Means and methods for spraying microdroplets from a chip

Abstract

The present invention relates to the field of microfluidic sample preparation, more specifically of biological sample preparation, in particular for spraying microdroplets from a microfluidic chip. In particular, the chip and methods for using the same are applicable in the field of protein sampling, structural biology, even more specifically for structural analysis of proteins by Cryogenic-electron microscopy (Cryo-EM). The invention provides for methods and devices for preparing aqueous protein samples for transmission electron microscopy, typically at millisecond time-resolution using a microfluidics-based integrated device. More specifically, the sampling means and methods of the invention relate to an improved dispensing module integrating distant actuation of the sample for droplet-on-demand microdroplet spraying from the microfluidic chip only requiring very limited sample amounts.

Description

[0001] ROEf / trEM-chip / 856

[0002] MEANS AND METHODS FOR SPRAYING MICRODROPLETS FROM A CHIP

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to the field of microfluidic sample preparation, more specifically of biological sample preparation, in particular for spraying microdroplets from a microfluidic chip. In particular, the chip and methods for using the same are applicable in the field of protein sampling, structural biology, even more specifically for structural analysis of proteins by Cryogenic-electron microscopy (Cryo-EM). The invention provides for methods and devices for preparing aqueous protein samples for transmission electron microscopy, typically at millisecond time-resolution using a microfluidics-based integrated device. More specifically, the sampling means and methods of the invention relate to an improved dispensing module integrating distant actuation of the sample for droplet-on-demand microdroplet spraying from the microfluidic chip only requiring very limited sample amounts.

[0005] BACKGROUND

[0006] To prepare cryo-EM grids, protein solutions may be sprayed on the cryo-EM grid as micrometer-sized droplets some of which rapidly spread on the surface of plasma-cleaned EM grids creating thin areas of buffer which is vitrified upon cryo-plunging, resulting in areas suitable for cryo-EM imaging (Berriman and Unwin, 1994; Feng et al., 2017). This method of sample application is different from the more common application by pipetting microliter volumes of the sample on EM grid followed by blotting with filter paper. However, said sample preparation method by spraying small droplets is the only method existing so far which is compatible with high (millisecond) time-resolution required to trap the intermediates of a biological process. Indeed, most reactions occur in very short millisecond time-scales, thereby complicating the sampling methods and means to prepare a biological sample for time-resolved structural analysis, in particular for time-resolved Cryogenic-electron microscopy (tr-Cryo-EM) of protein samples or protein complex samples. Recent attempts to improve the level of sophistication and automation for this type of sampling have shown that miniaturization of mixers and bioreactors allow to rapidly initiate and synchronize biochemical reactions, followed by spreading the mixtures onto a cryo- EM grid without the need for manual operation or blotting, which ultimately results in a faster sampling time than the lifetime of the structures of interest. Previously, a novel chip and method for fast mixing and droplet-on-demand sampling of small amounts of biological components such as proteins was described in Efremov et al. (WO2022 / 148859A1). The generation of airborne microdroplets using this ROEf / trEM-chip / 856 device involves direct 'on sample' actuation and has allowed high resolution time-resolved structural analysis for several proteins and complexes (Torino, et al. 2023, Nat Methods 20, 1400-1408).

[0007] Further approaches of droplet generation for time-resolved cryo-EM are based on atomization of the protein solution by a rapid stream of gas applied to the nozzle from which protein solution is extracted (Berriman and Unwin, 1994; Feng et al., 2017; Kontziampasis et al., 2019; Lu et al., 2014; Maeots et al., 2020). Alternatively, a spray can be created by ejecting a stream of droplets from a membrane vibrating with ultrasonic frequency (Ashtiani et al., 2018; Rubinstein et al., 2019), this approach however has not been combined with fast mixers to demonstrate its applicability to time-resolved cryo-EM. Other methods that aim at miniaturizing traditional sample preparation utilized a drop-on-demand (DOD) method to eject small droplets on an EM grid from a capillary followed by droplet thinning aided by metal nanowires covering the EM grid (Jain et al., 2012; US2014 / 0360286A1). The disadvantages of atomization using compressed air include the large sample consumptions (tens of microliters per grid) and poor control over droplet dimensions (Kontziampasis et al., 2019; Lu et al., 2014). These disadvantages can partially be overcome by more sophisticated 3D mixers and miniaturized nozzles fabricated by 3D two-photon photolithography (Knoska et al., 2020).

[0008] Alternative droplets on demand approaches known in the art are based on applying a piezoelectric actuator. Examples of such actuators integrated into microfluidic devices for microdroplet dispensing tend to replicate ink-print cartridges in a microfluidic form (Ahamed, et al. 2010), or having a microfluidic channel with a piezo-electric element separated from the sample chamber by a membrane that is placed between the actuator element and the channel or reservoir containing the target sample (Bransky et al., 2009). In the context of methods using laser-induced cavitation as actuation for ejection of droplets on demand, the Laser Induced Forward Transfer (LIFT) technology allows for indirect actuation where a donor is receiving the laser beam to later propel towards the receiving substrate, and is applied in the field of printing of all types of materials (Fernandez-Pradas, and Serra, 2020; Miksys, et al. 2019; Morales et al., 2018; Duocastella, et al. 2008). Finally, other technologies apply a fluid pump (Dijkinka and Ohlab, 2008) or water-in-oil droplets (Park et al., 2011) as distant actuation method as to generate a propulsion effect in a side channel in a microfluidic chip.

[0009] However, the current state of the art lacks a solution for microdroplet spraying of small amounts of samples, particularly prepared via microfluid chip devices applicable for purposes of time-resolved analysis, for instance through Cryo-EM. Moreover, the sampling small amounts of biological samples for time-resolved analysis would also benefit from a distant actuation of the microdroplet formation, whereby the biological sample itself retains its integrity, which is often a challenge when using heating, laser-absorbing dyes or further components that may affect the stability of the sample. ROEf / trEM-chip / 856

[0010] So, there is still a need for improving the microfluidic sampling methods and devices as to optimally prepare small amounts of biological samples in a droplet-on-demand (DOD) approach.

[0011] SUMMARY OF THE INVENTION

[0012] The present invention provides for novel means and methods for microdroplet spraying of biological samples prepared on microscale, such as for (time-resolved) Cryo-EM sampling by applying a method using a customized setup comprising a droplet-on-demand microfluidic chip which enables controllable DOD droplet generation involving a distant actuation mechanism, preserving the quality of the biological sample, which is especially beneficial for protein samples.

[0013] In a first aspect the invention relates to a novel microfluidic chip for microdroplet ejection from the chip, which chip comprises : an inlet module comprising at least one inlet for a fluid sample to enter the chip, said inlet being in connection with a main microchannel wherein the fluid sample merges towards the outlet of the chip, wherein the at least one inlet is controllable by a pressure control module configured to drive the fluid though the channel; and a microdroplet dispensing module, comprising: a nozzle channel in connection with one end of the main microchannel of the inlet module, and on the other end configured to end in one or more outlet openings to the outside of the chip for ejection of microdroplets from the chip; and an actuation chamber, which is in connected to the nozzle channel via a chamber opening ending in the nozzle channel wall, said chamber opening in said wall being at a location at a distance (d) from the nozzle channel outlet opening that allows for ejection of microdroplets from the outlet opening(s), and wherein the actuation chamber is configured to, upon actuation, transmit a pressure wave through the chamber opening transferring the wave into he nozzle channel liquid, thereby inducing the formation of microdroplets from the liquid present in the nozzle channel, resulting in droplets from said liquid being ejected through the outlet opening(s), and wherein said actuation chamber is configured to prevent any material from the actuation chamber from mixing with the liquid sample in the nozzle channel.

[0014] A further embodiment relates to said chip, wherein the actuation chamber comprises and inlet and an outlet for access of chamber material to the actuation chamber, and wherein said inlet and outlet are controllable via a pressure control module configured to prevent mixing of the chamber material with the liquid sample present in the nozzle channel when the chip is in operation mode, preferably wherein the width of the inlet and outlet each are not smaller than the width (w) of the nozzle channel. ROEf / trEM-chip / 856

[0015] Another embodiment relates to said chip, wherein the actuation chamber dimensions are provided as having a length (I) of at least 200 pm and maximum width (w) of at least 200 pm, a depth that is equal to or greater than the depth of the nozzle channel, and a chamber opening width which is at least the double of the width of the nozzle channel, and which is positioned at a distance (d) from the outlet opening(s) that is maximally 10-fold the width (w) of the nozzle channel.

[0016] Further embodiments relates to said chip wherein the actuation chamber material comprises a liquid or fluid solution suitable for actuation, preferably water, a fluorophore, or a dye solution.

[0017] Alternative embodiments relate to said chip, wherein the actuation chamber is configured to allow laser- induced cavitation, wherein the actuation chamber material comprises a dye for absorbing the laser light and generation of a cavitation bubble.

[0018] Additional embodiments relates to a chip for multiplexing of droplet spraying, wherein the nozzle channel is configured to end in multiple outlet openings, specifically containing 2, 3, 4, or 5 openings.

[0019] Further embodiments relate to said chip applied for mixing solutions, prior to droplet ejection, wherein the inlet module comprises: a mixer module for mixing liquid solutions within oil-encapsulated droplets comprising: at least 3 inlets, wherein each inlet ends in the main microchannel for combining the solutions from the inlets, said main microchannel being fluidly connected to a serpentine microchannel comprising at least 3 arms, wherein each of the inlets of the mixer module is further connectable to a pressure control module configured to control at least the pressure in the inlets; and a droplet merging module.

[0020] A second aspect of the invention relates to an integrated apparatus for preparation of a sample comprising the microfluidic chip described herein, connected to at least one pressure control module for controlling the flow rate of the solution in the channels of the chip when in operation mode, a plunger module, a droplet actuation module, a cryogenic module, and a microprocessor-based controller unit configured to synchronize and control the movement of the plunger arm via the arm controller unit, the pressure control module, and the thermostatic cryogenic module, wherein the components i. to iv. are mounted on one or more support structures configured to allow plunge-freezing of a grid held by the plunger module after droplets generated by the microfluidic chip have been sprayed on the grid. ROEf / trEM-chip / 856

[0021] Further embodiments relate to said integrated apparatus with a droplet actuation module configured for laser-induced actuation.

[0022] A final aspect of the invention relates to a method for ejecting microdroplets from a microfluidic chip, preferably on a grid, comprising the steps of: providing at least one liquid solution to the at least one inlet of the microfluidic chip described herein, and providing means connectable to the chip for driving the flow of said solution through the main microchannel, actuating the liquid solution in the nozzle channel by generating a pressure wave in the actuation chamber, which is transmitted through the chamber opening to the nozzle channel and triggers microdroplet formation in the solution in the nozzle channel which are consequently ejected from the at least one or more outlet openings of the chip.

[0023] DESCRIPTION OF THE FIGURES

[0024] The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes.

[0025] Figure 1. Schematic overview of the microfluidic chip with distant actuation. The microchip of the invention comprises an inlet module, which has at least one inlet channel (29) for supplying the sample to the chip, and a main microchannel (30) through which the sample flows as driven by means to control sample flow rate, such as a pressure control module, connectable to the inlet of the chip. The chip further comprises a dispensing module which is configured to generate airborne microdroplets at the outlet opening (40) at the end of the nozzle channel (38) of the chip, said microdroplets being formed upon actuation of the liquid solution in the nozzle channel via a distant actuation mechanism taking place in the actuation chamber (39). The actuation chamber may be configured for laser-induced actuation (A), with an inlet (7) and outlet (8) for circulation of a solution containing a laser-absorbing dye through the chamber. The actuation chamber (39) and the nozzle channel (38) are thus separated but physically connected by the presence of a chamber junction or opening (41) at the intersection of the actuation chamber and nozzle channel, as to allow transmission of the actuation signal that is initiated in the actuation chamber and propagates via the actuation chamber material (black) towards the liquid sample solution flowing through the nozzle channel. The in- and outlet channel (7, 8) of the actuation chamber (39) are connectable to means for driving the flow of the actuation chamber material, such as the dye solution, such as a pressure control module, in coordination with the means driving the flow in the nozzle channel preventing that the fluid material of the actuation chamber would flow into the nozzle channel. ROEf / trEM-chip / 856

[0026] A laser-containing actuation module with a laser beam focused in the dye solution of the actuation chamber (white star) triggers the formation of a cavitation bubble thereby generating a pressure wave in the actuation chamber material which propagates to the nozzle channel as to obtain droplets-on- demand from the liquid sample therein. So in general (B), independent from the type of actuation module, the actuation chamber (39) is configured to produce an actuating pressure wave towards the chamber junction (41) being transmitted to the solution in the nozzle channel (38) as to obtain ejection of microdroplets from the liquid sample out of the chip outlet opening (40).

[0027] Figure 2. Schematic overview of the design and operation of the microfluidic device for sample mixing and airborne droplets ejection through laser-induced cavitation. The microfluidic chip design reflects a particular embodiment wherein the chip as disclosed in Efremov et al., WO2022 / 148859 is applied with the droplet generation module being replaced with the novel dispensing module of the present invention. The chip allows for mixing of different solutes and thus has several inlet channels and a droplet merging module . The inlet module thus comprises an oil inlet channel (oil in- 28) and at least 2 sample inlets (29) to supply the sample for mixing to the mixing module to provide for mixed oil-encapsulated droplets. The mixing is obtained by the presence of a serpentine channel (31) with arms (32) and bending regions (33) resulting in droplets of mixed sample solution within the oil-fluid. The aqueous droplets flow through the main microchannel (30) in the droplet merging module, configured for extraction of the oil and merging of the droplets to a mixed solute. The oil-extraction is obtained by removing the oil via a side channel (35) separated from the main microchannel by an array of pillars (36), thereby lining the wall of the main microchannel (30); after extraction and merging, the liquid mixed solution proceeds in the main channel (30) towards the dispensing module, or goes via an optional additional microchannel (37) towards the dispensing module for actuation. The previously disclosed 'actuation module' is herein replaced with the current novel 'dispensing module' which comprises the nozzle channel (38) which is in connection on the one end with the main microchannel (30 or 37) of the inlet module for the merged aqueous fluid sample solution to pass through, and on the other end terminates in an outlet opening (40) at the end of the chip, where microdroplets are being ejected to the air upon distant actuation. In this particular exemplified design, the actuation occurs through laser-induced cavitation, which is initiated by focusing a laser beam (white star) on the actuation chamber fluid (black) which induces a cavitation bubble to propagate a pressure wave signal towards the chamber junction (41), where the transmission of said pressure wave reaches meniscus and induces the formation of microdroplets in the liquid sample solution in nozzle channel (38) allowing spraying through the outlet opening. The distance between the chamber junction and the outlet opening has to be equal or less than 10-times the width of the nozzle channel (38) as to obtain microdroplets upon pressure wave transmission. Furthermore, the dimensions of the actuation chamber require no cross-flow of the actuation chamber fluid to the ROEf / trEM-chip / 856 nozzle channel so that fluid is not mixed in the liquid sample solution, which is controlled by balancing the pressure control modules used for driving the flow rate of the liquid sample solution and the dye solution.

[0028] Figure 3. Schematic overview of an exemplified design of the dispensing module of the chip.

[0029] Figure 4. Visual explanation of the airborne droplets' ejection through laser-induced cavitation using a dedicated actuation chamber. Top panel: The actuation chamber (positioned here above the nozzle channel) is filled with the dye solution (dark grey area). When the laser is activated and focused on the dye solution (white area in the circle), an air-bubble is created. Lower panel: in the successive frame, the bubble is already fully collapsed (arrow). The generated propulsion effect is transmitted to the nozzle channel and an airborne droplet is ejected from the chip.

[0030] Figure 5. Screenshot from a recorded video of the operating chip. The actuation chamber (positioned here above the nozzle channel) is filled with the dye solution (dark grey area). The bright white spot represents the focused laser, while the surrounding black circular contour is the cavitation bubble. Exposure time 55 ms.

[0031] Figure 6. Schematic overview of the integrated apparatus. The integrated apparatus as shown here is composed of a microfluidic chip (1) as described herein, wherein the inlet channels (28 and / or 29) are connected to a pressure control module which is composed of a flow reservoir (3) for each inlet channel, connected via tubing to a flow meter (FM) between the reservoir and the inlet channel, and controlled by a pressure controller (4) for each inlet channel, optionally, an additional pressure controller (4) can be present for adding a negative pressure connected via a tube (3b) to the side channels for oil removal (35); optionally the pressure control module(s) are connected to a (personal) computer (PC); the chip (1) is mounted on a manual XYZ stage (24b), held by the holder (24), which is in its turn mounted on a motorized XY stage (23) which also has a plunger arm module (as shown for instance in WO2022 / 148859 Figure IB) and the cryogen module mounted on it, in such a relative position as to have the plunger arm (5) and grid-clip mechanism (6) for an EM grid less than 1mm from the outlet opening on the chip (1), and the outlet opening above the surface of the cryogen (at a distance preventing freezing of the chip by cryogen). The cryogen module as presented herein is composed of a cryogenic container (12) for liquid nitrogen, covered by a lid (15) with an opening above the reservoir (13) for ethane, and a grid box holder (14) and housed grid box (14b) for storing EM grids. The actuation module comprises the laser (17), with a shutter (27), after which the laser beam intensity is monitored by a power meter (26), going through a beam expander (21), and further comprises an optical module, which allows to focus the laser beam on the actuation chamber material for laser-induced cavitation, wherein said module as shown herein comprises a beam splitter (22), an optical microscope (19), and a camera (20), as well as an ROEf / trEM-chip / 856 objective lens (18). Optionally, after the beam splitter (22), a diffractive beam splitter (44) is introduced, to divide the single laser focus point into a linear array of equidistant focused laser spots.

[0032] Figure 7. Schematic showing the operation of the previous microfluidic chip for sample mixing with direct actuation on the sample to form airborne droplets using laser-induced cavitation. The design shown herein is disclosed and incorporated herein from Efremov et al., WO2022 / 148859.

[0033] Figure 8. Cryo-EM analysis of Respiratory Complex I sample. Representative cryo-EM micrographs of grids obtained after plunge-freezing are shown for (1) samples prepared using the previously designed chip as described in W02022 / 148859 and as shown in figure 7, in comparison to (2) samples prepared using the novel chip of the present invention comprising the novel dispensing module for distant actuation as shown in Figure 2. Single particle analysis of the cryo-EM dataset collected from grid (2) resulted in 2D class averages shown on the right panel, and 3D map of complex I resolved to resolution of 8 A from 5000 particles.

[0034] Figure 9. Schematics of a chip design with multiple nozzle outlet openings. The dimension indicated in the figure are an example on a design with multiple nozzle outlets allowing multiplexing of droplet ejection. In addition, the dimensions are exemplifying an option to apply a smaller actuation chamber as compared to the design of Figure 3, which goes along with a shorter length of the nozzle channel (110 pm for a chamber with an opening op about 50 pm) and allows for shortening the on-chip (mixing) time.

[0035] Figure 10. Schematics showing a multi-droplet-spraying chip with dedicated (laser-)actuation chamber in operation mode.

[0036] Figure 11. Two consecutive frames from a video of the multi-droplet-spraying chip operating according to the design of Figures 9-10, recorded at 50,000 fps.

[0037] DESCRIPTION

[0038] The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. Of course, it is to be understood that not necessarily all aspects or advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example those skilled in the art will recognize that the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other aspects or advantages as may be taught or suggested herein. The invention, both as to organization and method of operation, together with features and advantages thereof, may best be understood by reference to the following detailed ROEf / trEM-chip / 856 description when read in conjunction with the accompanying drawings. The aspects and advantages of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases 'in one embodiment' or 'in an embodiment' in various places throughout this specification are not necessarily all referring to the same embodiment but may.

[0039] Definitions

[0040] Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments, of the invention described herein are capable of operation in other sequences than described or illustrated herein. The following terms or definitions are provided solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4thed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016), for definitions and terms of the art relating to molecular biology or biochemistry. For the field of microscopy, particulars in the art are described in for instance: Heath, JP., Dictionary of Microscopy, 2005, Wiley; Hajibagheri M. A. Nasser, Electron Microscopy Methods and Protocols, 1999, Humana Press, vol 117. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g. in molecular biology, biochemistry, structural biology, computational biology, microscopy, and / or mechanics).

[0041] The terms "protein", "polypeptide", and "peptide" are interchangeably used further herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. A monomeric or protomer is defined as a single polypeptide chain from amino-terminal to carboxy-terminal ends. A "protein subunit" as used herein refers to a monomer or protomer, which may form part of a multimeric protein complex or assembly. The term "molecular complex" or "complex" refers to a molecule associated with at least one other molecule, which may be a protein or a chemical entity. The term "associating with" refers to a condition of proximity between a chemical entity or compound, or portions ROEf / trEM-chip / 856 thereof, and a binding pocket or binding site on a protein. As used herein, the term "protein complex" or "protein assembly" or "multimer" refers to a group of two or more associated macromolecules, whereby at least one of the macromolecules is a protein. A protein complex or assembly, as used herein, typically refers to binding or associations of macromolecules that can be formed under physiological conditions. Individual members of a protein complex, such as protein subunits or protomers, are linked by non-covalent or covalent interactions. The term "multimer(s)", "multimeric complex", or "multimeric protein(s) or assemblies" comprises a plurality of identical or heterologous polypeptide monomers. Polypeptides can be capable of self-assembling into multimeric assemblies (i.e.: dimers, trimers, pentamers, hexamers, heptamers, octamers, etc.) formed from self-assembly of a plurality of a single polypeptide monomers (i.e., "homo-multimeric assemblies") or from self-assembly of a plurality of different polypeptide monomers (i.e. "hetero-multimeric assemblies").

[0042] A 'microfluidic chip' is a set of micro-channels etched or molded into a material (e.g. glass, silicone or polymer). The micro-channels forming the microfluidic chip are connected together in order to achieve the desired features (mix, pump, sort, or control the biochemical environment). This network of microchannels trapped into the microfluidic chip is connected to the outside by inputs and outputs pierced through the chip, as an interface between the macro- and micro-world. The simplest current microfluidic device consists in micro-channels molded in a polymer that is bonded to a flat surface (such as a glass slide). The polymer most commonly used for molding microfluidic chips is PolyDimethylSiloxane (PDMS). PDMS is a transparent, biocompatible, deformable and inexpensive elastomer. It is easy to mold and bond on glass.

[0043] The term 'post' or 'post stand' or 'mounting post' as used herein is a type of stand often used with microscopes, or in the case described herein with equipment in a microscopical environment, such as the plunger arm module. The post stand consists of a single post rising vertically from the base with the capability to rotate the part mounted on top of the post around a horizontal axis, as a rotatable alternative option to the fixed arm stand.

[0044] A 'stage' as used herein is a flat plate where the modules can be mounted on and brought into relative position to each other as described for the integrated apparatus herein. A stage may be a 'mechanical stage' containing at least a plate and further also knobs to manually turn as to move the mounted part in a certain direction (e.g. a XY mechanical stage, to move in X and Y direction), or further electronic means to position the mounted parts on the 'motorized' stage in a certain direction.

[0045] Time-resolved Cryogenic-electron microscopy (tr-Cryo-EM) is a structural biology method which in principle allows high-resolution structural analysis of intermediates of biological reactions and other non-stationary processes occurring in soft matter or some nanomaterials. Time-resolved cryo-EM in fact ROEf / trEM-chip / 856 combines the visualization of the molecular structure at single-particle level with the ability to dissect the time progress of a reaction between molecules, and / or of a molecule in different conformational states during a biological reaction.

[0046] Detailed description

[0047] The present invention relates to a microscale or microfluidic application for sample preparation wherein the droplet-on-demand generation of airborne microdroplets acts via a distant actuation mode to maximize or ascertain the quality of the microdroplet components, which is critical especially for biological components such as proteins. Specifically, the invention may be considered as an adaptation of a time-resolved sampling method and microfluidic chip as described in Efremov et al., WO2022 / 148859, wherein the droplet generation module has been altered to provide for higher quality sampling of small biological samples wherein the integrity and stability of the materials is retained. The subject matter of Efremov et al., WO2022 / 148859, as relating to the present invention, is fully incorporated herein. The microfluidic chip and method for spraying microdroplets of the present invention is described in more details in the example section, and / or in combination with the drawings and figures as referred to herein, and as further explained in detail in the Example section.

[0048] The droplet generation of microdroplet dispensing module, as used interchangeably herein, and the chip as proposed herein, may as well serve for the purpose of protein sampling in other analytical methods, such as Cryo-EM sampling of a single protein, possibly involving additional features on the chip such as for protein purification, as well as for the purpose of microscopic techniques on further types of samples, or even in other fields such as for cell dispension in small drops, multidimensional FACS based on fast processes like Ca2+-signaling, nanoparticle synthesis, rapid mixing of materials in 3D printing applications, among others.

[0049] The first aspect of the invention relates to a microfluidic chip device for spraying of microdroplets, wherein droplet generation is controlled via a droplet-on-demand actuation mechanism with a dispensing module configured to allow distant actuation of a sample. With 'distant actuation' as used herein, it is referred to triggering the initiation of a pressure wave in a fluid, wherein said fluid is not the sample but wherein the pressure wave in said fluid is generated at a micrometer distance from the sample solution and consequently transmitted to the sample solution as to generate microdroplets from said sample solution for ejection out of the chip. So 'distant' herein refers to the pressure wave being at another location than where the sample solution that needs to be sprayed is present. The initial trigger or actuation of the pressure wave is obtained by applying an actuator outside of the chip, which is also the case for 'on sample' or 'direct' actuation. The dispensing module in the chip of the present invention ROEf / trEM-chip / 856 operates to obtain controlled droplet ejection combining controlled flow of liquid sample solution through the main channel (or nozzle channel), and controlled distant actuation in a physically separated but connected actuation chamber (Figure 1). The 'nozzle channel' of the dispensing module used herein refers to the main microchannel in which the liquid sample solution that needs to be sprayed as airborne droplets is present. The nozzle channel on one side fluidly connects to the inlet module it's main microchannel, from which the liquid sample solution is driven towards the dispensing module, said nozzle channel providing the connection between said main microchannel of the inlet module and the outlet opening(s) of the chip, through which the liquid sample solution is finally ejected under the form of microdroplets after actuation in a droplet-on-demand manner, for which a pressure wave is initiated at a distance from the liquid sample solution, namely in the actuation chamber of the dispensing module. The nozzle channel further contains a chamber junction, which is the point in the nozzle channel where a physical connection or opening is made to from one side of the wall of the nozzle channel to the inside of the actuation chamber. Said connection is required so that the actuation chamber material or fluid present in the actuation chamber, and the liquid sample solution flowing through the nozzle channel, are both in close proximity without in between materials so that the actuation signal, typically a pressure wave propelling through the actuation chamber fluid, can be transmitted to the liquid sample solution in the nozzle channel.

[0050] A nozzle is typically defined as a tool designed to control characteristics of the fluid flow, and is provided in the chip of the present invention as a simple channel tool, therefore called herein the 'nozzle channel', directing the fluid towards the chip's end, the outlet opening(s), where it is sprayed outside of the chip.

[0051] The present invention relates to a chip wherein the dispensing module comprises an actuation chamber for distant actuation. Specific embodiments of actuation mechanisms to trigger a pressure wave within the actuation chamber material are known in the art. The initial trigger comes from an actuator or actuator module that is not on the chip, but remotely present, such as a laser actuation module for laser- induced cavitation, or a piezoelectric element to trigger a pressure wave in the actuation chamber fluid. Another distant actuation mode may involve thermal actuation, wherein a remote actuator heats the actuation chamber fluid thereby generating an air bubble that collapses and generates a hydraulic pressure wave that propagates towards the chamber junction and the nozzle resulting in ejection of microdroplets from the nozzle. The operating principle is similar to the laser induced cavitation approach.

[0052] The actuation chamber is separated from the nozzle channel, as the location for distant actuation of the liquid sample in the nozzle channel, but has to be physically connected to the nozzle channel, to allow transmission of the pressure wave. It is however desired that the actuation chamber fluid and the liquid sample solution in the nozzle channel do not mix or at least that the actuation chamber fluid is prevented ROEf / trEM-chip / 856 from contaminating the solution in the nozzle channel. The chip inlets in the inlet module as well as in the actuation chamber are connectable to means for driving the flow of inlet sample solution and actuation chamber material / fluid, respectively. So the chip is configured thereby to allow control of the flow rates of both materials, inlet solution(s) and actuation chamber fluid, and the skilled person will thus be able to prevent such contamination or mixing at the chamber junction by adapting the pressure control module(s). Moreover, the relative dimensions of the actuation chamber, in particular its inlets and chamber junction, and the width of the nozzle channel, are set up as such that pressure control of flow rates allows for preventing that the sample solution get contaminated by the actuation chamber fluid. In a specific embodiment, the width of the actuation chamber inlet and outlet channels is at least the width of the nozzle channel.

[0053] A microfluidic chip for fast mixing of solutes and spraying of microdroplets configured for distant actuation

[0054] The invention provides for a microfluidic chip, exemplified in a specific embodiment shown in Figure 2, or as in Figure 9-10. The microfluidic chip as described herein functions to 1) merge two or more aqueous liquid solutions containing protein(s) of interest or further components or biological materials , and addition of an oil solution via one inlet channel to encapsulate the aqueous solution in droplets, 2) mix the aqueous liquids within the oil-encapsulated droplets within a characteristic time of a few milliseconds or preferably in less than 1 ms, by merging the solutions from the inlet channels in a microchannel and passing them through the serpentine microchannel and, after removal of the oil through the side channel outlets, allow merging of the aqueous droplets, and 3) deliver the mixed solution to the nozzle channel, wherein the actuation signal or trigger generated within the actuation chamber through an electrically-, optically-, or mechanically-controlled actuation mechanism is transferred to the mixed solution in the nozzle channel resulting in the generation of airborne droplets spraying droplets with well-defined controlled size, velocity and timing through the outlet opening(s), out of the chip, preferably on a grid.

[0055] In one embodiment it may thus be envisaged that the microfluidic chip is composed of at least the following two parts (e.g. as shown in Figure 2): an inlet module composed of a mixing module or mixer (as indicated in WO2022 / 148859) comprising at least 3 inlet channels configured to add at least two aqueous solutions via at least 2 inlet channels (28) and one oil composition via one inlet channel (29), to obtain merging of the inlet channel solutions in one microchannel (30) resulting in oil-encapsulated aqueous solution droplets. Very rapid mixing of aqueous liquids is achieved within each aqueous droplet through recirculation of liquid within the droplets as they pass from the microchannel (30) through a serpentine microchannel (31). The serpentine microchannel comprises at least 3 arms (32), or straight channel portions, connected by a bended region (33), typically at an angle of 30-45°, or a turn, typically ROEf / trEM-chip / 856 over an angle of 135-150°, as to allow efficient mixing. The second part of the inlet module concerns a droplet merging module or pillar-induced droplet merger, comprising a main microchannel (30) and at least one side channel (35), for extraction of the oil phase from the main channel, allowing to merge aqueous droplets present in the main microchannel (30) into one liquid phase by directing the oil into said side channel (35), which is positioned transversally to the main microchannel (34), and comprises an array of pillars (36) with a flat surface at the intersection of the main micro- and side channel, constructed to extract the oil from the main microchannel. To do so, the row of pillars from the side channel form the wall of the microchannel along its length, or at least along part of its length, wherein said row of pillars have openings at a distance that is smaller the diameter of the droplets (i.e. smaller than the width of the main microchannel) between the pillars to let the oil be extracted into the side channels. The chip in operation with liquids result at a continuous aqueous phase at the end of the main microchannel (30) of the droplet merging module after the oil removal occurred through the side channel. In one embodiment the chip optionally comprises a further microchannel (37) connected to the main microchannel (30), as a connection to the dispensing module for distant actuation. The dispensing module, as described herein, comprises the nozzle channel, fluidly connected to the main microchannel (30 or 37) and terminates in at least one outlet opening (or nozzle channel tip 40) at the end of the chip, providing the point where sample is sprayed or ejected out of the chip, and further comprises the actuation chamber, as described herein, configured and positioned at a distance to the outlet opening(s) that is maximally 10-fold the distance of the width of the nozzle channel, fluidly connected to the nozzle microchannel as to generate airborne droplets upon actuation to fly through the outlet(s) (40) towards preferentially a cryo-EM grid. In order to activate droplet spraying, a DOD actuator is required, which may for instance be based on laser-induced-cavitation, as known to the skilled person and as demonstrated herein using an optical module, or may as well be based on alternative droplet actuator mechanisms known in the art, such as a miniaturized piezoelectric transducer (essentially a MEMS - micro-electro-mechanical-system), or a heat-induced actuation similar to for example those described in this patents US7364275, US7445314, US7988247, US6183067, US5598196, US6758544.

[0056] The nozzle channel may end in an outlet with a single outlet opening, for ejecting sequentially single droplets upon distant actuation, as provided in Figure 1-3, or alternatively may constitute several outlet openings by a split of the nozzle channel beyond the chamber junction, as exemplified in Figure 8-9, which allows for parallel multiplex droplet ejection upon distant actuation.

[0057] So, the invention provides for a microfluidic chip for rapid mixing of protein solution and generation of air-born droplets from a protein mixture in a controllable manner, through distant actuation as to secure high quality and integrity of the sample, which is preferably a biological sample, wherein said chip, comprises: an inlet module comprising a mixer module for mixing solutions by encapsulating them in oil ROEf / trEM-chip / 856 droplets, said mixer module comprising at least 3 inlet channels, ending in a microchannel for merging the solutes of the inlet channels, forming the oil phase encapsulated droplets of aqueous solutions, and wherein the mixer module ends in a serpentine microchannel, that contains at least 3 arms, sequentially connected by a bended region or turn; and a droplet merging module comprising a main microchannel connected to the other end of the serpentine microchannel of the mixing module, and further comprising one or more side channels transversal to the main microchannel, wherein said side channels are configured as transversal outlets from the main microchannel walls, wherein the width of the side channel is at least half of the length of the main microchannel, and wherein said side channel comprises an array of pillars (36), which align the main microchannel wall, and wherein the pillars of an array are distributed evenly over the width of the side channel and separated from each other by a distance (dl) that is smaller than the droplet diameter, or preferably at least two times smaller than the width of the main channel (d2). More specifically, when two side channels are present on opposite sides of the main microchannel walls, both arrays of pillars, which line the wall of the main microchannel by a flat surface, are apart at a distance d2 of at least the width of the main microchannel, the arrays being positioned on each side of the intersection with the main channel, replacing the main microchannel walls; finally the oil extraction chamber is continued by the dispensing module as described herein, for continuous spraying of droplets upon distant actuation, said module comprising a nozzle channel connected to the end of the main microchannel of the merging module, and with at least one outlet opening at the end of the chip for ejecting the generated droplets, which are formed by a DOD actuation that is triggered and transmitted from the actuation chamber via the chamber junction to the solution in the nozzle channel. The modules of the microfluidic chip are composed of a silicone elastomer, and mounted on a flat surface, wherein the microchannels of the chip preferably have a rectangularly-shaped cross-section with an aspect ratio below 2, and a maximum height of 100 pm ± 10 pm, 80 pm ± 10 pm, or most preferably 50 pm ± 10 pm, and wherein the inlet channels of the mixer module are further connectable to a pressure control module configured to control the pressure in each of the at least 3 inlet channels of the mixer module. The 'height' for features of the chip is defined as the distance in the direction perpendicular to the chip surface. The 'width' as defined herein is the distance in the direction of the same plane as the chip surface.

[0058] In more detail, the mixer module of the microfluidic chip contains a minimum of three inlet channels (28,29), of which through one inlet channel the oil phase can be injected (29), and through two or more additional inlet channels (28) the aqueous solutions to be mixed may be injected. Said inlet channels may be of any shape, and may of their length change in shape, but typically these inlet channels are wider at the side where the solution comes in, as compared to the width of the microchannels (30, 37, 38) in the mixing, drop merging and nozzle region, as to reduce the total hydraulic resistance of the ROEf / trEM-chip / 856 microfluidic chip. In a specific embodiment, said inlet channels initially have a width of 350-400 pm, and then narrow down towards the end where they merge into the microchannel (30). Within the 'functional' regions of the chip, the microchannels' (30, 37, 38) height is preferably 40-60 pm or smaller with a rectangular-shaped cross section with an aspect ratio of preferably below 2. The 'aspect ratio' is defined as the ratio of the height over the width.

[0059] So, in the mixer module the aqueous droplets encapsulated in the oil continuous phase are formed, when the three liquids come in contact with each other in the first microchannel (30), at which point the aqueous solutions are confined in the oil phase, and the size of the aqueous droplets is defined by the ratio of the flow rates of oil phase in the inlet channel (29) and the aqueous phases in its inlet channels (28). Next, the serpentine mixer is made of three or more sections each of which has an arm (32), which is constituted by a straight microchannel with a length of at least twice the microchannel width or longer, which is joined with the next arm by a turn or bending region, preferably at an angle in the range 30-45°. Mixing of aqueous solutions within the droplets is accelerated by the liquid recirculation in a droplet passing through the serpentine microchannel (31) where at each bending (33) of the channel, asymmetric circulation is introduced, by reorienting direction of recirculation within the droplet. This creates a 'complex' pattern of sandwiched thin liquid lamellas within which the mixing is accomplished by diffusion.

[0060] Second, the droplet merger separates the oil and aqueous phases to avoid the presence of oil in the nozzle (38) and on the EM grids. The drop merger module following the droplet mixing module, has a main microchannel (30) length preferably in the order of 200 pm (or longer), which is the shortest length enabling reliable droplet merging using the system described herein, since reliable merging is defined further by the size of the droplets and the gap width or distance (dl) between the pillars within the array, as to obtain optimal oil extraction via the side channels (35). For further details on the droplet merging module setup as proposed previously, it is referred herein to WO2022 / 148859.

[0061] Finally, the microfluidic chip may be fabricated using a standard soft lithography technique, as known in the art, and whereby the different chip modules are interconnectably composed of silicone elastomer, which is sealed on a flat surface. In a specific embodiment, the microfluidic chip is made using a silicone elastomer (such as polydimethylsiloxane (PDMS)) bonded to a glass slide. However, the chip can also be fabricated in other materials, including but not limited to thermoplastic polymers through injection molding or hot embossing fabrication processes, or glass or a combination of silicone and glass, or quartz. At least, the channel surfaces require a hydrophobic nature, as to ensure optimal wettability of the polymeric channel walls with the oil phase when flowing through the channel. To obtain the required hydrophobicity for the PDMS material, the module is kept on a hot-plate at 180°C for 4 hours. In a specific embodiment, where laser-induced cavitation is applied as actuation mechanism, the chip surface ROEf / trEM-chip / 856 material comprises an optically transparent flat surface material, such as glass. In a further specific embodiment, said glass has a thickness of maximally 250 pm to allow the laser beam reaching the focus point in the chip. In an alternative embodiment, when piezoelectric actuation is desired, the chip is made of at least two materials whereby the piezoelectric actuation is obtained by a specifically designed miniature actuator therein, such as for instance piezoelectric ceramic, as known by the skilled person.

[0062] In a specific embodiment, the manufacturing of a PDMS-glass chip involves cutting the PDMS chip transversal to the channel to make the nozzle outlet opening(s). After, the polymer device is sealed with a glass slide, by carefully aligning the two parts to obtain a sharp rectangular opening in the chip. In a preferred embodiment, showing the best reproducibility of the chip fabrication and alignment of the laser focus to the nozzle, a thin glass slide, with a thickness 250 um or less, was used to seal the PDMS chip. This significantly simplifies the accurate alignment (offset between aligned surfaces is below 5 pm) of the glass slide to the nozzle channel outlet opening(s) embedded in the PDMS part. So the outlet opening is made by cutting off the chip material and surface material in the same plane, transversal to the channel direction, and preferentially perpendicular to the channel direction. The fabrication to obtain a flat nozzle outlet opening at the nozzle tip is critical for reliable generation of the air-borne droplets.

[0063] A method for operating the microfluidic chip to spray aqueous droplets of (mixed) solutes.

[0064] Another aspect of the invention relates to the method to operate the microfluidic chip by adding fluids in the inlet channels (28, 29), whereby the flow of the liquid solutions in the chip is controlled for its pressure with an external pumping system operating manually, for instance by using a syringe pump, or preferably through an automated pressure controlling module (such as a Fluigent). The pressure control module as demonstrated herein provides for sample reservoirs (3) pressurized with air in order to control the pressure drop between each of the inlets (28, 29, 7, 8) and the outlets (40) of the microfluidic system. The flow rate depends on the applied pressure and the hydraulic resistance of the microfluidic chip device, which varies between different chips, depending on the size and material of the channels and the chip, and the number of inlet channels. In order to control the flow rate and maintain stable constant flow rates, as well as for better reproducibility of the results generated using different microfluidic chips (slight difference in hydraulic resistance in different chips is likely), each pressure controller is coupled to a flow meter (2) through a feedback loop. For controlling the pressure when operating the integrated device or the microfluidic chip as used herein, each of the at least three inlet channels (28, 29) is connected to a reservoir (3), which may be a 1.5 mL Eppendorf tube or any other reservoir suitable for the solution of interest, comprising the solutions for each inlet channel, further connected to the inlet channel via a flow meter using for instance PTFE tubing. ROEf / trEM-chip / 856

[0065] The operation of the device using a pressure control module connected to the inlet channels of the chip allows to vary the applied pressure via the pressure controllers (4), which may be computer-controlled by the microprocessor-based control unit as driven by a computer (PC), and with a pressure < 1 bar, preferably below 500 mbar, most preferably between 50 and 220 mbar, as to result in corresponding flow rates of the solutions in said inlet channels between 1 and 100 pL / min, or preferably between 2 and 60 pL / min, more preferably between 2 and 10 pL / min, depending on the pressure as well as on the chip geometry and reaction delay time. A single pressure controller (4) is connected to each one of the inlet channels (dedicated to oil and aqueous phases). The flow in the microfluidic chip is started by first setting the pressure values for each channel and then synchronously initiating the flow using custom written LabVIew software.

[0066] In a specific embodiment, an at least fourth pressure control module is present in the pressure controller (4), which is connected to the chip side channel's (35) entrance by tube (3b), as to apply a negative pressure, controlled by the control unit, when oil needs to be extracted from the chip channel.

[0067] In a specific embodiment, an at least further pressure control module is present in the pressure controller (4), which is connected to the chip's actuation chamber inlet (7) and outlet (8) entrance by a tube, as to apply appropriate pressure controlling the circulation of the actuation chamber fluid in a balanced manner with the flow rate of the liquid solution in the nozzle channel, as to prevent contamination of said actuation fluid to the nozzle channel.

[0068] The integrated apparatus for time-resolved sample preparation of a sample.

[0069] In another aspect, the present invention provides for an integrated apparatus comprising three main components commonly known to be required for time-resolved (cryo-EM) sample preparation on a grid: a) a microfluidic chip for generation of airborne microdroplets; b) a droplet actuation system for spray generation of airborne droplets; c) a plunger, designed or readapted, for time-resolved plunging of the sample grid.

[0070] The present invention provides for a solution to several of the above problems by further customizing the specific needs for tr-Cryo-EM sample preparation, wherein the methods and devices as presented herein allow for tunable DOD spraying applying a distant actuation mode of action to maintain high sample quality, controlled and coordinated vitrification, and a controlled reaction delay time regime, all controllable at a low flow rate to prepare grids using very low amounts of sample. As further discussed below, the reaction delay time may be selected for as a short, medium or long regime time, which is the time between initiation of mixing and grid plunge-freezing. The microfluidic chip as described herein functions to provide for rapid mixing: a mixing time down to 1 ms is possible using this device, and at ROEf / trEM-chip / 856 least 20 -30 ms has been demonstrated in combination with the other modules in the integrated apparatus of the present invention. Moreover, the chip provides for a regulated spraying of droplet on grid by DOD. The method and microfluidic chip for mixing and DOD as described herein thus combines specific steps and features, respectively, leading to controllable spraying on a grid, using low amounts of sample, without affecting the components of the sample, a combination that has not been used so far in time-resolved sampling. Furthermore, the integrated device comprising the microfluidic chip for mixing and generation of airborne microscopic droplets using a DOD approach (possibly actuated by laser-induced cavitation in the exemplified prototype), in a time needed for mixing (tChiP) as controlled by the flow rate in the channel, and a time required for the droplets to be ejected on the grid (tfiy), further provides for the combined use with a customized cryo-plunger in which the plunging time regime tpiUnger can be programmed, and resulting in reaction delay times (td= sum of tChiP+ tfiy+ tpiUnger) in the range between the shortest achievable reaction delay time (ca. 1 ms) and a few seconds time interval, according to the sampling needs. Moreover, the microfluidic chip functions at low pressures (< 1 bar) and due to controlled generation of every single drop for spraying, and controlled plunging, the sample consumption per grid can be reduced to below 1 pl, preferably below 0.1 pl even more preferably below 1 nl.

[0071] A further aspect of the invention relates to an integrated apparatus or device including the previously aspects, the customized microfluidic chip and a plunger module, as part of the solution for improved sample preparation in time-resolved cryo-EM analysis of protein structures, allowing to prepare EM- grids with vitrified protein sample, wherein the proteins were mixed in milliseconds, and processed at high speed, within a millisecond sampling time resolution, and requiring only picoliter to nanoliter protein amounts.

[0072] The integrated apparatus as described herein comprises the microfluidic chip as described herein (and as present in Figures 1-3) connected to a pressure module, as known in the art and / or as described and / or exemplified herein, a plunger module, and further comprises a droplet actuation module for controlled droplet on demand ejection of drops from the outlet opening(s) of the chip to the grid, as known in the art and / or as described and / or exemplified herein, and a microprocessor-based controller unit for synchronizing and controlling movement of the plunger arm, the pressure control unit and the thermostat of the reservoir of a cryogenic module as known in the art and described herein. The integrated apparatus and its components are configured to function in time-resolved sampling and vitrification as exemplified herein and as for instance (but not limited to) the setup shown in Figure 6.

[0073] More specifically, one embodiment relates to the integrated apparatus wherein the microfluidic chip (1) is mounted on a solid structure, such as a X-Y-Z stage (24b) which is positioned on a holder or housing (24), on its turn mounted on another solid structure, such as a mechanical X-Y stage (23). If motorized, ROEf / trEM-chip / 856 said X-Y stage should preferably have an accuracy below 1 mm, and preferably also has the plunger module and cryogenic module of the integrated apparatus mounted on it. Said setup is configured to relatively position the plunger module to allow plunger arm (5) movement parallel to the plane of the nozzle outlet of the chip (1), and the position of the grid clip mechanism (6) for holding a grid with its grid surface parallel to the surface of the nozzle outlet(s) (40) at a distance below 1 mm, which is a distance suitable for receiving droplets from the nozzle tip.

[0074] To obtain spraying of airborne microdroplets droplets on demand from the microfluidic chip (1) upon actuation in the nozzle and ejected from the outlet opening(s) or nozzle channel tip (40), a DOD droplet actuation module is required to mediate and control the DOD process. The droplet actuation may be mediated by different approaches as to generate DOD, such as for instance, but not limited to, actuation means known in the art as piezo-electric actuation, which is well established and characterized for ink jet printers in particular of mark EPSON (us 7,445,314 B2), or alternatively, laser-induced cavitation. While the first one is the mostly preferred in commercialized devices, as it is cheaper and easier to manage droplet generation, the fabrication of the device is more difficult. So, therefore the proof of concept prototype of the integrated device that was build and developed on laboratory scale as described herein, has made use of an optical module for laser-induced cavitation, which is compatible with simple chip fabrication strategies. However, the integrated device described herein, in particular the microfluidic chip, allows integration of an actuation module of the piezo-electric type as well, for which the geometry of the actuation chamber may require adjustment, depending on the exact implementation.

[0075] To generate droplets at the outlet opening(s) or nozzle channel tip (also referred to as nozzle tip in WO2022 / 148859) (40) via laser-induced cavitation, it is important to focus the laser on the fluid filling the actuation chamber (containing a laser-absorbing dye). The laser power used to produce droplets for the tr-Cryo-EM that is needed to induce droplet ejection (e.g. up to 18 pJ / pulse) can damage the material of the chip's actuation chamber, e.g. the PDMS, and consequently deteriorate its optical properties required for precise focusing the laser beam. In a specific embodiment as provided herein, the optimal conditions for droplet generation require the laser being focused in the chamber, and the chamber junction being positioned at a distance (d) from the outlet opening (40) that is defined by the width or cross-section of the nozzle channel, with a maximum distance of 10-times said width, and maximum laser power intensity usable to induce the cavitation effect without damaging the device. The accurate control of laser intensity has to be in line with the distance of the chamber junction and the outlet opening, and known how to determine by the skilled person. Laser flashes of higher energy ablate the glass or PDMS surface deteriorating optical properties of the glass and reducing cavitation effect. ROEf / trEM-chip / 856

[0076] In the actuation module setup as described herein, cavitation is generated using a focused laser beam from the second harmonic of a pulsed NdYAG laser (wavelength of 532 nm and pulse duration 6 nm). The operation principle of laser-induced cavitation is to have the laser light absorbed by the target media (i.e. the dye present in the actuation chamber fluid), which in the focal point results in heating of the fluid that induces boiling and thereby producing a short-lived air bubble within the chamber, where the air bubble grows and then collapses with a lifetime of a few microseconds. This collapse induces a pressure wave that propels towards the chamber junction where it is transmitted to the aqueous sample solution jetting from the nozzle channel as to produce airborne droplets from the outlet opening.

[0077] In the exemplified integrated device as shown herein, DOD ejections are created by laser-induced cavitation, requiring an actuation module comprising a laser (17), in a particular embodiment this may be a Q-switched NdYAG laser (AO-L-532, CNI Optoelectronics Tech. Co., Ltd.), which is coupled to an optical module, said optical module being meant to control and monitor the positioning of the laser to the nozzle of the microfluidic chip. In a particular embodiment, said optical module is composed of a focusing objective lens (18), in particular this may envisage a 5x objective LMH-5X-532 Thorlabs objective, which is mounted on an optical microscope (19), such as for instance, a Cerna Mini Microscope, Thorlabs Inc. Furthermore, the optical module may comprise a (high-speed) camera (20), such as for instance a Phantom VEO410L camera, which may be mounted on the microscope as to allow recording fast processes taking place on the microfluidic chip (1) and to monitor and control production and deposition of droplets on the EM grid during plunging. The laser beam is expanded with a 10X beam expander (21) (e.g. BE10-532 10X , Thorlabs Inc.) and directed with mirrors to the microscope through a side entry port where it is brought onto the optical axis of the objective lens (18) by a beam splitter (22) (e.g. BSW4R-532, Thorlabs Inc). The laser beam intensity is continuously monitored using a power meter (26) and the laser beam can be blanked by a fast, automated shutter or aperture (27).

[0078] So in a specific embodiment, the integrated apparatus as described herein comprises a droplet actuation module for laser-induced cavitation, said droplet actuation module comprising: a. a pulsed laser (17), preferably with an automated aperture (27) and power meter (26), and b. an optical module for focusing the laser on the nozzle, wherein the pulsed laser is focused on the material in the actuation chamber of the dispensing module of the microfluidic chip (1) at a focusing point suitable for induction of a cavitation bubble and transmission of a pressure wave resulting thereof towards the chamber junction into the nozzle channel, preferably at distance from the outlet opening(s) which is less than 10-times, more preferable 8-times or lower from the outlet opening(s) (40). ROEf / trEM-chip / 856

[0079] To start operating the droplet actuation module for laser-induced cavitation as described herein, the (NdYAG) laser (17) is warmed up and its energy, as detected by the power meter (26), is adjusted to 10- 12 pJ per pulse*per nozzle. This corresponds to the laser power per focal point of around 4-6 pJ on the microfluidic chip (1) surface, which is the power needed to induce the cavitation. The laser as described and used herein operates at the frequency between 1 and 5 kHz.

[0080] In a specific embodiment, said optical module, may thus comprise an objective lens (18) for focusing the laser beam on the nozzle of the chip (1), whereby the chip is aligned using an XY stage (24) and the objective lens (18) can be moved in Z direction for laser focusing, and (optionally) further comprises optical elements including a beam expander (21), a prism, mirrors and beam splitter (22) to bring the laser beam on the optical axis of the objective lens (18), an optical microscope (19) and / or a fast video recorder or camera (20) for recording droplet mixing, merging and spraying on the moving EM grid.

[0081] In a specific embodiment, the integrated apparatus as described herein comprises a cryogenic module which comprises a cryogenic container for liquid nitrogen (12), wherein said container is configured to allow a minimal flow of cold gas above the lid (15) surface, holding a reservoir (13) for liquid ethane, optionally thermostated, and optionally a holder for a grid box (14).

[0082] For longer reaction delay times (see below), the plunger arm may need to be parked in a waiting position for a few seconds in time, this period though requires additional measures to avoid evaporation of microdroplets with protein on the EM grid. The reduction of the evaporation rate may for instance be achieved by constructing a miniaturized environmental chamber, as known by the skilled person, creating local high humidity around the grid.

[0083] In order to assemble a functional integrated apparatus for tr-Cryo-EM sample preparation, using laser- induced cavitation as described and exemplified herein, certain points need to be taken into account, such as referred to in Efremov et al. (WO2022 / 148859, incorporated by reference herein).

[0084] Finally, in order to operate the integrated apparatus as described herein in an automated manner, a computer connected to the microprocessor-based controller as well as an electrical connection to the other components of the setup as indicated in Figure 6 are required. For a description of the requirement on synchronizing and controlling the integrated apparatus and its components, see Efremov et al. (WO2022 / 148859, incorporated by reference herein).

[0085] The setup as shown in Figure 6 applying the chip of Figure 2-3, applying 3 reservoirs connected to an inlet channel, one filled with an oil composition, one with an aqueous solution containing protein of interest, and one with an aqueous solution containing another protein, has been tested and confirmed to allow stable and controlled generation of droplets, vitrification of the proteins within the samples and high resolution cryo-EM analysis. ROEf / trEM-chip / 856

[0086] A method for spraying of mi on a Cryo-EM

[0087] Another specific embodiment relates to said method as described herein wherein the actuation is obtained via laser-induced cavitation, preferably by using the integrated apparatus comprising the chip as described herein, the method comprising the steps of : a. placing a grid in the grid-clip mechanism (6) of the plunger arm of the integrated apparatus as described herein, and apply at least 2 aqueous solutions to at least 2 inlet channels (28) and an oil composition to at least 1 inlet channel (29) of the chip (1) or the apparatus as described herein, b. switching on the laser, setting the pressure control using the microprocessor-based control unit of the integrated apparatus for mixing of the solutions and subsequent droplet generation in the chip at a constant flow rate, by selecting a pressure ratio for the oil / aqueous phases in the inlet channels below 1 bar, and c. setting the positive pressure control to regulate the inflow of the laser absorbing-dye solution entering the actuation chamber, and the negative pressure control to regulate the outflow, by setting pressures below 1 and -1 bar, so that when switching on the laser, at each new pulse the actuation chamber in consistently fully filled d. synchronizing the pressure control with the opening of the laser aperture (or shutter) using the microprocessor-based controller unit, as to focus the laser and induce cavitation in the actuation chamber for forming droplets, and activating the plunger arm of the plunger module after a specified reaction delay time, preferably within less than 10 ms, to allow rapid plunge freezing of the grid in the cryogen after the droplets are sprayed on the grid, e. after the droplets are deposited on the grid and plunger reaches the cryogen, stop the plunger arm movement, and close the laser aperture, wherein the specified reaction delay time is in in the range of around 1 ms to a few seconds, wherein the oil composition comprises fluorinated oil and a surfactant in the range of 1-10 % (VJ / V), and wherein the pressure is set to a value < 1 bar with the ratio between the oil and aqueous solution being in a range 0.5-1.5, and wherein the material applied in the inlet channel (7) of the actuation chamber (39) in the chip contains absorbing material at the emission wavelength of the laser.

[0088] In the specific embodiment where laser-induced cavitation is applied as actuating module for the DOD generation, the cavitation is only generated upon absorption of laser energy, which requires the presence of an absorbing material being a dye solution having an Extinction Coefficient > 10 mW1cm'1at 532 nm, such as Amaranth Acid red 27 or Direct Red 81. ROEf / trEM-chip / 856

[0089] The emission wavelength of the laser should correspond to the excitation wavelength of the absorbing material that is applied in the aqueous solution in the inlet channel. For instance, said laser may have an excitation wavelength of 532 nm and absorbing material absorbing at 532 nm, such as Amaranth Acid red 27 (CAS Number 915-67-3; extinction coefficient at 532nm of 25000 M-lcm-1), preferably present in a concentration in a range of 8-20 mM, more preferably at 10-15 mM, specifically at 12 mM in the solution prior to mixing, with a final concentration in the nozzle being optimal at 3-10 mM, 5-8 mM, preferably 6 mM; or alternatively Direct Red 81 (extinction coefficient at 532nm of 15000 M-l cm-1).

[0090] In another specific embodiment, said method described herein using laser-induced cavitation means for droplet generation applies a pulsed laser that is operating with pulse duration of 6 ns and a frequency of 2500Hz for forming droplets of 7-150 pL.

[0091] The method as described herein may desire to apply a short reaction delay time, which may be obtained by applying a pressure of 220 mbar to the oil composition and to the aqueous solution, using a laser with frequency of 5000 Hz (at 5 pulses / ms) and a plunger arm transferring the grid between nozzle and cryogen solution in less than 8 ms preferably less than 1ms. Alternatively, a sampled grid may be obtained using a method wherein a mid-term delay reaction time is selected by continuous unidirectional movement of the plunger arm but at lower speed; using a laser with frequency of 5000Hz. Furthermore, the time resolved sampling method wherein a long reaction delay time is attained is obtained by reversing the direction of the arm movement and plunging the EM grid after user-specified delay.

[0092] Finally, the invention relates to the use of the microfluidic chip as described herein, and / or the integrated apparatus as described herein, and or the methods as described herein, for time-resolved sample preparation, preferably sample preparation for Cryo-EM analysis, most preferably for time-resolved structural analysis.

[0093] Further alternative embodiments relating to the use of the microfluidic chip or apparatus or method as disclosed in the different embodiments herein are envisaged for a multiplicity of applications that would benefit from 1) fast mixing on nanoscale and / or 2) microdroplet spraying through distant actuation, so for instance but not limited to the use for applications in: cell dispensing in microdroplets (e.g. of about 20-50 pm); multidimensional Fluorescence Assisted Cell Sorting (FACS) wherein sorting is based on a fast processes like molecular signaling (e.g. Ca2+signaling, ...) wherein the sorting requires cell separation in more than 1 dimension or more than 2 buckets; alternative microdroplet spraying under time resolution of milliseconds to seconds, such as for instance nanoparticle synthesis or chemical synthesis; rapid mixing of materials in 3D printing applications, where multi-material components are needed allowing for more complex and functional parts. ROEf / trEM-chip / 856

[0094] It is to be understood that although particular embodiments, specific configurations as well as materials and / or molecules, have been discussed herein for methods, and apparatus according to the disclosure, various changes or modifications in form and detail may be made without departing from the scope of this invention. The following examples are provided to better illustrate particular embodiments, and they should not be considered limiting the application. The application is limited only by the claims.

[0095] EXAMPLES

[0096] The examples presented herein provide for non-limiting proof of concept for a functional integrated apparatus for applying the chip disclosed herein, and method for time-resolved sample preparation using the same, and further supports the controllable working range for sample preparation for different applications of sample preparations, such as specifically demonstrated herein for (time-resolved) Cryo- EM sampling.

[0097] Example 1. A microfluidic chip for spraying microdroplets through direct / on sample actuation.

[0098] In a previously designed microfluidic chip for time-resolved sample preparation in the field of Cryo-EM (Efremov et al., WO2022 / 148859; Torino, et al. 2023, Nat Methods 20, 1400-1408), a sample (mixed) solution is directed into a nozzle channel where airborne droplets are generated through laser-induced cavitation and ejected from the chip outlet opening on a grid. However, this type of actuation in the sample solution requires the laser to be directly focused on the liquid sample flowing through the nozzle channel, and laser-absorbing material needs to be provided within the mixed solution (Figure 7).

[0099] Said chip design as shown in Figure 7 and WO2022 / 148859 has therefore three main limitations in its actuation of the liquid sample to spray microdroplets: (1) the laser-absorbing dye is mixed with the (protein) sample solution in the main / nozzle microchannel of the chip and consequently, and especially in the case of membrane protein samples, the presence of such laser-absorbing components may cause interference with the sample components, such as prohibition of protein complex formation or dissociation of protein complexes; (2) upon actuation, the laser pulses are focused directly on the main microchannel of the chip through which (protein) sample flows, which may cause local heating of the chip and denaturation of temperature-sensitive components such as certain proteins; and / or (3) in cases where a detergent is present in the sample, the dye may form fibers that interfere with the contrast of protein particles visible on the micrographs. All these limitations affect the sample quality and the consequent analysis, especially in the case of protein samples, with potentially impacting for instance the resolution of the final protein structure obtained from analysis of the sprayed microdroplets. Although the initially designed chip configuration allowed for collection of high-resolution time-resolved cryo-EM data and high-resolution 3D reconstruction for several proteins, the resolution as well as the ROEf / trEM-chip / 856 sampling potential was limited to the types of protein(s) that are not affected by the direct actuation, such as the laser or the presence of a dye, in the sample.

[0100] Example 2. A novel microfluidic chip for spraying microdroplets through distant actuation.

[0101] In the present application, we describe an improved chip design which is based on the design as exemplified in Example 1, but in which the above limitations of direct or 'on sample' actuation for microdroplet formation have been overcome by adapting the chip to a design configured for distant actuation (Figure 1 and 2). So, the novel microfluidic chip presented herein comprises at least an 'inlet module', for applying the liquid sample solution to the chip, and which may comprise the additional features necessary for being able to mix solutions from several inlets (Figure 2), and the chip comprises a novel 'dispensing module', which allows actuation of the liquid sample from a distance, specifically from within an actuation chamber that is separated from, but in connection with, the main microchannel or nozzle channel through which the liquid (protein) sample solution flows. This distant actuation mechanism thus allows microdroplet generation within the nozzle channel at the outlet opening upon ejection of the liquid sample solution. The distant actuation however requires a precise setup in which the actuation chamber and the junction forming the connection between said chamber and the nozzle channel in said 'dispensing module' is positioned so that the distance between said junction and the outlet opening of the chip allows for controlled spraying of microdroplets from the chip. This improved dispensing module, as part of a microfluidic chip for sample preparation, was shown herein to function in the controlled formation and spraying of microdroplets (on grid) allowing to establish (time-resolved) sampling in very small volumes and using very small quantities of sample. In addition, the novel chip is advantageous in that the liquid (protein) sample is not mixed or contaminated with actuation material, such as the laser-absorbing material, and the spatial separation of the actuation (i.e. distant actuation) from the nozzle channel containing the protein solution.

[0102] As shown in Figure 1, said new dispensing module of the chip is mainly composed of a main microchannel or nozzle channel (38), which receives the liquid sample solution by being in fluid connection with the main microchannel (30) connected to the inlet channels (29), and which terminates at the outlet opening (40) of the chip, where microdroplets can be ejected; and an actuation chamber (39), which is in connectivity with the nozzle channel (38) through a chamber junction (41). The liquid sample solution flows within the nozzle channel in the direction of the outlet opening, of which the flow is typically controlled by a pressure control module. The actuation chamber is typically filled with a material or fluid required for transmission or propagation of a pressure wave which is triggered upon actuation. Said pressure wave is further transmitted towards the chamber junction, where induction of microdroplet formation is consequently triggered in the liquid sample solution, hence via distant actuation. The actuation may be performed through laser-induced cavitation which requires a laser-absorbing dye as ROEf / trEM-chip / 856 actuation chamber material. As shown in Figure 1A for instance, such a dye is envisaged herein to circulate through the actuation chamber via an inlet (7) and outlet (8) for the fluid solution comprising the dye, connectable to means for controlling the actuation chamber circulation flow during chip operation. For this purpose, the actuation chamber thus functions to generate the laser-induced cavitation bubble(s), by focussing the laser on one of the points within the chamber, on the actuation chamber material, being a solution containing a laser-absorbing dye. The resulting pressure pulse propagates towards the nozzle channel and reaches meniscus without a significant attenuation at the chamber junction where it is in fluid connection with the liquid sample solution flowing through the nozzle channel.

[0103] As for the distant actuation through laser-induced cavitation, the larger size of the actuation chamber permits formation of larger laser-induced cavitation bubbles and hence for more stable droplet generation allowing to apply laser pulses hundreds of microns away from the nozzle channel thereby limiting potential heating of the liquid solution or nozzle channel chip material.

[0104] Most importantly, the chamber junction (41), which is the opening that fluidly connects the inside of the actuation chamber with the nozzle channel, is positioned so that the pressure wave will be transferred from the actuation chamber material to the liquid solution in the nozzle channel (38), which thereby generates a microdroplet ejected at the outlet opening (40), as a result of the pressure wave decay occurring in close enough distance (d) to said outlet opening (40).

[0105] During chip operation, the flow of liquid solution in the nozzle channel is balanced such that actuation chamber fluid does not flow inside the nozzle channel, and so contamination of liquid sample solution with the actuation chamber material is prevented. The fluid material in the actuation chamber thus preferentially flows to the dye-out channel of the actuation chamber (8). The control of the fluid flow in the actuation chamber, as well as sample flow through the main microchannel (30) and nozzle channel (38) of the chip is driven by means such as pressure control modules that balance the flow as such that no fluid mixing occurs at the chamber junction, though allowing the transmission of the pressure pulse generated by the cavitation bubble in the chamber, as to decay in the liquid sample solution upon microdroplet ejection from the chip.

[0106] Alternatively, the distant actuation mechanism in the actuation chamber may be initiated through alternative actuation modules as known in the art, typically resulting in a pressure wave generated within the actuation chamber material (Figure IB), such as for example through piezoelectric actuation inducing a pressure wave in a liquid such as water as actuation chamber material. ROEf / trEM-chip / 856

[0107] Example 3. A microfluidic chip designed for protein sample microdroplet spraying.

[0108] A particular chip was designed for the experiments as shown in Figures 3-5, with an actuation chamber having 2D dimensions of approximately 450 x 550 pm, and with a depth equal to or greater than that of the proteins' (liquid sample solution) channel (~25-50 pm) as to avoid fluid material from the chamber mixing with the solution in the nozzle channel. Upon laser-induced cavitation, the cavitation bubble, being unrestrained by the wall of the microfluidic system in at least 2 dimensions, reached 110-120 pm in diameter, as measured using high-speed video recording (Figure 4 & 5). It is envisaged herein that to ensure consistent operation, the actuation chamber has in-plane dimensions at least twice that of the generated cavitation bubble to minimize effect of the channel wall on the bubble expansion. Hence the chamber should have 2D dimensions of at least 200 x 200 pm, through preferably slightly larger, such as 300 pm -450 pm (also see Figure 3).

[0109] Furthermore, during chip operation using pressure control modules for driving the fluid flow, the pressure wave generated by the cavitation bubble collapse propagates to the proteins' solution nozzle channel through the chamber junction physically connecting the two regions, wherein the width (w) of the chamber junction should not be too large, to prevent actuation chamber material from flowing preferentially in the direction of the nozzle channel instead of to the actuation chamber outlet channel (8), nor too narrow, to avoid excessive attenuation of the pressure wave transmitted from the actuation chamber material to the liquid solution in the nozzle channel. For a nozzle channel with a width of 25 pm, as used herein, a chamber junction with a width of 25-50 pm is therefore required (Figure 3).

[0110] A continuous circulating flow of dye solution within the actuation chamber must be maintained by letting the dye solution in through an inlet channel (7), and driving the dye solution to an outlet channel (8) in the actuation chamber, by applying means that control the flow of actuation chamber material or fluid, which is taking into account the pressure / flow of the liquid solution in the nozzle channel as to prevent dye solution from flowing into the nozzle channel. For instance, the width of the nozzle channel will be important for the design of the inlet and outlet of the actuation chamber, so that the flow rate of the protein solution in the nozzle channel is not too low or lower than the flow rate of the fluid circulating in the actuation chamber. As a rule, the width of the nozzle channel should thus be smaller than the width of the inlet channel (7) and the outlet channel (8) of the actuation chamber. Both the dye inlet (7) and outlet (8) channels have a width of 50 pm in the exemplified chip design of Figure 3, with a nozzle channel width of 25 pm. Using a pressure control module as means for circulating and controlling the flow of dye solution, a positive pressure between 50 and 80 mbar is applied to the dye inlet channel (7), while a negative pressure ranging from -10 to -100 mbar is applied to the dye outlet channel (8). ROEf / trEM-chip / 856

[0111] The pressure wave will experience some attenuation, which depends primarily on the properties of the propagation medium or material, and on the composition material of the microfluidic chip. To ensure reliable ejection of airborne droplets, the distance (d) between the actuation chamber's junction to the liquid protein solution flow and the outlet opening, where the sample is ejected, should be less than 200 pm (Figure 3), or as considered relative to the width of the nozzle channel, this distance (d) should be maximally 10-times the width of the nozzle channel, preferably maximally 8-times the width, or even less. This was verified experimentally using the chip device wherein the large actuation chamber was tested in relation to the nozzle channel with protein solution for its ejection as microdroplets through the outlet opening.

[0112] Example 4. Resolving Protein 3D structures using time-resolved cryo-EM analysis.

[0113] The integrated apparatus setup as described in Efremov et al. (WO2022 / 148859; and as shown in Figure 6 herein) was used to compare time-resolved sampling of both chips, the previously designed with direct actuation and the currently designed with distant actuation, for a protein sample containing protein Respiratory Complex I (EC 7.1.1.2 also known as NADH: ubiquinone oxidoreductase, Type I NADH dehydrogenase and mitochondrial complex I).

[0114] The left panels of Figure 8 demonstrates typical micrographs obtained by plunge-freezing the respiratory Complex I sample using both chips. In (1), applying the previously described chip of WO2022 / 148859, proteins were observed on the cryo-EM grids, though their structure was not well-preserved, and hardly any intact complexes could be identified among the particles. Likely, the actuation on the sample and / or presence of the dye in the sample negatively affected the protein integrity. In contrast, the application of the microfluidic chip with the distant actuation mode as described herein allowed to achieve grids where the full protein complex is clearly visible. The micrographs in figure 8 also show improved contrast, with a much cleaner background. From the collected data (right panels of Figure 8), high-quality 2D classes were generated and a 3D reconstruction at 8 A resolution using just 5000 particles was obtained. In conclusion, this resulting structure highlights the high quality of the obtained data while and due to the preservation of protein integrity during the sample preparation.

[0115] Example 5. Multiplex droplet spraying device.

[0116] In addition to the chip designed and applied in time-resolved Cryo-EM as exemplified above, we have developed an alternative chip design with a smaller actuation chamber and a multiple nozzle outlet openings for parallel droplet spraying. The chip as shown in Figure 9-10 has been designed and tested for its ability of multidroplet spraying of a protein sample. ROEf / trEM-chip / 856

[0117] The application of an actuation chamber with smaller dimensions as compared to the previous exemplified design allowed for reducing the nozzle channel length, and therefore resulted in the advantage that protein samples have a shorter on-chip (mixing) time.

[0118] In addition, the multiple nozzle outlet openings in fluid connection with the actuation chamber, as previously explained herein for the single outlet opening nozzle channel, resulted in multiple droplets being ejected in parallel from the chip, as shown in Figure 11, with the advantage of allowing multiplexing of droplets on a grid.

[0119] Methods.

[0120] Pressure control

[0121] Control of the liquid flow in the chip is obtained by using a custom software interface written using LabView to fine-tune the device in its use for low sample amounts by controlling the liquid flow in the chip as to only induce flow temporarily and specifically during plunging time. The software interface control also enables synchronization of the laser pulse exposure with the sample flow in the actuation chamber. The interface allows to set the desired pressure both for the plunging and standby mode. The integrated device exemplified herein is controlled through the software interface to regulate the following parameters:

[0122] Laser frequency and intensity: these values influence the number of droplets to be ejected on the grid and their size distribution.

[0123] The plunging time: the time between the moment the sample is applied on the grid (grid passes in front of the outlet opening) and the moment it is plunged in liquid ethane.

[0124] Pressure applied to the sample and oil channels during plunging: the pressure applied to the channel is set to have liquid flowing only during actual plunging (plunging mode); when other operations are performed, the pressure applied to the proteins channel is set to a value for which no sample is consumed , so that no water in oil droplets are formed (stand-by mode).

[0125] Positive and negative pressure applied to regulate the dye solution flow: the positive pressure control regulates the inflow of the laser-absorbing dye solution into the actuation chamber, while the negative pressure control manages the outflow. Proper device operation is achieved by balancing these pressures, ensuring that the actuation chamber is fully filled with each new laser pulse when the laser is activated.

[0126] The device as exemplified herein thus has the possibility to control the sample flowing into the microfluidics chip in a way that the sample is consumed only during actual plunging. The typical operating RoEf / trEM-Chip / 856 flow rate is in a range 1-4 pL / min. The software-controlled transient application of pressure module and laser beam enables to induce the flow and spray only during plunging while maintaining the setup in standby mode in between plunging events by reducing the applied pressure to minimize sample flow in standby regime. A certain priming of the device is needed to stabilize the flow and droplet formation which results in application of high pressure over a period of approximately 500 ms and corresponding sample consumption of below 100 nl per plunging.

[0127] ROEf / trEM-chip / 856

[0128] REFERENCES

[0129] Ahamed, et al. 2010. A Piezoactuated Droplet-Dispensing Microfluidic Chip. JOURNAL OF MICROELECTROMECHANICAL SYSTEMS, VOL. 19, NO. 1.

[0130] Ashtiani, D., Venugopal, H., Belousoff, M., Spicer, B., Mak, J., Neild, A., de Marco, A., 2018. Delivery of femtolitre droplets using surface acoustic wave based atomisation for cryo-EM grid preparation. Journal of Structural Biology 1-26. doi:10.1016 / j.jsb.2018.03.012

[0131] Berriman, J., Unwin, N., 1994. Analysis of transient structures by cryo-microscopy combined with rapid mixing of spray droplets. Ultramicroscopy 56, 241-252.

[0132] Bransky, Korin, Khoury and Levenberg, 2009, Lab Chip, 9, 516 —520.

[0133] Dijkinka and Ohlab, 2008. Laser-induced cavitation based micropump. Lab Chip, 8, 1676-1681.

[0134] Duocastella, Fernandez-Pradas, Dominguez, et al. 2008. Printing biological solutions through laser- induced forward transfer. Appl. Phys. A , 93, 941-945.

[0135] Feng, X., Fu, Z., Kaledhonkar, S., Jia, Y., Shah, B., Jin, A., Liu, Z., Sun, M., Chen, B., Grassucci, R.A., Ren, Y., Jiang, H., Frank, J., Lin, Q„ 2017. A Fast and Effective Microfluidic Spraying-Plunging Method for High-Resolution Single-Particle Cryo-EM. Structure / Folding and Design 1-12. doi:10.1016 / j.str.2017.02.005

[0136] Fernandez-Pradas, J.M. and Serra, P. 2020. Laser-Induced Forward Transfer: A Method for Printing Functional Inks. Crystals, 10, 651.

[0137] Jain, T., Sheehan, P., Crum, J., Carragher, B., Potter, C.S., 2012. Spotiton: A prototype for an integrated inkjet dispense and vitrification system for cryo-TEM. Journal of Structural Biology 179, 68-75. doi:10.1016 / j.jsb.2012.04.020

[0138] Knoska, J., Adriano, L., Awel, S., Beyerlein, K.R., Yefanov, O., Oberthuer, D., Murillo, G.E.P., Roth, N., Sarrou, I., Villanueva-Perez, P., Wiedorn, M.O., Wilde, F., Bajt, S., Chapman, H.N., Heymann, M., 2020. Ultracompact 3D microfluidics for time-resolved structural biology. Nature Communications 11, 1-12. doi:10.1038 / s41467-020-14434-6

[0139] Kontziampasis, D., Klebl, D.P., ladanza, M.G., Scarff, C.A., Kopf, F., Sobott, F., Monteiro, D.C.F., Trebbin, M., Muench, S.P., White, H.D., 2019. A cryo-EM grid preparation device for time-resolved structural studies. lUCrJ 6, 1024-1031. doi:10.1107 / S2052252519011345

[0140] Lu, Z., Barnard, D., Shaikh, T.R., Meng, X., Mannella, C.A., Yassin, A.S., Agrawal, R.K., Wagenknecht, T., Lu, T.-M., 2014. Gas-assisted annular microsprayer for sample preparation for time-resolved cryoelectron microscopy. J. Micromech. Microeng. 24, 115001. doi:10.1088 / 0960-1317 / 24 / ll / 115001

[0141] Maeots, ME., Lee, B., Nans, A. et al. Modular microfluidics enables kinetic insight from time-resolved cryo-EM. Nat Commun 11, 3465 (2020). https: / / doi.org / 10.1038 / s41467-020-17230-4

[0142] Miksys, J., Arutinov, G. & Romer, G.R.B.E. 2019. Pico- to nanosecond pulsed laser-induced forward RoEf / trEM-Chip / 856 transfer (LIFT) of silver nanoparticle inks: a comparative study. Appl. Phys. A 125, 814.

[0143] Morales, Munoz-Martin, Marquez, Lauzurica, and Molpeceres, 2018, Chapter 13 - Laser-Induced Forward Transfer Techniques and Applications, In Woodhead Publishing Series in Welding and Other Joining Technologies, Advances in Laser Materials Processing (Second Edition), Woodhead Publishing, Pages 339-379, ISBN 9780081012529.

[0144] Park, Wu, Chen, Teitell and Chiou (2011). High-speed droplet generation on demand driven by pulse laser-induced cavitation (Communication) . Lab Chip, 11, 1010-1012.

[0145] Rubinstein, J.L., Guo, H., Ripstein, Z.A., Haydaroglu, A., Au, A., Yip, C.M., Di Trani, J.M., Benlekbir, S., Kwok, T., 2019. Shake-it-off: a simple ultrasonic cryo-EM specimen-preparation device. Acta Crystallogr D Struct Biol 75, 1063-1070. doi:10.1107 / S2059798319014372

Claims

ROEf / trEM-chip / 856CLAIMS1. A microfluidic chip (1) for spraying microdroplets comprising: a) an inlet module comprising at least one inlet (29) for a liquid solution to enter the chip, said inlet fluidly connected to a main microchannel (30) for passing the liquid solution through the chip, wherein the at least one inlet is connectable to a pressure control module (4) configured to drive the liquid solution through the chip; and b) a microdroplet dispensing module, comprising:- a nozzle channel (38) fluidly connected on one end to the main microchannel (30) of the inlet module, and on the other end forming one or more outlet openings (40) to the outside of the chip for ejection of microdroplets from the chip;- an actuation chamber (39), which is in connection with the nozzle channel (38) through a chamber junction (41), which is positioned at a distance (d) from the outlet opening (40) allowing generation of microdroplets, and wherein the actuation chamber (39) is configured to, upon actuation, transmit a pressure wave through the chamber junction (41) towards the nozzle channel (38), thereby inducing the formation of microdroplets from the liquid solution present in the nozzle channel (38), for ejection through the outlet opening (40), and wherein said actuation chamber (39) is configured to prevent any material from the actuation chamber (39) from contaminating the liquid solution in the nozzle channel (38).

2. The chip (1) of claim 1, wherein the actuation chamber (39) comprises and inlet (7) and an outlet (8) channel for circulation of chamber material within the actuation chamber (39), and wherein said channels are connectable to a pressure control module (4) configured to prevent contamination of material from the actuation chamber (39) to the nozzle channel (38), preferably wherein the width of the inlet (7) and outlet (8) channels is not smaller than the width (w) of the nozzle channel (38).

3. The chip of claims 1 or 2, wherein the actuation chamber (39) has a length (I) and maximum width (w) of at least 200 pm x 200 pm, a depth that is equal to or greater than the depth of the nozzle channel (38), and a chamber junction (41) width which is at least the double of the width of the nozzle channel (38), and which is positioned at a distance (d) from the outlet opening (40) that is maximally 10-fold the width (w) of the nozzle channel (38).

4. The chip of claims 1 to 3, wherein the actuation chamber material is a liquid suitable for actuation, preferably water, a fluorophore, or a dye solution.

5. The chip of claims 1 to 4, wherein the actuation chamber (39) is configured to allow laser-induced cavitation, wherein the actuation chamber material comprises a dye for absorbing the laser light and generation of a cavitation bubble.34ROEf / trEM-chip / 8566. The chip of claims 1 to 5, wherein the nozzle channel ends in multiple outlet openings, for multiplex droplet ejection from the chip.

7. The chip of claims 1 to 6, for mixing solutions, wherein the inlet module comprises:- a mixer module for mixing liquid solutions within oil-encapsulated droplets comprising:■ at least 3 inlets (28, 29), wherein each inlet ends in the main microchannel (30) for combining the solutions from the inlets,■ said main microchannel (30) being fluidly connected to a serpentine microchannel (31) comprising at least 3 arms (32), wherein each of the inlets (28, 29) of the mixer module is further connectable to a pressure control module (4) configured to control at least the pressure in the inlets (28, 29); a droplet merging module comprising:■ a main microchannel (30) fluidly connected via one end with the serpentine microchannel (31),■ at least one side channel (35), serving as oil outlet from the main microchannel (30), wherein said side channel (35) is transversely intersecting the main microchannel (30), and comprises an array of pillars (36), wherein said pillars each comprise a flat surface that aligns substantially with the wall of the main microchannel (30) and is spaced from one another at a distance smaller than the droplet diameter, or at least two times smaller than the width of the main microchannel (30), and wherein said array of pillars is positioned at a distance from the opposite wall of the main microchannel (30), which is substantially the same as the width of the main microchannel (30), and the pillars extend into the side channel (35).

8. An integrated apparatus for time-resolved preparation of a sample comprising: i. the microfluidic chip (1) of claims 1 to 7, connected to at least one pressure control module (4) for controlling the flow rate in the channels of the chip (1), ii. a plunger module, ill. a droplet actuation module, iv. a cryogenic module, and v. a microprocessor-based controller unit configured to synchronize and control the movement of the plunger arm (5) via the arm controller unit, the pressure control module (4), and the thermostatic cryogenic module,35ROEf / trEM-chip / 856 wherein the components i. to iv. are mounted on one or more support structures configured to allow plunge-freezing of a grid held by the plunger module after droplets generated by the microfluidic chip (1) have been sprayed on the grid.

9. The integrated apparatus of claim 8, wherein the droplet actuation module comprises:- a pulsed laser (17) connected to an automated aperture (27) and to a power meter (26), and- an optical module for focusing the laser (17) inside the actuation chamber (39) of the chip (1).

10. A method for spraying microdroplets from a microfluidic chip comprising the steps of: a) providing at least one liquid solution to the at least one inlet (29) of the microfluidic chip (1) of any one of claims 1 to 7 , and providing means connectable to the chip for driving the flow of said solution through the main microchannel (30), b) actuating the liquid solution in the nozzle channel (38) by generating a pressure wave in the actuation chamber (39), which is transmitted through the chamber junction (41) to the solution in the nozzle channel (38) for induction of microdroplets when ejected from the at least one or more outlet openings (40) of the chip, c) spraying of the induced microdroplets out of the chip, preferably on a grid.

11. The method of claim 10, wherein the means for driving the flow rate of the solution through the main microchannel (30) comprises a pressure control module (4).

12. The method of claims 10 or 11, wherein the means for driving the flow rate of the solution through the main microchannel (30) is coordinated with the configuration of the actuation chamber (39) thereby preventing contamination of the actuation chamber material (39) to the nozzle channel (38) via the chamber junction (41).

13. The method of claims 10 to 12, wherein the actuation is initiated by laser-induced cavitation wherein the laser (17) is focused within the actuation chamber (39) for generation of a pressure wave in the actuation chamber material.

14. A method for time-resolved preparation of a sample on a grid, comprising the steps of: a) combining two or more liquid solutions and an oil composition in the microfluidic chip (1) of claim 7, forming oil-encapsulated droplets of the liquid solutions in the chip (1), b) mixing said liquid solutions within said oil-encapsulated droplets in the chip (1), c) extracting the oil composition for merging the droplets of mixed liquid solution in said chip (1), d) actuating the mixed liquid solution in the nozzle microchannel through induction of a pressure wave in the actuation chamber (39), which is transmitted through the chamberRoEf / trEM-Chip / 856 junction (41) to the solution in the nozzle channel (38) for generation of microdroplets when ejected from the one ore more outlet openings (40) of the chip, e) spraying of the microdroplets out of the chip, preferably on a grid.

15. The method of claim 14, wherein the actuation in step d. is initiated through laser-induced cavitation, said method further comprising the steps of:- switching on a laser (17), synchronizing the pressure with the opening of the laser shutter (27) using the microprocessor-based controller unit, as to focus the laser (17) within the actuation chamber (39) of the chip (1),- closing the laser shutter (27) after microdroplet generation, wherein the actuation chamber material comprises a laser absorbing material for laser-induced cavitation at the emission wavelength of the laser (17), and wherein the laser (17) is focused within the actuation chamber (39) to induce formation of a cavitation bubble which transmits a pressure wave to the solution in the nozzle microchannel (38) through the chamber junction (41) allowing spraying of microdroplets from the chip.

16. Use of the microfluidic chip (1) of claims 1-7, the integrated apparatus of claim 8-9, or use of the method of any one of claims 10-15 , for time-resolved cryo-EM sample preparation.

Images