WO2014108323A1 - Procédé et appareil de dépôt de gouttelettes sur un substrat - Google Patents

Procédé et appareil de dépôt de gouttelettes sur un substrat Download PDF

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Publication number
WO2014108323A1
WO2014108323A1 PCT/EP2014/000005 EP2014000005W WO2014108323A1 WO 2014108323 A1 WO2014108323 A1 WO 2014108323A1 EP 2014000005 W EP2014000005 W EP 2014000005W WO 2014108323 A1 WO2014108323 A1 WO 2014108323A1
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WIPO (PCT)
Prior art keywords
droplets
fluid
substrate
sample
sample spots
Prior art date
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PCT/EP2014/000005
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English (en)
Inventor
Petra Dittrich
Martin Pabst
Simon KUESTER
Original Assignee
Eth Zurich
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Application filed by Eth Zurich filed Critical Eth Zurich
Publication of WO2014108323A1 publication Critical patent/WO2014108323A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/02—Burettes; Pipettes
    • B01L3/0241—Drop counters; Drop formers
    • B01L3/0262—Drop counters; Drop formers using touch-off at substrate or container
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0673—Handling of plugs of fluid surrounded by immiscible fluid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/14—Process control and prevention of errors
    • B01L2200/142—Preventing evaporation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5088—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above confining liquids at a location by surface tension, e.g. virtual wells on plates, wires
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/80—Fraction collectors
    • G01N30/82—Automatic means therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/08—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
    • H01J49/0409—Sample holders or containers
    • H01J49/0418—Sample holders or containers for laser desorption, e.g. matrix-assisted laser desorption/ionisation [MALDI] plates or surface enhanced laser desorption/ionisation [SELDI] plates

Definitions

  • 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.
  • a fundamental problem when handling very small liquid samples 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.
  • oils often tend to dissolve lipophilic constituents of the droplets, and many such oils are not chemically inert.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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.
  • 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.
  • the first fluid can be a liquid or a gas.
  • the second fluid is preferably a liquid.
  • the first fluid and the second fluid are miscible.
  • the first fluid and the second fluid are immiscible.
  • 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.
  • 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.
  • both the first and the second fluid are preferably a low-viscosity oil.
  • the oil is a perfluorinated oil, in particular, a perfluorocarbon such as the family of perfluorocarbons offered by 3M Company under the trade name FluorinertTM.
  • a perfluorocarbon such as the family of perfluorocarbons offered by 3M Company under the trade name FluorinertTM.
  • suitable perfluorocarbons are perfluorohexane, available under the trade name FluorinertTM FC-72; a mixture of perfluorotri-n-butylamine with perfluoro-n-dibutylmethylamine, available under the trade name FluorinertTM FC-40; perfluoro(2-butyl-terrahydrofurane), available under the trade name FluorinertTM FC-75; and perfluorodecalin (octadecafluoronaphthalene).
  • 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.
  • 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.
  • the substrate surface 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.
  • 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.
  • the sample spots are preferably more hydrophilic than the surface portion that surrounds each sample spot.
  • the substrate can be microstructured.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the distance d should be chosen as follows:
  • 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).
  • 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:
  • Observation of the products 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.
  • the method allows for combining two or more droplets on the substrate.
  • the method can further comprise:
  • step (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.
  • the droplet(s) deposited in step (c) or the further droplet(s) deposited in step (h) can comprise a MALDI matrix.
  • the further droplet(s) deposited in step (h) can comprise a reagent for inducing a (bio-) chemical reaction or process in the droplets.
  • 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.
  • 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.
  • 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.
  • 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.
  • the droplets that are subsequently deposited in step (c) or step (h) can contain a chemical reagent for reaction with the pre-loaded samples.
  • the pre-loaded samples can comprise at least one protein or peptide
  • the chemical reagent deposited in step (c) or step (h) can comprise at least one enzyme, or vice versa.
  • 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.
  • 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).
  • 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).
  • 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.
  • a separation technique such as LC or CE
  • MS another analytical technique
  • post-translational modifications e.g., glycosylation and phosphorylation as well as methylations, acetylation or any kind of esterifications
  • 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.
  • 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.
  • a glycopeptidase e.g. PNGAseA and/or PNGAseF
  • O-glycosidase e.g. O-glycanase
  • an endoglycosidase e.g. ENDO F2
  • an exoglycosidase e.g. neuraminidases
  • 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.
  • the present invention provides a method for interfacing a separation technique with an analytical technique, the method comprising:
  • sample spots on a substrate 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;
  • a reagent 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,
  • depositing the droplets comprises:
  • step (e) repeating steps (c) and (d).
  • 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.
  • 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.
  • this fluid can be the same as for covering the substrate during deposition of the reagent droplets, or it can be a different fluid.
  • 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 present invention also relates to a method for interfacing a separation technique with an analytical technique, the method comprising:
  • 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:
  • 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.
  • the eluate can comprise at least one protein or peptide
  • the chemical or biochemical reagent can comprise at least one enzyme.
  • the enzyme can act to remove a post-translational modification from the peptide or protein.
  • the enzyme can be selected from the group consisting of glycopeptidases, O- glycosidases, endoglycosidases, exoglycosidases, alkaline phosphatases, acid phosphatases, sulfatases and esterases.
  • the enzyme can be selected from the group consisting of transferases, synthetases, and synthases.
  • 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 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);
  • 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).
  • 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 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.
  • the substrate are transparent (in particular, the part where the droplets are deposited), optical surveillance of the spotting process from below is facilitated.
  • 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
  • Fig. 7 shows results of a N-glycosylation analysis of the human IgG Fc region.
  • Figure 1 illustrates, in a highly schematic manner, an apparatus according to the present invention and the corresponding method.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 mm 3 ), which was coated with a 10 ⁇ 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 ⁇ 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 ⁇ , 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 cm 2 . 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.
  • perfluorodecalin As the oil phase, perfluorodecalin (ABCR, Düsseldorf, 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 ⁇ ). Both the aqueous droplet phase and the oil phase were driven by neMESYS syringe pumps (cetoni GmbH, Korbussen, Germany) equipped with 250 ⁇ glass syringes (Agilent Technologies, Basel, Switzerland). FEP capillaries with 250 ⁇ 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 ⁇ O.D., 100 ⁇ I.D.; Upchurch), which formed the capillary 20, using a microsleeve (F-242x, Upchurch).
  • PFA perfluoroalkoxyalkane
  • 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, Düsseldorf, 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 ⁇ 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 Computerwiee 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.
  • 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 ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the separation technique is LC
  • the eluent often comprises an organic solvent such as acetonitrile
  • the separation technique is CE
  • evaporation of the carrier might not be necessary.
  • the columns have a width of two sample spots each, leaving out a distance of two sample spots each between adjacent vertical columns.
  • 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.
  • MALDI matrix is deposited to all sample spots in the above-described manner.
  • 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.
  • samples can contain a peptide or protein, and the reactant can be an enzyme.
  • PTMs post-translational modifications
  • 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.
  • 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.
  • 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.
  • 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 ⁇ particle size). The separation was performed using a 20-minute water- acetonitrile gradient at a total flow rate of 500 nL/min.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • G0F, GIF and G2F three most abundant glycans

Abstract

La présente invention concerne un procédé permettant de déposer des gouttelettes (24) séparées par un premier fluide d'un canal (20) sur un substrat (10). Afin d'empêcher l'évaporation des gouttelettes et pour permettre l'association des gouttelettes, le substrat est recouvert d'un second fluide, et l'extrémité formant l'orifice de sortie (26) du canal est immergée dans le second fluide. Le procédé peut être utilisé comme interface entre une technique de séparation, par ex., la chromatographie liquide, et une technique analytique telle que la spectrométrie de masse MALDI. Une dimension chimique supplémentaire peut être ajoutée en fractionnant un pic unique de la technique de séparation sur une pluralité de points d'échantillon et en soumettant uniquement les points d'échantillon sélectionnés à une réaction chimique. Les applications comprennent l'analyse de modifications post-traductionnelles de protéines et de peptides.
PCT/EP2014/000005 2013-01-10 2014-01-06 Procédé et appareil de dépôt de gouttelettes sur un substrat WO2014108323A1 (fr)

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US11142791B2 (en) 2016-08-10 2021-10-12 The Regents Of The University Of California Combined multiple-displacement amplification and PCR in an emulsion microdroplet
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