WO2014196856A1

WO2014196856A1 - Methods and means for performing microdroplet-based reactions

Info

Publication number: WO2014196856A1
Application number: PCT/NL2014/050353
Authority: WO
Inventors: Samaneh MASHAGHI TABARI; Elisabeth Maria Johanna VERPOORTE; Antonius Martinus VAN OIJEN
Original assignee: Rijksuniversiteit Groningen
Priority date: 2013-06-05
Filing date: 2014-06-04
Publication date: 2014-12-11

Abstract

The invention relates to methods and means for performing microdroplet-based reactions, and in particular to droplet based microfluidics. Provided is a method for performing a microdroplet-based reaction comprising the steps of : (i) providing a carrier fluid and a plurality of droplets, the droplets containing reactants and being immiscible with and surrounded by the carrier fluid, and (ii) introducing into the carrier fluid a first agent that is (a) soluble in both the carrier fluid and the droplets and (b) capable of modulating at least one reaction inside the droplets. Also provided is a microfluidic device designed for performing a method of the invention.

Description

Title: Methods and means for performing microdroplet-based reactions.

The invention relates to methods and means for performing microdroplet-based reactions, and in particular to droplet based

microfluidics.

Droplet based microfluidics is a rapidly growing interdisciplinary field of research combining soft matter physics, biochemistry and

microsystems engineering. "Droplet microfluidics" enables the manipulation of discrete fluid packets in the form of microdroplets that provide numerous benefits for conducting biological and chemical assays. Among these benefits are a large reduction in the volume of reagent required for assays, the size of sample required, and the size of the equipment itself. Such technology also enhances the speed of biological and chemical assays by reducing the volumes over which processes such as heating, diffusion, and convective mixing occur. Precise control of droplet volumes and reliable manipulation of individual droplets such as coalescence, mixing of their contents, and sorting in combination with fast analysis tools allow us to perform chemical reactions inside the droplets under defined conditions. US2011/0180571 is directed to droplet actuators, modified fluids and methods for enhancing droplet operations. Microdroplet-based microsystems are a valuable tool for analytical chemistry, synthetic chemistry, biochemistry, microbiology, medical diagnostics or molecular diagnostics. See for example The et al. (Lab Chip, 2008, 8, 198-220)

Typically, a droplet based microfluidic system includes a microfluidic channel, a fluid in microfluidic channel, and micro droplets in the

microfluidic channel, wherein an emulsion is formed of the micro droplets in the fluid. An emulsion is a mixture of two immiscible liquids. One liquid (the dispersed phase) is dispersed in the other (the continuous phase). A commonly used emulsion is a water-in-oil emulsion, where aqueous droplets are dispersed in an oil. In other cases, oil droplets are dispersed in an aqueous mixture, which is a oil-in-water emulsion.

The combination of microfluidic manipulation of 'water-in-oil' emulsion droplets and compartmentalization of reactions in small volumes presents the foundation of many modern lab-on-a-chip biological and chemical assays. Such two-phase flow microfluidics are developing as a widespread technology for a wide range of applications involving, for example, high-throughput encapsulation, chemical synthesis and

biophysical assays. See for instance Guo et al. (Lab Chip, 2012, 12, 2146- 2155). Within such a droplet-based platform, the formation and control of the aqueous-phase droplets inside an immiscible carrier fluid are two critical steps: the emulsification step should lead to a very well controlled drop size and the use of droplet as micro-reactors requires a reliable control on the properties of droplets inner medium, with a minimum manipulation of the droplet boundaries.

Droplet formation processes have been extensively researched with significant success. However, for droplet control after formation, the only reported method to change the environment inside microdroplets after they are formed, is to merge a droplet with another one with different filling to obtain a larger droplet having the final desired contents.

The disadvantage of this droplet fusion method is that the surface area of the droplet expands and therefore concentrations of all reagents decrease compared to their initial concentrations. In droplet reaction chamber based assays, it is often desirable to change the inside of a droplet many times over, resulting in an ever-increasing droplet size and

concomitant dilution of the reactants. Further, the actual merging of droplets is challenging when droplets are stabilized for example by interfacial surfactants. The state of the art of droplet merging protocols relies on electric fields to force merging of droplets. The drawbacks are the usage of high voltage and the possibility of un desired coalescence. It would therefore be desirable to have a method to repeatedly change chemical conditions of the droplet contents, without changing the volume of the droplets or having to merge them.

Accordingly, the present inventors set out to develop a novel microdroplet- based system that allows to alter the environment within the droplets essentially without a change in volume of the droplets. In particular, they aimed at a system which is suitably used in a high throughput setting. Furthermore, the new system should ideally allow for kinetic analysis of the reaction(s) taking place inside the droplets.

It was surprisingly found that at least some of these goals can be met by the use of reactants that are soluble in both the oil and water phase. By changing the concentration of the reactant in the continuous phase, its concentration will also be changed inside the droplet by diffusion. As is shown herein below, this principle is illustrated by changing the pH of the droplet contents in a reversible and repeated fashion. By injecting oil-water soluble acid or base to the continuous oil phase, one can modulate the pH of picoliter or nanoliter size droplets, and thereby trigger an acid- or base- inducible reaction within them. Notably, the volume change of droplets for a pH change from 7 to 4 is about 0.004% of droplet volume, which is negligible. The new method is rapid and can be applied to a large number of static and dynamic microdroplets in parallel. The ability to trigger the reactions in a controlled fashion allows to observe the kinetic details of the fusion reactions, e.g. providing much more pharmacologically relevant information than conventional, static screening assays.

Therefore, in one embodiment the invention provides a method for performing a microdroplet-based reaction comprising the steps of : (i)

providing a carrier fluid and a plurality of droplets, the droplets containing reactants and being immiscible with and surrounded by the carrier fluid, and (ii) introducing into the carrier fluid a first agent that is (a) soluble in both the carrier fluid and the droplets and (b) capable of modulating at least one reaction inside the droplets. A method of the invention comprises providing a carrier fluid and a plurality of droplets, wherein the droplets are immiscible with and surrounded by the carrier fluid and wherein the droplets contain reactants participating in the reaction of interest. Droplets can be static droplets or dynamic droplets.

In one embodiment, a method of the invention comprises providing static drops, for example in immobilized arrays of nanoliter-scale

microfluidic drops allowing to execute many chemical reactions in parallel. For example, wells of a multiwall plate are first filled with oil after which well-defined volumes of aqueous phase containing reactants are injected into the oil to create aqueous droplets. After formation and stabilization of the droplets, at least one water-oil soluble agent is injected into the oil phase to change the environment (e.g. pH or redox potential) and modulate a reaction taking place inside the droplets. Automated multichannel pipettes connected to a microfluidic pressure control system are

advantageously used to achieve a high accuracy.

In another embodiment, a method of the invention comprises providing dynamic drops e.g. using a microfluidic device. Methods and devices for producing dynamic microdroplet emulsions are well known in the art. There are basically two means to generate atomized droplets. One is using the vibration of a piezoelectric material to squeeze a liquid out of a nozzle disc to generate microdroplets. Another means is using a piezoelectric material to vibrate a nozzle disc, and using the vibration of the nozzle disc to atomize liquid into microdroplets. In some cases, the latter means adopts a liquid-vibration plane. For example, a bundle of capillary tubes is used to transport a liquid, and the terminals of the capillary tubes are fabricated into a plane. The liquid flowing to the plane is vibrated and atomized into microdroplets. Alternatively, a compressor is used to transport a liquid to a nozzle disc, and the vibrating nozzle disc atomizes the liquid into

microdroplets. In the latter means, whether the vibration energy is effectively transmitted to the nozzle disc depends on whether the

piezoelectric material and the nozzle disc are joined well.

Microfluidic devices can be used to compartmentalize reactants by using inert carrier fluid, usually oil, to encapsulate small volumes of aqueous reagents in droplets and separate the fluidics from the droplet contents. Such devices can produce monodisperse droplets, ranging in volume from 0.05 pL to 1 nL, or from 5 μιη to 120 μιη in diameter. The volume of a particular microdroplet is substantially unlimited in terms of minimal volume. The microdroplets can be as small as desired, so long as they can be manipulated. The maximum volume of a microdroplet is typically

determined by the particular volume limit past which the microdroplet is no longer substantially spherical. Droplets can encapsulate any type of desired constituent, ranging from molecules and complexes to particles, liposomes and cells.

In a specific aspect, a method of the invention involves surfactant stabilized droplets. Suitable additives for reducing loss of droplet phase components from the droplet phase and/or for improving droplet operations include non-ionic low HLB (hydrophile-lipophile balance) surfactants. The HLB is preferably less than about 10, or less than about 5. Exemplary additives include Triton X-15 (HLB 4.9), Span 85 (HLB 1.8), Span 65 (2.1), Span 83 (HLB 3.7), Span 80 (HLB 4.3), Span 60 (HLB 4.7) and fluorinated surfactants. Preferred surfactants that may be added to the continuous phase fluid include, but are not limited to, surfactants such as sorbitan- based carboxylic acid esters (e.g., the "Span" surfactants, Fluka Chemika), including sorbitan monolaurate (Span 20), sorbitan monopalmitate (Span 40), sorbitan monostearate (Span 60) and sorbitan monooleate (Span 80), and perfluorinated polyethers (e.g., DuPont Krytox 157 FSL, FSM, and/or FSH). Other non-limiting examples of non-ionic surfactants which may be used include polyoxyethylenated alkylphenols (for example, nonyl-, p- dodedyl-, and dinonylphenols), polyoxyethylenated straight chain alcohols, polyoxyethylenated polyoxypropylene glycols, polyoxyethylenated

mercaptans, long chain carboxylic acid esters (for example, glyceryl and polyglycerl esters of natural fatty acids, propylene glycol, sorbitol,

polyoxyethylenated sorbitol esters, polyoxy ethylene glycol esters, etc.) and alkanolamines (e.g., diethanolamine-fatty acid condensates and

isopropanolamine-fatty acid condensates). In addition, ionic surfactants such as sodium dodecyl sulfate (SDS) may also be used. However, such surfactants are generally less preferably for many embodiments of the invention. For instance, in those embodiments where aqueous droplets are used as nanoreactors for chemical reactions (including biochemical reactions) or are used to analyze and/or sort biomaterials, a water soluble surfactant such as SDS may denature or inactivate the contents of the droplet. Combinations of two or more distinct surfactants may also be used. See US2011/0180571, paragraph 7.2.1 for details. In one embodiment, the method involves the use of a water-in-oil emulsion i.e. wherein the carrier fluid is an oil or a mixture of oils and wherein the droplets have an aqueous content. This includes a water-in oil-in-oil emulsion i.e. wherein the carrier fluid is oil phase and wherein aqueous droplets are surrounded by a second oil (2 oils are immiscible) are dispersed. In another embodiment, the method involves the use of an oil -in- water emulsion i.e. wherein the carrier fluid is aqueous and wherein oil droplets are dispersed. This includes a water-in-oil-in water emulsion i.e. wherein the carrier fluid is an aqueous phase and wherein aqueous droplets are surrounded by an oil. Preferably, the method uses an water-in-oil emulsion wherein the droplets have an aqueous content and the carrier fluid forms an oil phase. The droplet forming aqueous liquid is typically an aqueous buffer solution, such as ultrapure water (e.g., 18 mega-ohm resistivity, obtained, for example by column chromatography), 10 mM Tris HCI and 1 mM EDTA (TE) buffer, phosphate buffer saline (PBS) or acetate buffer.

The fluid in which the droplets are formed is one that is immiscible with the droplet forming fluid. The fluid passing can be a non-polar solvent, most preferably decane (e g., tetradecane or hexadecane), fluorocarbon oil or another oil (for example, mineral oil). Preferred oils include

octamethylsiloxane and perfluorinated oils, such as C7F15OC2H5 and the like.

The fluids used in the invention may contain one or more additives, such as agents which reduce surface tensions (surfactants). Surfactants can include Tween, Span, fluorosurfactants, and other agents that are soluble in oil relative to water. Surfactants can aid in controlling or optimizing droplet size, flow and uniformity, for example by reducing the shear force needed to extrude or inject droplets into an intersecting channel. This can affect droplet volume and periodicity, or the rate or frequency at which droplets break off into an intersecting channel. Furthermore, the surfactant can serve to stabilize aqueous emulsions in fluorinated oils from coalescing

A method of the invention is characterized among others by the use of a first agent that is (a) soluble in both the carrier fluid and the droplets and (b) capable of modulating at least one reaction inside the droplets.

"Solubility" is the property of a chemical substance called solute to dissolve in a solid, liquid, or gaseous solvent to form a homogeneous solution of the solute in the solvent. As will be understood, the agent is soluble under the conditions (e.g. temperature, pressure) used when performing a method of the invention. Thus, the agent is soluble both in the water and oil phase. In the oil phase, solubility is based on hydrogen bonding or based on nonpolar hydrophobic interactions. The molecular agent can be dissolved in oil as individually solvated molecules or even as solvated molecular clusters (micelles).

Typically, the partition coefficient -which is defined as the ratio of concentrations of a compound in a mixture of two immiscible phases at equilibrium-- is used as a measure of the difference in solubility of the compound in these two phases. Normally one of the solvents chosen is water while the second is a hydrophobic liquid such as octanol.

Partition coefficients are useful in estimating distribution of agents within the droplet based microfluidic platform. The agents with high octanol/water partition coefficients are preferentially distributed to oil phase while low octanol/water partition coefficients preferentially are found in aqueous droplets.

, _n , [solute octanol

log P oet = log- ϋ¾ί [solute] water

Solubility and partition coefficient defines the dynamic range of pH. For acetic acid and propylamine we tested the dynamic range and we found out that we can readily and reproducibly change the pH in the full range. As used herein, the term "modulating"' is meant to encompass both the initiation or triggering of a reaction, as well as inhibition or quenching of a reaction. The modulatory effect is achieved by introducing into the carrier fluid an agent which alters the chemical environment inside the droplets such that the at least one reaction is influenced. In one

embodiment, the invention provides a method to externally trigger a chemical reaction inside a microdroplet by using a compound that is soluble in water and in oil. The agent can be any type of compound capable of modulating at least one reaction inside the droplets. Combinations of different agents may also be used, optionally in a consecutive manner.

As will be appreciated by the skilled person, the concept underlying the invention can be applied to any reaction that requires a component that can be dissolved in both water and oil. The reaction can be chemical or biochemical reaction. For example, it is an organic synthetic reaction requiring one or more components that can be delivered to the interior of the droplets via diffusion from the continuous phase.

In one embodiment, the agent is a reactant which participates in the reaction. In another embodiment, it is a reaction catalyst or an enzyme co- factor. Suitable agents include those which alter the ionic strength and/or redox potential inside the droplets. Exemplary agents comprise acids, bases and ionic species like mono- or divalent metal ions, for example selected from the group consisting of Na⁺, K⁺, Ca²⁺, Zn²⁺ and Mg²⁺ or any

combination thereof. In one embodiment, the reaction inside the droplets is a Mg²⁺-dependent reaction, such as a reaction catalysed by a Mg²⁺- dependent enzyme like a kinase or a phosphatase. Other examples include phosphate, sulphate, nitrate ions.

The agent or combination of agents may be used such. Alternatively, the agent(s) can be complexed to, conjugated to, encapsulated by, associated with or attached to another entity or supramolecular structure to induce or enhance its water-oil miscibility. For example, reverse micelles are suitably used to make a reagents soluble in oil which otherwise only displays aqueous solubility. Likewise, regular micelles can be used for the reverse situation i.e. to make oil-soluble compounds soluble in water.

In a specific aspect, the process occurring in the droplets is an acid- or base-triggered reaction and the agent is capable of altering the pH within the droplets. In one embodiment, the agent is a water-oil soluble acid, preferably acetic acid. In another embodiment, the agent is a water-oil soluble base, preferably propylamine or ethylamine. In one embodiment, a method of the invention further comprises the step of monitoring the at least one reaction and/or a reaction product. As used herein, the term 'monitoring" comprises continuous and discontinuous (e.g. with intervals of several seconds, minutes or hours) analysis of the reaction. Reactants and/or resulting products may be analysed. Monitoring may involve any suitable technology. In one embodiment, monitoring is performed using optical detection, preferably microscopy, more preferably fluorescence microscopy. For example, one or more fluorescent compounds are used to monitor the progress of the reaction.

The concept of externally modulating the reaction taking place in the droplets can be applied more than once in a method of the invention. For instance, the modulating effect of the first agent may be reversed by introducing a second agent. Alternatively, one or more reactions in parallel or subsequent to the reaction modulated by the first agent may be

modulated by a second, third etc. reagent. Therefore, in one embodiment, a method of the invention further comprises introducing into the carrier fluid a second agent that is (a) soluble in both the carrier fluid and the droplets and (b) capable of modulating a second reaction inside the droplets. As an exemplary embodiment, the first agent causes a pH decrease in the droplets to induce an acid-dependent reaction, followed after a predetermined time period by introducing a water-oil soluble base into the carrier fluid to rapidly reverse the pH switch.

The skilled person will appreciate that the invention can be practised for any type of microdroplet-based reaction that requires or can be modulated by a component which is soluble in an oil and an aqueous environment. In one embodiment, it is a chemical reaction. Performing reactions in the microscale conserves expensive and precious reagents, reduces exposure to hazardous chemicals and allow multiple reactions to be carried out in highly parallelized experiments. In batch process, there is a risk involved when performing exothermic reactions where large excess amounts of heat can be released. By scaling down the reaction in microdroplets, parallel reactions can be performed with minimized risk. Also, reactions can be done much quicker due to shorter diffusion and heat and mass transfer distances.

Exemplary chemical reactions include organic compound synthesis, redox reactions, precipitation reactions, crystal growth and particle synthesis. In one aspect, the invention provides a method for performing a

microdroplet-based redox reaction comprising the steps of : (i) providing a carrier fluid and a plurality of droplets, the droplets containing reactants and being immiscible with and surrounded by the carrier fluid, and (ii) introducing into the carrier fluid a first agent that is (a) soluble in both the carrier fluid and the droplets and (b) capable of modulating at least one reaction inside the droplets. In one embodiment, an oil-soluble agent is used which can initiate a redox reaction in the water phase. For example 2,3- dichloro-5,6-dicyano-l,4-benzoquinone (DDQ) dissolves in n-butyronitrile-oil and can oxidize Fe(II) to Fe(III) in the aqueous droplet. Knowing that many biological phenomena are based on the oxidation of Fe, a method of the invention allows to control (either trigger or inhibit) those reactions in the droplets by DDQ-Oil. An exemplary reaction is inducing conformational changes within HbpS protein by oxidation of Fe(Q). Another example of oil- soluble agents that can initiate redox reactions in aqueous droplets is ferrocene methanol, an electrochemical redox mediator that can be introduced to the aqueous droplet. Using reverse micelles to make water- soluble compounds soluble in oil, one can use ATP in reverse micelles to trigger any ATP -based reaction in the droplets. Such a system is

advantageously used to visualize the kinetics of a biochemical reaction, for instance nucleic acid replication and/or translation in the presence of candidate antibiotic compounds to be screened for inhibiting replication or translation.

In a further aspect, the invention relates to a method for performing a microdroplet-based biochemical reaction. In nature, chemical and biological operations are carried out in micron-sized spaces such as in cells and their organelles. Droplet microfluidics offers the capability to form femto- to picoliter sized droplets and to compartmentalize and mimic reactions and molecular processes within individual droplets. With the development of tools for the transport and manipulation of droplets and particles, a number of possibilities exist for combining these fluidic elements to carry out synthesis and functionalization of particles for biomedical applications. For this reason, droplet-based microfluidic platforms, with the ability to transport, mix, split, and sort droplets, are being applied to particle synthesis for therapeutic delivery, biomedical imaging, drug discovery, biomolecule synthesis, and diagnostics.

In a further specific aspect, the invention provides a method for performing a microdroplet-based enzymatic reaction comprising the steps of : (i) providing a carrier fluid and a plurality of droplets, the droplets containing reactants and being immiscible with and surrounded by the carrier fluid, and (ii) introducing into the carrier fluid an enzyme and/or enzyme modulator that is (a) soluble in both the carrier fluid and the droplets and (b) capable of modulating at least one enzymatic reaction inside the droplets. Such method has important industrial applications. For example, in the field of chemistry there is a growing interest to modify enzymes to make them suitable for reactions in organic solvents. In nonaqueous solvents, enzymes exhibit remarkable new properties, including the ability to catalyze reactions impossible in water and radically altered selectivity. Researchers have found (e.g. peroxidase) or engineered enzymes with increased apparent oil-water partition coefficient (e.g. Superoxide dismutases (SOD)) which function in both aqueous and organic solvent. In one embodiment of a method of the invention, an (engineered) enzyme is dissolved in the organic oil phase so that it can trigger a reaction in the aqueous droplet phase.

Of particular interest for this purpose are enzymes that are naturally found in oils as well as in water. Interestingly, many of these oils can be as the carrier oil. Natural oils such as soybean oil and sunflower oil contain enzymes that can be (directly be used or extracted) used to trigger a reaction in aqueous droplets containing other reaction agents needed for the reaction. For example, phospholipase A, which is an enzyme in soybean oil is a prospective enzyme to use in place of phosphoric acid as a degumming agent. Droplet microfluidics can also be used for the synthesis of biological molecules such as protein and DNA. Droplet microfluidics with its ability to rapidly create highly uniform aqueous droplets with controlled contents, could serve as an important component for the creation of artificial cells. Since droplets can be made micron-sized or smaller, encapsulation of a single template copy of DNA can be realized. The integration of heating elements and the ability to precisely control droplet movement allow these vesicles to serve as microreactors for in vitro protein expression, DNA amplification, and other biochemical reactions. Although

biomolecule synthesis is done well in living cells, synthesis in droplets is advantageous due to its ability to isolate and control specific reactions, increase effective concentrations of reagents, parallelize experiments, synthesize proteins lethal to cells, and for its potential in high throughput molecular engineering. Many cell-free biological reactions have been carried out in droplets, such as ATP synthesis using microbubbles, and protein expression in emulsions, and membrane fusion and transport has been studied in micelles and liposomes.

In one embodiment, the invention provides a method for performing a microdroplet-based fusion reaction between lipid bilayers. The bilayers can be artificial or biological membranes. A lipid bilayer, also known as the phospholipid bilayer, is a sheet of lipids two molecules thick, arranged so that the hydrophilic phosphate heads point "out" to the water on either side of the bilayer and the hydrophobic tails point "in" to the core of the bilayer. This arrangement results in two "leaflets" which are each a single molecular layer. Lipids self-assemble into this structure because of the hydrophobic effect which creates an energetically unfavorable interaction between the hydrophobic lipid tails and the surrounding water. Biological membranes typically include several types of lipids other than phospholipids. A particularly important example in animal cells is cholesterol, which helps strengthen the bilayer and decrease its permeability. Cholesterol also helps regulate the activity of certain integral membrane proteins. Integral membrane proteins function when incorporated into a lipid bilayer. Because bilayers define the boundaries of the cell and its compartments, these membrane proteins are involved in many intra- and inter-cellular signaling processes. Certain kinds of membrane proteins are involved in the process of fusing two bilayers together. This fusion allows the joining of two distinct structures as in the fertilization of an egg by sperm or the entry of a virus into a cell

In one embodiment, the invention provides a method for performing a microdroplet-based fusion of biological membranes. Specific fusion of biological membranes is a central requirement for many cellular processes varying from exocytosis and intracellular trafficking to zygote formation. Membrane fusion is also a critical event in the entrance of membrane- enveloped viruses (e.g., HIV and influenza viruses) into the cell and represents an important target for antiviral therapeutics.

Accordingly, in one embodiment, the invention provides a method for investigating acid-induced viral fusion with a target membrane, preferably fusion with a lipid vesicle. To that end, an oil carrier fluid comprising a plurality of aqueous pH-neutral droplets is provided wherein the droplets contain (i) a labelled, enveloped particle containing one or more viral proteins, and (ii) a target membrane. Preferably, the target membrane is an artificially-prepared vesicle composed of a lipid bilayer, hke a liposome. For example, the droplets contain virus, virosome or virus-like particles prebound to liposomes. Upon introducing into the carrier oil an acid that is miscible with both the carrier fluid and the droplets, the pH in the droplets drops and fusion is initiated.

The membrane fusion or hemi-fusion kinetics can be monitored by analysis of a detectable label used to label one or more components comprising constituents of the reaction under investigation e.g. one or more of the lipid vesicles, virosomes, target membrane, etcetera. In the case of pH-triggered fusion (e.g., influenza), a dye such as, e.g., a pH-sensitive fluorophore can be incorporated into the liposome and/or a particle containing one or more viral proteins to monitor the local pH. Hence, the novel method makes it possible to trigger, observe, and quantify membrane fusion.

As used herein, the term virus includes DNA or RNA animal viruses. As used herein, RNA viruses include, but are not limited to, virus families such as picornaviridae (e.g., pohoviruses), reoviridae (e.g., rotaviruses), togaviridae (e.g., encephalitis viruses, yellow fever virus, rubella virus), orthomyxoviridae (e.g., influenza viruses), paramyxoviridae (e.g.,

respiratory syncytial virus, measles virus, mumps virus, parainfluenza virus), rhabdoviridae (e.g., rabies virus), coronaviridae, bunyaviridae, flaviviridae, filoviridae, arenaviridae, bunyaviridae, and retroviridae (e.g., human T-cell lymphotropic viruses (HTLV), human immunodeficiency viruses (HIV)). As used herein, DNA viruses include, but are not limited to, virus families such as papovaviridae (e.g., papilloma viruses), adenoviridae (e.g., adenovirus), herpesviridae (e.g., herpes simplex viruses), and poxviridae (e.g., variola viruses).

In certain exemplary embodiments, one or more enveloped viruses are used in a method described herein. As used herein, the term "enveloped virus" refers to a virus that contains a membrane that envelopes the virion. As used herein, the term "virion" refers to a virus particle which typically comprises a nucleic acid surrounded by a capsid. The membrane is derived from the outer membrane of an infected host cell or from host cell internal membranes. Proteins (e.g., viral glycoproteins) embedded in the envelope serve to bind to receptor sites on the host cell membrane (e.g., viral attachment proteins). Proteins (e.g., viral fusion proteins) also mediate fusion between the virion and the host cell. Viral attachment proteins and viral fusion proteins may be separate proteins (e.g., H/HN/G proteins and F proteins of paramyxoviruses). Alternatively, a single viral protein can function to bind one or more receptors and mediate membrane fusion (e.g., HA proteins of orthomyxoviruses). Examples of enveloped virus families include, but are not limited to, togaviridae, flaviviridae, bunyaviridae, arenaviridae, coronaviridae, herpesviridae, orthomyxoviridae,

paramyxoviridae, poxyiridae, retroviridae and rhabdoviridae.

In certain exemplary embodiments, one or more particles containing one or more viral proteins are used.. Such particles include, for example, virosomes and virus-like particles. As used herein, the term "virosome" refers to vesicles (e.g., vesicles comprising a phospholipid bilayer) that can contain one or more viral proteins (e.g., fusion proteins). Virosomes are typically devoid of genetic material. As used herein, the term "virus-like particle" refers to particles having one or more viral structural proteins (e.g., capsid proteins). Virus-like particles may optionally contain a lipid bilayer and/or viral fusion proteins. Like virosomes, virus-like particles typically lack genetic material. Particles containing one or more viral proteins also include viruses and/or virions. Viruses, virions, particles containing one or more viral proteins, viral fusion proteins and their receptors are further described in Fields Virology 5th Ed., Knipe et al.

(2007) Lippincott, Williams & Wilkins.

In certain exemplary embodiments, a target membrane, e.g., a liposome, can be used whose composition can be controlled to mimic a cellular membrane. Optionally, specific receptors can be included to allow viral particles to bind to the surface preceding fusion.

In one embodiment, the viral particle has a detectably labelled envelope and/or a detectably labelled internal region, In one embodiment, the envelope and/or the internal region each have a fluorescent label. In other aspects, the envelope and the internal region each have a different detectable label. In yet other aspects, the envelope contains a lipophilic, detectable dye (e.g., Rhl lOCl8) and/or the internal region contains a water soluble, detectable label (e.g., sulforhodamine B). In certain aspects, viral fusion is monitored for a single, enveloped virion, such as an intact virion. In certain aspects, the labelled, enveloped particle containing one or more viral proteins is a virion, a virosome or a virus-like particle.

In certain aspects, hemifusion and/or formation of a fusion pore is monitored by observing an increase or decrease in one or more photophysical properties (e.g., fluorescence intensity, fluorescence lifetime, emission wavelength, absorption wavelength, polarization and the like) of the lipophilic, detectable label or the water soluble, detectable label,

respectively. In certain aspects, hemifusion is monitored by observing an instantaneous increase in brightness of the lipophilic, detectable label, and in other aspects, the instantaneous increase in brightness of the lipophilic, detectable label is followed by a decrease in brightness of the of the lipophilic, detectable label. In still other aspects, formation of a fusion pore is monitored by observing a decrease in brightness of the water soluble, detectable label.

In a preferred embodiment, the membrane fusion process is

monitored using a self-quenching fluorescent dye. The dye may be present in the viral particle or in the target membrane, like a liposome. Prior to fusion, the dye displays a reduced fluorescence intensity when packed in the particle. However, upon fusion the dye is spatially redistributed. The dye concentration is reduced and fluorescence is increased via the disruption of intramolecular dequenching of the dye. Fusion events can thus be inferred from dequenching. Of particular interest for use in the present invention are hpophilic, cationic fluorescent dyes such as R18, Rhl lOCl8 and DID

(DilCl8(5). Because a method of the invention allows for a rapid, external triggering of a microdroplet-based reaction, it is especially suitable for kinetic studies e.g. to obtain pharmacologically relevant kinetic data on a biochemical or biological process such as viral fusion.

Furthermore, the method format easily allows for including one or more additional components in the droplets which can influence the reaction under investigation. In one embodiment, the droplets contain at least one test compound to be evaluated for its effect on the at least one reaction. The test compound can be a small molecule. In the fields of pharmacology and biochemistry, a small molecule is a low molecular weight (<800 Daltons) organic compound that may serve as an enzyme substrate or regulator of biological processes. In pharmacology, the term is usually restricted to a molecule that binds to a biopolymer such as protein, nucleic acid, or polysaccharide and acts as an effector, altering the activity or function of the biopolymer. Small molecules may function across a variety of cell types and species (e.g. mice and humans) and their study can lead to the development of new therapeutic agents. Some can inhibit a specific function of a multifunctional protein or disrupt protein-protein interactions. The upper molecular weight limit for a small molecule is approximately 800 Daltons which allows for the possibility to rapidly diffuse across cell membranes so that they can reach intracellular sites of action.

In one aspect, the test compound is a proteinaceous compound, such as a peptide or an antibody, or a nucleic acid molecule (DNA, RNA, PNA, aptamer).

In an exemplary embodiment, the invention provides assays for screening candidate or test compounds which modulate (e.g., modulators that inhibit or stimulate) virus-mediated membrane fusion. For example, the test compound is a potential anti-viral compound. This development will aid in the screening of large small-molecule libraries to identify novel candidate (drug) compounds e.g. drugs effective in inhibiting viral fusion. The test compounds can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. A pipetting robot is suitably used during droplet formation to ensure that each of the plurality of droplets contains a distinct test compound, e.g. a different compound from a compound library.

Of particular interest is the screening of candidate compounds in a high -throughput set-up. Conventional high-throughput screens use microtiter plate wells to store a large number of unique elements; each element is then tested for some chemical or biological property. However, high throughput screens in droplet microfluidics require that the unique elements be stored, manipulated, and tested in droplets. Accordingly, in droplet-based screens each test compound must be encapsulated in droplets prior to performing the screen itself. Although it is possible to emulsify and screen each element individually, this would take more time than performing a conventional screen; instead, after encapsulation, droplets of different elements can be pooled into a "droplet library," ready for

subsequent use in a single screening assay that includes all library elements. Since each element is emulsified into many droplets, the same library can be used for multiple screens, where each assay uses an aliquot, or small portion, of the library, and each aliquot includes many droplets of each library element. As an example, small compounds dispensed in microtiter plates, totalling tens of microlitres of each element, may be encapsulated to form a droplet library of billions of droplets at a rate of several minutes per plate. This is suitably achieved by parallelizing microfluidic devices and interfacing them with microtiter plates. An aliquot of one-thousandth of this library contains thousands of copies of each library element and this constitutes sufficient statistics for one assay. To perform a screen, the aliquot of droplets may be reinjected into a microfluidic device, a target such as a microbial organism is added to each drop, and after some reaction occurs during an incubation step, the droplets are detected and sorted at a typical rate of 1000 Hz. In this fashion, a library of one million compounds can be screened in a day as opposed to months using

conventional screens, while using one-thousandth of the amount of reagents that is conventionally used.

When the contents of a microtiter plate are emulsified and pooled to form the droplet library, sample labeling encoded by spatial positioning in the original plate is lost; thus, some other means must be developed to track the library contents. One solution is to associate a unique barcode with each element and include it with the element in the plate prior to encapsulation. Each droplet then contains a barcode that indicates which element is encapsulated in the droplet. Potential barcodes include nucleic acid sequences and fluorophore combinations. See for example Meyer et al.

(Nucleic. Acids Res., 2007, 35, e97) or Han et al. (Nat. Biotechnol., 2001, 19, 631- 635). Preferably, fluorophore combinations are used as barcode for droplet libraries because they can be read in real-time concurrently with the result of the assay. However, the number of distinguishable fluorophore combinations is limited by the dynamic range of the optical setup that detects the fluorophores. In contrast, nucleic acid sequences cannot be read in real-time but can accommodate arbitrarily large library sizes.

The invention finds important applications in drug discovery.

Typically, the first step in drug discovery is to screen libraries of small- molecule compounds, peptides, or antibodies for their ability to modulate a reaction, for example to inhibit membrane fusion. Typically, promising compounds are identified in a primary screen and then more fully

characterized in a dose-response analysis with 7-10 data points per compound. However, identifying compounds that inhibit (viral) fusion has proven to be a challenging task. One of the key problems is the lack of a fast and quantitative fusion assay that allows the screening of large numbers of candidate compounds and reports on their mechanism of inhibition. In particular, no assays exist that allow the observation of the kinetics of fusion in a high-throughput fashion. Thus, also provided is a method for conducting a high-throughput screen, comprising performing a

microdroplet-based reaction as disclosed herein. In a particular

embodiment, the invention provides a lab-on-a-chip' approach, wherein the novel method of external triggering of microdroplet-based reactions is used. In particular, it is used to enable the highly multiplexed observation of the kinetics of membrane fusion events.

The invention also relates to a microfluidic device adapted for, yet not limited to, performing a method of the invention. Provided is a microfluidic device comprising (i) one or more inlet modules that have at least one inlet channel adapted to carry a dispersed phase fluid (ii) at least one main channel adapted to carry a continuous phase fluid, wherein the inlet module is in fluid communication with the main channel such that the dispersed phase fluid is immiscible with the continuous phase fluid and forms a plurality of droplets in the continuous phase fluid; (iii) one or more outlet modules and (iv) downstream from the inlet module at least one reservoir adapted to contain a first modulating agent.

The reservoir can have any design and configuration that allows said reservoir and the main channel being fluidically connected to one another while preventing via an obstruction structure, droplets in the main channel from entering the reservoir. As used herein, the term "reservoir" is meant to refer to any structure suitable to contain modulatory agent. For example, it can be a confined chamber to receive a predetermined volume of agent or it can be a part of a channel capable of receiving the agent via a continuous inlet stream.

Typically, a microfluidic device for performing dynamic microdroplet- based reactions as provided herein (also referred to a "Chemical Modulator or "CM") is used in combination with a droplet generator and a detector module. The system can be driven by one or more controllable pumps.

Figure 1 shows an exemplary design of a microfluidic pH switch structure.

The droplet generator can be any type of known or yet to be developed design that can generate microdroplets. Droplets can either be generated on the chip or generated separately and stored. Later the stored droplets can also be used in a structure with chemical modulator but without generator to perform a reaction inside them. The detection section can also be a section wherein droplets are no more dynamic but standing still. For droplets with long reaction incubation time, the detector module is suitably made for static droplets and for reactions that take place within less than a minute dynamic detection sections with delay cavities is preferred.

The reservoir and main channel are configured to be fluidically connected to one another while droplets in the main channel are prevented from entering the reservoir while allowing fluid to pass. The specific design of the obstruction structure connecting reservoir and main channel can vary. In one embodiment, it is a grid or other type of structure which physically blocks droplet entry into the reservoir while enabling efficient introduction of the modulatory agent into the main channel comprising the droplet emulsion.

The obstruction structure provides for a relatively large contact surface between the reservoir and the main channel such that there is a rapid exchange between the fluid contents of the reservoir and the main channel while loss of droplets is prevented by a porous obstruction at the interface between the reservoir and the main channel. In this manner, the medium in the passing droplets can be efficiently changed without the need for droplet merging. Thus, in contrast to known microfluidic devices the reservoir connection to the main channel is not configured as a fork structure (Y-junction). For example, the obstruction is a porous structure, e.g. formed by a sheet or membrane comprising a plurality of pores of any suitable shape and size. Preferably, the surface area of the porous structure is at least 2, preferably at least 3, more preferably at least 4 times that of the cross section of the main channel. The surface area of the porous part should be at least about 1.5 times of the cross section of the main channel. The surface area of the membrane should be bigger depending on the porosity. The porous obstruction is suitably fabricated of a polymer conventionally used in microfluidic device manufacture, such as

polydimethylsiloxane.

Other preferred designs include so-called "comb-like" obstruction structures comprising a plurality of narrow channels feeding into the droplet path in the main channel. Figure 2 shows different variants of a comb-like structure. The small channel cross section is preferably about one- third of that of the main channel to avoid capillary effect of the CM- channels. The channels of the obstruction structure may have a component in the direction of the droplet path to further ensure droplet entry in the reservoir. For example, the plurality of narrow channels intercept the main channel at an angle <90 degrees relative to the main channel (see Figure 2 panel E for an exemplary embodiment) Also, proper parameters for driving pressures used for droplet preparation and CM liquid can avoid droplet penetration into the reservoir. The fluidic connectivity between reservoir and main channel may be regulated by a first valve and a control unit configured to control the valve. For example, the valve is configured to be pneumatically actuated.

The inlet module generally comprises a junction between the sample inlet channel and the main channel such that a solution of a sample (i.e., a fluid containing a sample such as molecules, cells, small molecules (organic or inorganic) or particles) is introduced to the main channel and forms a plurality of droplets. The sample solution can be pressurized. The sample inlet channel can intersect the main channel such that the sample solution is introduced into the main channel at an angle perpendicular to a stream of fluid passing through the main channel. For example, the sample inlet channel and main channel intercept at a T-shaped junction; i.e., such that the sample inlet channel is perpendicular (90 degrees) to the main channel. However, the sample inlet channel can intercept the main channel at any angle, and need not introduce the sample fluid to the main channel at an angle that is perpendicular to that flow. The angle between intersecting channels is in the range of from about 60 to about 120 degrees. Particular exemplary angles are 45, 60, 90, and 120 degrees. A device may contain more than one inlet module if desired. The inlet module is in fluid

communication with the main channel. The inlet module can include a junction between an inlet channel and the main channel of a device of the invention. The junction can permit the introduction of a pressurized fluid to the main channel. The inlet channel can be at an angle perpendicular to the flow of fluid in the main channel. The fluid introduced to the main channel through the inlet module is "incompatible" (i.e., immiscible) with the fluid in the main channel so that droplets of the fluid introduced through the inlet module are formed in the stream of continuous fluid in the main channel. A microfluidic device of the invention may further comprise one or more micromixers. Micromixers can be integrated in a microfluidic system or work as stand-alone devices. Rapid mixing is essential in many of the microfluidic systems and often used in biochemistry analysis, drug delivery and sequencing or synthesis of nucleic acids. Biological processes such as cell activation, enzyme reactions and protein folding often involve reactions that require mixing of reactants for initiation.

Micromixers have been used in the art as a tool for dispersing immiscible liquids and forming microdroplets or to work as a separator for particles based on their different diffusion coefficients. However, the application of a micromixer to facilitate the delivery of a substance via the continuous phase into dispersed droplets has not been disclosed. This is because the art has primarily focussed on modulating the content of droplets by merging or fusion with other droplets. In contrast, according to the invention the droplet interior is modulated by an agent that is delivered without changing the volume of the droplets or having to merge them.

Accordingly, the invention provides a microfluidic device comprising (i) one or more inlet modules that have at least one inlet channel adapted to carry a dispersed phase fluid (ii) at least one main channel adapted to carry a continuous phase fluid, wherein the inlet module is in fluid communication with the main channel such that the dispersed phase fluid is immiscible with the continuous phase fluid and forms a plurality of droplets in the continuous phase fluid; (iii) one or more outlet modules; (iv) downstream from the inlet module at least one reservoir adapted to contain a first modulating agent; and (v) downstream from the reservoir a micromixer to enhance diffusion of the modulating agent into the dispersed droplets.

In general, micromixers can be categorized as passive micromixers and active micromixers (see for a review Nguyen and Wu, J. Micromech. Microeng. 15 (2005) R1-R16). Passive micromixers do not require external energy and the mixing process relies entirely on diffusion or chaotic advection. Passive mixers can be further categorized by the arrangement of the mixed phases: parallel lamination, serial lamination, injection, chaotic advection and droplet. Active micromixers use the disturbance generated by an external field for the mixing process.

Because of its simple concept, the passive mixer is preferred. Due to the dominating laminar flow on the microscale, mixing in passive micromixers relies mainly on molecular diffusion and chaotic advection. In one

embodiment, a microfluidic device of the invention comprises a passive micromixer designed for effective mixing of both the content of droplets and the liquid at the oil-water interface caused by a change of shape of the droplets. The design of the micromixer can vary. Micromixer designs for mixing with chaotic advection are known in the art and include a modified Tesla structure, a C-shape, an L-shape, connected out-of-plane L-shapes, twisted microchannel and other three-dimensional designs of a twisted conduit. In one embodiment, the passive mixer design is as shown in Figure 3 showing a larger width of about three times of the main channel and smaller width of about one time of the main channel. The angle of groove here is about 90 degrees.

In a preferred aspect, a device of the invention contains a passive micromixer which is placed immediately downstream of the reservoir adapted to contain a first modulating agent in order to facilitate diffusion of the modulating agent (e.g. acid or base) at the droplet interface. Moreover, grooves at the walls of the micromixer may facilitate mixing and diffusion at the oil- water interface. The change in the shape of the main channel (larger width typically) at the micromixer helps the mixing inside the droplet. The change in the shape of main channel can however be any change that results in change in the shape of the droplet when passing through the micromixer. Grooves on the walls of the micromixer facilitate mixing and diffusion at the oil- water interface.

It is to be noted that the presence of a micromixer does not require the presence of an obstruction structure, and vice versa. In fact, a physical obstruction is not a necessity, as long as there is no way the droplets from the main channel can get into the reservoir. Likewise, the necessity of the presence of the micromixer depends on the type of the reservoir that is in the microfluidic platform. For example, if the reservoir contain an

obstruction structure(s) that enhance the mixing, no micromixer is needed.

To allow for monitoring of the microdroplet-based reaction modulated by the first agent, the microfluidic device advantageously includes, or is used in combination with, a detection module positioned downstream from the at least one reservoir module. A "detection module" is a location within the device, typically within the main channel where droplets are to be detected, identified and/or measured on the basis of at least one

predetermined characteristic. The detection module can be in

communication with one or more detection apparatuses. The content of the droplets can be examined one at a time, and the characteristic is detected or measured optically, for example, by testing for the presence or amount of a reporter molecule. This type of "snap shot" detection of individual droplets passing by a certain point in the device is for instance suitable used in a setup for compound screening. In another embodiment, the detection module is set up to follow one or more individual droplets as the reaction occurs. This may be achieved using a delay line like a serpentine channel. The delay line may be provided with one or more delay structures to further slow down the droplets as they pass the detection module. The delay structure can consist of an increase in the diameter of the channel over a few millimetres, e.g. gradually over 2 times of the main channel. A design wherein the detection module comprises a serpentine channel comprising a series of delay structures (see items 7 and 8 in Figure 4) was found very useful to obtain kinetic information about fast reactions, which has heretofore been very difficult to achieve on a microfluidic scale. For example, the detection module is used to make a movie of one or more droplets as the reaction progresses.

The detection apparatuses for use in or in combination with a microfluidic device of the invention can be optical or electrical detectors or combinations thereof. Examples of suitable detection apparatuses include optical waveguides, microscopes, diodes, light stimulating devices, (e.g., lasers), photo multiplier tubes, and processors (e.g., computers and software), and combinations thereof, which cooperate to detect a signal representative of a characteristic, marker, or reporter, and to determine and direct the measurement or the sorting action at the sorting module. In a specific aspect, the detection module comprises means for detecting an increase or decrease in one or more photophysical properties of a detectable label. For example, the one or more photophysical properties are selected from the group consisting of fluorescence intensity, fluorescence lifetime, emission wavelength, absorption wavelength and polarization. In a preferred embodiment, the detection module comprises a fluorescence detector. In one embodiment, the detection module is in fluid

communication with a discrimination region and is at, proximate to, or upstream of the discrimination region.

A "discrimination region" or "branch point" is a junction of a channel where the flow of droplets can change direction to enter one or more other channels, e.g., a branch channel, depending on a signal received in connection with an examination in the detection region. Typically, a discrimination region is monitored and/or under the control of a detection region, and therefore a discrimination region may "correspond" to such detection region. The discrimination region is in communication with and is influenced by one or more sorting techniques or flow control systems, e.g., electric, electro-osmotic, (micro-) valve, etc. A flow control system can employ a variety of sorting techniques to change or direct the flow of droplets into a predetermined branch channel.

An "outlet region" is an area that collects or dispenses droplets after sorting. An outlet region is typically downstream from a detection module region, and may contain branch channels or outlet channels. A device may contain more than one outlet region if desired.

In a specific embodiment, the microfluidic device is configured to contain downstream from a first said detector module a second reservoir module adapted to contain a liquid comprising a second modulating agent, the latter being distinct from the first modulating agent, and wherein said second reservoir is connected to the main channel by a porous obstruction to prevent droplets from entering the reservoir. This specific device

configuration is particularly suitable to perform a microdroplet-based reaction wherein a first external trigger is followed by a second, distinct external trigger. Also here, the reservoir may be provided with a valve to control fluid communication with the main channel, and/or a second detection module may be present downstream from said second reservoir.

The device of the present invention can further include one or more delay modules. The "delay module" can be a delay line e.g. comprising a serpentine channel. A delay line allows to increase the residence time within the device. For example, in case a reaction within a droplet initiated by a first agent entering the main channel from a first reservoir is allowed to occur for a non-trivial length of time before being monitored, the delay module can be located downstream of the reservoir and upstream of the detection module. For reactions demanding extensive residence time, longer or larger delay lines are required. The channel dimensions and

configurations can be designed to accommodate the required residence time with minimum pressure drops across the device.

In another embodiment, the device is configured to contain a detection module immediately downstream the reservoir connection with the main channel to allow monitoring of rapid reactions taking place in the droplets passing the reservoir upon diffusion of the first agent via the fluid carrier to the inside of the droplets.

The fluid stream containing droplets is passed through the detection region, such that on average one droplet occupies the detection region at a given time. The droplets can be sorted into an appropriate branch channel based on the presence or amount of a detectable signal such as an optical signal, with or without stimulation, such as exposure to light in order to promote fluorescence.

A method, a microfluidic device according and/or a kit according to according to the invention are highly suitable for use in conducting a high- throughput screen. A microfluidic device of the invention is advantageously used in drug discovery, e.g.to screen libraries of small-molecule compounds, peptides, or antibodies for their ability to modulate the microdroplet-based reaction. Following the identification of promising compounds in a primary "snap-shot" screen, they can be more fully characterized in a dose-response analysis with multiple (e.g. 5-15 data points) per compound. Prior to the invention, the identification of compounds that inhibit viral fusion was a highly challenging task because of the lack of a fast and quantitative fusion assay that allows the screening of large numbers of candidate compounds and reports on their mechanism of inhibition. In particular, no assays were available that allow the observation of the kinetics of fusion in a high- throughput fashion. The present invention provides a lab-on-a-chip' approach, wherein the novel method of external triggering of microdroplet- based reactions is advantageously applied to enable the highly multiplexed observation of the kinetics of membrane fusion events.

For example, a microdroplet-based microfluidic system is used in which the oil phase is a carrier of aqueous droplets containing single or multiple liposomes (or cells) and a single or multiple copies of a virus (or virus-like) particles. Aqueous droplets containing virus particles that are prebound to liposomes will be introduced into a microfluidic flow channel. Before introduction into the microfluidic devise, each of these droplets will also carry a different compound from a compound library that we wish to screen for its ability to inhibit fusion. Using the external triggering approach described herein, the pH within the droplets is changed when they pass the first reservoir on the microfluidic chip, which can be seen as a 'trigger point'. Following the trigger, the droplets will pass through a long serpentine channel. The relative position in the channel corresponds to a well-defined time period elapsed since the rigger. By wide-field fluorescence microscopy, it is detected at what position in the channel fusion occurs, allowing to extract the kinetics of the fusion process for every individual droplet i.e., for every individual compound from the library. The droplet- based microfluidic platform of the invention allows the rapid injection (seconds) of small volumes (nanoliters) of virus-containing solutions and hposome-containing solutions into many (hundreds) microdroplets as nanoreactors. Using typical length scales of wide-field microscopy and microfluidic chip design, a throughput of up to 1-10 compounds per second (up to 10,000 compounds per hour) can be reached.

Also provided is a method for the manufacture of a microfluidic device of the invention. The channels of the invention are microfabricated, for example by etching a silicon chip using conventional photolithography techniques, or using a micromachining technology called "soft lithography", developed in the late 1990's. These and other microfabrication methods may be used to provide inexpensive miniaturized devices, and in the case of soft lithography, can provide robust devices having beneficial properties such as improved flexibility, stability, and mechanical strength. Devices according to the invention are relatively inexpensive and easy to set up. They can also be disposable, which greatly relieves many of the concerns of sterilization. A microfabricated device of the invention is preferably fabricated from a silicon microchip or silicon elastomer. The dimensions of the chip are those of typical microchips, ranging between about 0.5 cm to about 5 cm per side and about 1 micron to about 1 cm in thickness. A microfabricated device can be covered with a material having transparent properties, e.g., a glass coverslip to permit detection of a reporter molecule for example by an optical device, such as an optical microscope.

The dimensions of the channels and in particular of the detection region are influenced by the size of the droplets under study. Accordingly, detection regions used for detecting droplets have a cross-sectional area large enough to allow a desired droplet to pass through without being substantially slowed down relative to the flow of the fluid carrying it. To avoid "bottlenecks" and/or turbulence, and promote single-file flow, the channel dimensions, particularly in the detection region, should generally be at least about twice, preferably at least about five times as large per side or in diameter as the diameter of the largest droplet that will be passing through it.

The channels of the device are preferably rounded, with a diameter between about 2 and 100 microns, preferably about 60 microns, and more preferably about 30 microns. This geometry facilitates an orderly flow of droplets in the channels. Similarly, the volume of the detection module in an analysis device is typically in the range of between about 10 femtoliters (fl) and 5000 fl, preferably about 40 or 50 fl to about 1000 or 2000 fl, most preferably on the order of about 200 fl. In preferred embodiments, the channels of the device, and particularly the channels of the inlet, are between about 2 and 50 microns, most preferably about 30 microns.

A further aspect of the invention relates to a kit of parts comprising a microfluidic device according to the invention, and a container holding a labeled reagent, preferably a fluorescent dye. LEGEND TO THE FIGURES

Figure 1. pH switch in microfluidics. Inlet (1) is the inlet for oil and (2) is the inlet for aqueous sample in the main channel (3). Reservoirs A and B contain water-oil soluble acid in oil and water-oil soluble base in oil respectively. Porous (grid-like) obstruction structures (4, 5) prevent droplets from entering the reservoir. A detector module is arranged downstream of each reservoir. The outlet is indicated by (6). Figure 2. Schematic drawing of exemplary comb-like obstruction structures for introducing an agent from the reservoir into the main channel.

Figure 3. Schematic drawing of the design of a passive micromixer. Figure 4. Schematic drawing of an exemplary design of a microfluidic device. Inlet of an oil phase (1) and inlet of an aqueous phase (2) merge into the main channel (3). Reservoir (4) provided with a comb-like obstruction structure (5) is positioned upstream of a passive micromixer (6). A

serpentine channel (7) provided with multiple delay units (8) is present to slow down dispersed droplets while they pass a detection module (not shown in the figure) and leave the device via outlet (9).

Figure 5. pH switching in bulk using an emulsion of aqueous droplets comprising fluorescein in silicon oil.

Figure 6. Hemifusion traces of individual viruses in a single droplets. EXPERIMENTAL SECTION

In the following section, two representative embodiments of the invention are shown wherein a pH-dependent reaction inside microdroplets is modulated by external agents. Two high throughput setups are described, one for static and the other for dynamic microdroplet systems. In the following designs, the droplets have an aqueous content and are embedded in the oil phase. However, the same designs can be used for oil droplets in aqueous medium.

Example 1: Static-Droplet Reaction Chamber

This exemplary embodiment is based on the parallel execution of many chemical reactions in aqueous droplets in oil phase, one droplet in each well of a multi-well plate. An automated multichannel pipette that is connected to a microfluidic pressure control system is used to achieve high accuracy. The wells are first filled with oil (mineral oil, silicon oil, etc. with a neutral pH of 7). Injecting well-defined volumes of the aqueous phase into the oil then creates the aqueous droplets. Also present in these droplets is an amount of Phenol Red, a pH indicator which is yellow at a pH < 6.8 and red at a pH of > 7.4. Since the volume of the drops might be very small, a certain amount of oil is injected for pushing the droplets in to the oil phase to disconnect the drops from pipet tips. When the system is stabilized and droplets are localized by optical detectors (e.g., plate reader), an oil/water-soluble acid is injected into the oil phase. As a result, the pH of the oil phase rapidly decreases and with it the pH inside the aqueous droplets. At this point, the pH-dependent biochemical reaction will take place in the droplet and its kinetics can be read out by optical detection (plate reader). The change in colour of the Phenol Red upon the H change provides an accurate readout of the 't=0' point at which the reaction starts. Subsequent addition of an oil/water-soluble base can bring up the pH back. Exemplary a (bio)chemical reactions that can be investigated by such a method include the pH-induced fusion of a virus, such as HIV, influenza or dengue virus, with a lipid vesicle. The virus and the lipid vesicles are present in the pH-neutral droplets, after which the pH is dropped and the fusion kinetics read out by fluorescence means (e.g., dequenching of a lipid dye). The abihty to study such a reaction in a highly multiplexed way would allow for the screening of small-molecule and antibody libraries in their ability to inhibit fusion and represents a novel drug-screening tool.

Example 2: Dynamic-Droplet Reaction Chamber

In this design, the concept of modulating conditions in the aqueous droplets through the oil phase is integrated into a microfluidic platform. A flow channel is connected to a microfluidic control system for accurate pressure control. Figure 1 shows an exemplary design of a microfluidic pH switch structure. "A" represents a reservoir containing "acidic oil" (water-oil soluble acid is dissolved in oil) and "B" is a reservoir containing "basic oil" (water-oil soluble base is solved in oil). The reservoirs are separated from the main channel (3) partially with PDMS blocks to prevent droplets from going inside the reservoirs. The reservoirs can be closed or open by pneumatic valves (shown in figure 1 by dash lines). Inlet (1) is the inlet for oil as continuous carrier fluid and inlet (2) is the inlet for the aqueous sample injection. The outlet is indicated by (6).

Similar to the static case above, Phenol Red was used as an indicator for the pH in the aqueous droplet. The oil can be mineral oil, silicon oil, etc. At the beginning of the experiment both valves are closed, sealing off reservoirs A and B. At the T junction (top of figure 1) droplets are formed. As the droplets reach the acidic reservoir, the reservoir valve is opened. After droplets pass the reservoir, they are detected by the optical detector that is placed right after the reservoir. The change in the colour of the droplets from red to yellow indicates that the pH of medium inside droplets has dropped.

Then, the acidic reservoir is closed and the basic reservoir is opened. When droplets pass the basic reservoir, they become basic, caused by the diffusion of the base in the continuous oil phase and then into the droplets. If the droplets are detected at a position downstream of the basic reservoir, a change in the colour of the droplets from yellow to red is observed.

In this way one can readily control the medium in nanoliter or picoliter chambers from bulk without the need for droplet merging. This pH controller has a very simple structure and can be fabricated in most microfluidic structures to have any desired pH.

To demonstrate the feasibility of droplet pH switching through the oil phase, the following experiment was performed. First, an emulsion of 1 mM aqueous fluorescein solution in 2 ml oil was prepared by sonication.

Fluorescein is a pH indicator, allowing to track the pH of the aqueous phase by monitoring the fluorescence intensity of the emulsion over time as we induce various pH changes (figure 5). At T= 245s acetic acid was injected to the cuvette containing the emulsion. Clearly visible is a reduction of the intensity (after settling) corresponding to a decrease in pH. Subsequent injection of an oil-water soluble base to the system, in this case

propylamine, at T = 600s, an increase in fluorescence intensity of the system is detected. Acetic acid was again added to the system at T=850 to reverse the pH switch. Example 3: Method to perform a microdroplet-based viral

membrane fusion reaction.

This example demonstrates how a pH-triggerable biochemical process can be controlled and investigated by application of water-oil soluble pH modulator in a method of the invention.

Influenza (X31) viral fusion with liposomes was performed in static microdroplets in oil. The experiments were performed under conditions that allowed the observation of fusion of individual viral particles, thus demonstrating that the procedure can be down-scaled to a point at which microdroplets can be studied containing only single molecules of reactants.

A large droplet of the oil phase was placed on top of a microscope cover slip, in which nano-liter sized aqueous droplets were injected containing pre-docked virus-liposome complexes at neutral pH. Lowering of the pH of the aqueous phase is predicted to initiate fusion. The viral particles were stained with R18, a dye that displays reduced intensity when packed in the viral membrane before fusion, but will increase in

fluorescence upon fusion. Using a fluorescence microscope the evolution of the fluorescence in single droplets was monitored. The pH drop was induced by injecting acetic acid, which is an oil-water soluble acid, to the oil phase. The resulting pH inside droplets was 4.9, which is the proper pH for viral fusion induction. The oil used for this experiment is 100%

octamethyltrisiloxane.

Figure 6 shows 5 examples of fluorescence traces obtained from single particles within a single droplet. The kinetics of fusion are consistent with the viral fusion studies performed at the single molecule level on a supported lipid bilayer (Floyd et al. PNAS Oct 2008, vol. 105 no. 40 15382- 15387). Whereas these preliminary experiments were all done on static aqueous droplets in an oil phase, this technique is readily integrated in microfluidic-based platforms to allow for the rapid manipulation and observation of hundreds of droplets.

Claims

1. A method for performing a microdroplet-based reaction comprising the steps of :

(i) providing a carrier fluid and a plurality of droplets, the droplets

containing reactants and being immiscible with and surrounded by the carrier fluid, and

(ii) introducing into the carrier fluid a first agent that is (a) soluble in both the carrier fluid and the droplets and (b) capable of modulating at least one reaction inside the droplets.

2. Method according to claim 1, further comprising the step of (iii) monitoring the at least one reaction and/or a reaction product.

3. Method according to claim 2, wherein monitoring the reaction is performed using optical detection, preferably microscopy, more preferably fluorescence microscopy.

4. Method according to any one of the preceding claims, wherein the volume of the droplets remains essentially unchanged.

5. Method according to any one of the preceding claims, wherein the droplets are dynamic droplets.

6. Method according to any one of the preceding claims, wherein the droplets have an aqueous content and the carrier fluid forms an oil phase.

7. Method according to any one of the preceding claims, further comprising following step (ii), a step (iii) of introducing into the carrier fluid a second agent that is (a) soluble in both the carrier fluid and the droplets and (b) capable of modulating a second reaction inside the droplets.

8. Method according to any one of the preceding claims, wherein the first and/or second agent is a reactant or a catalyst.

9. Method according to any one of the preceding claims, wherein the first or second agent is a water-oil soluble acid or a water-oil soluble base.

10. Method according to any one of the preceding claims, wherein the microdroplet-based reaction is a biochemical process

11. Method according to claim 10, wherein the biochemical process comprises fusion or hemi-fusion of lipid biolayers

12. Method according to claim 11, wherein the process is acid-induced viral fusion with a target membrane, preferably acid-induced viral fusion with a target lipid vesicle.

13. Method according to any one of the preceding claims, wherein droplets contain at least one test compound to be evaluated for its effect on the at least one reaction.

14. Method according to claim 13, wherein the test compound is a small molecule, a proteinaceous compound, such as a peptide or an antibody, or a nucleic acid molecule.

15. A microfluidic device comprising (i) one or more inlet modules that have at least one inlet channel adapted to carry a dispersed phase fluid; (ii) at least one main channel adapted to carry a continuous phase fluid, wherein the inlet module is in fluid communication with the main channel such that the dispersed phase fluid is immiscible with the continuous phase fluid and forms a plurality of droplets in the continuous phase fluid; (iii) one or more outlet modules and (iv) downstream from the inlet module at least one reservoir adapted to contain a first modulating agent, wherein said reservoir and the main channel are configured to be fluidically connected to one another via an obstruction structure to prevent droplets in the main channel from entering the reservoir.

16. A microfluidic device comprising (i) one or more inlet modules that have at least one inlet channel adapted to carry a dispersed phase fluid (ii) at least one main channel adapted to carry a continuous phase fluid, wherein the inlet module is in fluid communication with the main channel such that the dispersed phase fluid is immiscible with the continuous phase fluid and forms a plurality of droplets in the continuous phase fluid; (iii) one or more outlet modules; (iv) downstream from the inlet module at least one reservoir adapted to contain a first modulating agent; and (v) downstream from the reservoir a micromixer arranged to enhance diffusion of the modulating agent into the dispersed droplets.

17. Microfluidic device according to claim 16, wherein said reservoir and the main channel are configured to be fluidically connected to one another via an obstruction structure to prevent droplets in the main channel from entering the reservoir.

18. Microfluidic device according to claims 15 or 17, wherein the obstruction structure is a porous obstruction structure, preferably fabricated of a polymer such as polydimethylsiloxane, or a comb-like structure comprising a plurality of narrow channels intercepting the main channel.

19. Microfluidic device according to any one of claims 15-18, furthermore including a detection module positioned downstream from the at least one reservoir module.

20. Microfluidic device according to claim 19, comprising downstream from said detection module a second reservoir module adapted to contain a liquid comprising a second modulating agent which is distinct from the first modulating agent, wherein said second reservoir is connected to the main channel by an obstruction structure to prevent droplets from entering the reservoir, preferably wherein the reservoir is provided with a valve to control fluid communication with the main channel, and wherein a second detector module is present downstream from said second reservoir.

21. Kit of parts comprising a microfluidic device according to any one of claims 15-20, and a container holding a detection reagent, preferably a fluorescent dye.

22. Use of a method according to any one of claims 1-14, a microfluidic device according to any one of claims 15-20 and/or a kit according to claim 21 in conducting a high-throughput screen.

23. Use according to claim 22, in conducting a high-throughput screen for identifying a viral fusion inhibitor.