, wherein a solvent of the separation technique is evaporated after pre-loading the sample spots and before depositing the droplets that contain the reagent.

7. The method of any one of claims 2-6, wherein the pre-loaded samples comprise at least one protein or peptide, and wherein the chemical reagent deposited in step (c) comprises at least one enzyme.

8. The method of claim 1 ,

wherein, before depositing the droplets (24), selected sample spots are preloaded with a chemical or biochemical reagent,

wherein the droplets (24) that are deposited in step (c) are droplets of an eluate of a separation technique, the droplets being deposited in such a manner that a fraction of the eluate that corresponds to a single analyte peak is fractionated over a plurality of sample spots.

9. The method of claim 8, where the selected pre-loaded sample spots are interleaved with sample spots that are not pre-loaded with said chemical or biochemical reagent.

10. The method of claim 8 or 9, wherein a different reagent is pre-loaded or subsequently applied to^" those sample spots that are not pre-loaded with the chemical or biochemical reagent.

1 1. The method of any one of claims 8-10, wherein the eluate deposited in step (c) comprises at least one protein or peptide, and wherein the chemical or biochemical reagent that is pre-loaded to the sample spots comprises at least one enzyme.

12. The method of claim 7 or 1 1, wherein the enzyme acts to remove a post- translational modification from the peptide or protein.

13. The method of claim 7 or 1 1, wherein the enzyme is selected from the group consisting of glycopeptidases, O-glycosidases, endoglycosidases, exoglycosidases, alkaline phosphatases, acid phosphatases, and esterases.

14. The method of claim 7 or 11, wherein the enzyme is selected from the group consisting of transferases, synthetases, and synthases.

15. The method of any one of claims 2-14, wherein the separation technique is liquid chromatography, in particular, nano-LC with a flow rate below 1000 nanoliters per minute.

16. The method of any one of claims 2-14, wherein the separation technique is capillary electrophoresis.

17. The method of any one of claims 2- 16, further comprising:

(f) allowing a (bio-) chemical reaction or process to take place in the droplets or at their interface after their deposition; and

(g) observing products from said (bio-) chemical reaction or process.

18. The method of claim 17, wherein step (g) is carried out by mass spectrometry, by microscopy and/or by spectroscopy.

1 . The method of any one of the preceding claims, wherein the second fluid and the first fluid are of the same type.

20. The method of any one of the preceding claims, wherein the second fluid and/or the first fluid is a perfluorocarbon.

21. The method of any one of the preceding claims, wherein the first and/or second fluid has a higher density than the droplets.

22. The method of any one of the preceding claims, wherein the sample spots are wettable by the droplets, whereas the sample spots have a lower affinity for the first and second fluid.

23. The method of any one of the preceding claims,

wherein a continuous stream of the first fluid containing the droplets (24) is delivered during steps (c)-(e), and

wherein the change in relative position takes place only during time intervals when the first fluid that separates the droplets (24) exits the outlet end of the channel (20), or wherein the moving speed of the outlet end (26) relative to the substrate (10) is reduced during time intervals in which the droplets exit (24) the outlet end (26).

24. The method of any one of the preceding claims, wherein an essentially constant distance is maintained between the outlet end of the channel (20) and the substrate (10) during the entire deposition of droplets (24) to the sample spot (1 1) in step (c).

25. The method of any one of the preceding claims, further comprising:

(h) depositing at least one further droplet to each of a set of selected sample spots.

26. The method of claim 25, wherein the further droplet is deposited while the substrate (10) remains covered by the second fluid, so as to create a combined droplet comprising a mixture.

27. The method of claim 25 or 26, wherein the further droplet comprises a MALDI matrix.