GB2427689A

GB2427689A - Radiometric fluorescently labelled carrier particles and detection methods

Info

Publication number: GB2427689A
Application number: GB0612371A
Authority: GB
Inventors: Simon Webster
Original assignee: Avacta Ltd
Current assignee: Avacta Ltd
Priority date: 2005-06-23
Filing date: 2006-06-22
Publication date: 2007-01-03
Also published as: WO2006136824A3; GB0612371D0; WO2006136824A2; GB0512769D0

Abstract

The present invention provides ratiometrically labelled lipid or lipophilic carrier particles having at least two fluorescent signals and mixtures thereof, each carrier particle having incorporated in or on its shell surface at least one target receptor. The invention also includes a method of screening the particles with a range of probe ligands in sorting or binding assays and in a ratio-metric multicolour fluorescence correlation spectroscopy screen and a method of making such particles. The carrier particles are especially useful for carrying membrane proteins as the target receptors. The carrier particle may be a vesicle such as a liposome. The methods of screening may also include screening with a labeled test ligand to simultaneously determine both binding and the level of binding of the ligand.

Description

Ratiometric Multicolour Fluorescence Correlation Screening; Agents for use

therewith and Methods of Production thereof The present invention relates to ratio-metrically labelled carrier particles and mixtures thereof, each carrier particle having a lipid outer surface or shell and incorporated in or on its shell surface at least one target receptor, methods of producing said particles and a method of screening said particles with a range of probe ligands in sorting or binding assays and especially, but not exclusively, in a ratio-metric multicolour fluorescence correlation spectroscopy screen. The carrier particles and the method of screening said particles are of particular, but not exclusive, use in biochemical assays, clinical diagnosis and especially for the case of membrane proteins as the target receptors.

Background to the Invention

Two dimensional array technology (2D arrays) or bio-chip technology systems for the detection of protein or DNA interactions with ligands in solution are well known in the art [1-4]. These methods are particularly well suited for water soluble proteins and nucleic acids, and are routinely employed in high throughput screening of candidate therapeutics and other biochemical or diagnostic assays.

An alternative approach is to dispense with the 2D array which requires a pattern of immobilised receptor molecules (protein or nucleic acids) and to use coded beads as carrier particles for the receptors [5,6]. Such an approach is sometimes described as a multiplex assay' or a homogeneous assay'. In one method of the prior art, polymeric beads or microspheres have been dyed with semi-conducting nanocrystals which fluoresce at different wavelengths [7]. These beads are subjected to surface derivatisation enabling the coloured beads to be coupled to soluble oligonucleotide probes and which, when illuminated with UV or visible light, causes the coloured beads to emit a characteristic wavelength of light encoding the bead/spheres for the particular oligonucleotide that has been attached to its surface. Each colour of bead can then be used as a substrate for a specific biological assay. However, a major disadvantage of this system is that it is unsuitable for insoluble (or membrane) proteins as attachment to the bead surface renders the insoluble protein in a foreign environment resulting in detrimental impairment of the function of the insoluble protein attached thereto.

Accordingly, any results obtained from binding or non-binding of a target ligand to the

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surface agent could result in false positives and negatives and so render such a method of little value as a biological assay or diagnostic test.

In another example from the prior art ratiometrically labelled microspheres have been internally labelled with fluorescent dyes that are completely enclosed within their cores [8]. The microspheres may be adapted to further include surface carboxyl groups for attachment of ligands or biomolecules or are modified to have a surface layer of avidin for binding of biotin or biotinolyted ligands or they may be chemically pre-coupled with oligonucleotide sequences. Laser excitation is used to excite the fluorescence from the internal dye labels so that the identity of each microsphere, and thereby the identity its surface immobilised biomolecule, can be determined. As mentioned above, the attachment of a protein that is insoluble in water to the surface of a particle may result in an alteration of its functionality thereby resulting in false readings in a biochemical assay or diagnostic test.

Membrane proteins are therefore particularly problematic to use as targets in multiplexed assays because of their insolubility in aqueous solutions. However, they represent a potentially very important class of target for biochemical assays and diagnostic tests.

Membrane proteins constitute -30% of alt proteins in the human body and are responsible for a wide range of physiological functions essential for life. As such they represent a particularly important target for therapeutic treatments and diagnostic tests.

If insoluble proteins are to be incorporated in a biochemical assay and retain their function they need to be incorporated within a lipid or lipophilic environment and thus require a lipid bilayer or lipophilic layer in which to be inserted. Typically, if such methods also employ 2D multiplex array construction then the lipophilic layer must be patterned to spatially encode the identities of the target molecules [9]. This is very difficult to achieve; robotic spotting and microfluid handling techniques are required making such technology quite complicated and time consuming.

Thus, a system which could obviate the need for a patterned tipophilic layer to host and immobilise a multitude of different insoluble proteins, yet provide a method of uniquely identifying each of these as a target receptor in a binding assay would offer immediate advantage to fundamental biological science, the pharmaceutical industry and clinical diagnosis.

In the present invention, we have made agents (carrier particles) suitable for a generic, multiplexed high throughput screening methodology. We have also developed a method for detecting not only these new carrier particles but existing ratiometrically labelled particles based on sorting or binding assays, for example fluorescence detection of binding events which may be applied to diverse targets including nucleic acids and soluble proteins. Most importantly, however, the agents and method of the present invention are applicable to insoluble (membrane) proteins. The receptor targets are incorporated in or carried on the surface shell of a carrier particle without the requirement of chemical attachment. This advantageously minimises any possible impairment of the functionality of the target molecule itself and presents a more natural or native' environment to both the receptor target and a ligand and thus encourages a natural binding event thereby reducing any false readings.

Statement of the Invention

According to a first aspect of the invention there is provided a carrier particle comprising a lipid or lipophilic outer surface or shell and attached thereto or incorporated therein or contained within, at least two different fluorescent labelling agents present in defined ratiometric amounts, the at least two different carrier particles being distinguishable by the different ratios of their labelling agents, the carrier particle further comprising at least one receptor target, being either attached to or incorporated in the outer surface or shell of said carrier particle.

Throughout the description and claims of this specification, the words "comprise" and ucontain and variations of the words, for example "comprising" and "comprises", means uincluding but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

The essence of the present invention resides in the use of two or more dyes in varying ratios in a liposome to allow the liposome to be uniquely identified by the ratio of the fluorescent signal from the dyes. This allows each liposome with its own unique fluorescent code to be identified in a homogenous mixture of labelled liposomes. A membrane bound target molecule (receptor) within the liposome can therefore also be identified by this fluorescence code allowing a homogeneous (solution based) assay of insoluble target molecules (membrane proteins). In the present invention we avoid a solid support which is a particularly attractive feature as it is extremely problematic to get membrane protein into a supported bilayer and keep them native because of the proximity of the solid support which upsets/disrupts their structure.

Preferably, the lipid or lipophilic shell of the carrier particle comprises a lipid or mixture of lipids selected, for example and without limitation, from the group comprising glyco-, sphingo-, phospho- or any other type of lipid, and may also preferably contain cholesterol, proteins, peptides or any other moiety that will reside in a lipid membrane.

Thus the shell of the carrier particle provides a substantially or predominantly lipid or lipophilic environment.

It will be appreciated that the outer shell or surface of the carrier particle provides a lipid environment in or on which at least one target receptor can be presented. The at least one target receptor is incorporated into the lipid environment of the carrier particle so that it may be carried on its surface in a "semi-immobilised" fashion, either by insertion into the shell or attachment via a lipophilic anchor or any other method of immobilisation that is to say the at least one receptor target may be capable of movement within the lipid shell of the carrier particle, however it will be unable to detach from or leave the lipid shell. It will also be appreciated that the surface receptor target may also be immobilised in another manner so that it is unable to move freely within the lipid surface.

Whether the surface receptor target is semi-immobilised or fully immobilised is a matter of choice of the user's requirements.

Reference herein to "ratiometric" refers to the ratio of the number, quantity or intensity of a first fluorescent labelling agent as compared to a second or further fluorescent 4 labelling agent. The two or more different fluorescent labelling agents are incorporated into, or attached to, or contained within the shell of the carrier particle and by varying the ratios of the two or more fluorescent labelling agents associated with the shell, individual carrier particles can be "colour coded" or "encoded" so that they may be subsequently identified in a biological assay.

Preferably, the carrier particles are ratiometrically labelled with the fluorescent labelling agents. That is to say that that the carrier particle will incorporate a different proportion of a first fluorescent label compared to a second fluorescent label or a different proportion to any further fluorescent label or dye attached to, incorporated in or contained within the carrier particle. It will thus be appreciated that the fluorescent labelling agents can each be carried on the surface of the shell, in the shell itself or be enclosed within the shell. Equally, one fluorescent labelling agent may be carried in a different part of the shell compared to the at least one other, thus any combination of positioning the at least two fluorescent labelling agents is intended within the scope of the present invention, for example a carrier particle having at least one fluorescent labelling agent attached to its shell surface and also having at least one further fluorescent labelling agent enclosed within the shell is within the scope of the application.

Preferably, the ratiometric fluorescent labelling agent attached to or incorporated in or contained within the outer shell of the carrier particle is selected from the group comprising a fluorophore (a fluorescent molecule), such as a fluorescent dye or an inorganic fluorescent entity, such as a quantum dot.

Preferably, the fluorophore or inorganic fluorescent entity is lipophilic or conjugated to a lipophilic agent and capable of integration into a lipid membrane. For example, the fluorescent moiety may be selected from any one of the following non-exhaustive list comprising NBD, Oregon Green, Texas Red, Tetramethyirhodamine, Fluorescein, BODIPY, BODIPY Fluorescein, the AlexaFluor family of dyes or from the family of fluorescent quantum dots.

Preferably, the intensity or level of labelling with the at least two fluorescent labelling agents may be varied. In this way a carrier particle may carry a specified radiometric amount of fluorescent labelling agents of a first distinct intensity level which allows discrimination from a similarly labelled carrier particle with second distinct intensity level, thereby allowing the same ratiometric fluorescent code to be used to identify multiple carrier particles. In this respect the carrier particles can be compared analogously to electric light bulbs having 20, 40, 60,80 or 100 Watts.

Preferably, the carrier particle is a microspherical particle or microsphere. However, the carrier particle could be any other shape providing it retains the functional capability of carrying at least one target receptor and at least two or more ratiometrically labelled fluorescent agents.

In one embodiment of the invention the microspherical particle or microsphere is in the form of a vesicle, that is to say a small anatomically normal sac or bladder like structure that contains a fluid, in an alternative embodiment of the invention, it may be a lipid or lipophilic shell that contains or surrounds a solid or fluid or gel.

Reference herein to a "vesicle" is intended to include an artificially made microscopic hollow container that is fluid-filled or it may also be a liposome i.e. a spherical particle in aqueous medium formed by a lipid bilayer enclosing an aqueous compartment. A lipid bilayer is the basic structure of biological membranes and so the environment in the shell of a liposome is similar to that of a biological membrane, for this reason liposomes as carrier particles are the preferred embodiment of the present invention.

Preferably, in the embodiment where the carrier particle is a vesicle it is a lipid vesicle or liposome.

Preferably, the fluid filled interior of the lipid vesicle or liposome carrier particle comprises any one or more of the following interiors selected from the group comprising a buffer, an aqueous solution which optionally may contain a biochemically sensitive agent, for example and without limitation an ion sensitive dye, a hydrophilic environment or a hydrogel.

In the example where the carrier particle is a lipid vesicle it may carry a calcium ion channel as the at least one of the target receptors and the fluid filled interior may include an ion sensitive dye, so that if an appropriate ligand binds to the calcium ion channel receptor the binding event and functional consequence may simultaneously be detected.

The interior of the carrier particle encompassed by the lipid shell may, in an alternative 6 embodiment, be solid. Thus the core or solid interior surrounded by the lipid shell may be in the form of a solid particle made of latex or glass or a synthetic polymer material or any other material that is known in the art for the construction of microspheres.

Preferably, the in the embodiment where the microspherical particles or microspheres comprise a solid interior or core they are coated with a lipid layer so that the lipid layer may be impregnated with a mixture of ratiometric dyes or have them incorporated into the lipid layer or the lipid surface may be labelled with a mixture of ratiometric dyes.

In this embodiment of the invention it is possible to modify prior art microspherical particles or microspheres to the desired characteristics of the carrier particles of the present invention.

Preferably, in the instance of the carrier particle being a lipid vesicle or liposome it is uni- lamellar or multi-lamellar, that is to say it may comprise a single lipid bilayer, or it may comprise a number of bilayers of differentially increasing diameter so that the outer surface represents the greatest diameter so that the carrier particle is similar to the layers of an onion. It will be appreciated that the embodiment of the invention wherein the microspherical particle or microsphere core is solid, it may also be encompassed by or coated with at least one or more lipid layers in this manner.

Preferably, the carrier particle whether it is a lipid vesicle with a fluid filled interior or it is a lipid coated solid cored microsphere it has a diameter in the region of lOnm to 5Opm, and more preferably is in the region of 0.1 to 10 pm in diameter and more preferably still is about 1 pm in diameter.

Preferably, the carrier particles of the present invention may comprise a mixture of particles with solid or hollow cores of the same or different diameters or may comprise a mixture of only hollow core particles of the same or different diameters or only solid carrier particles of the same or different diameters.

It will be appreciated that the lipid vesicle or the surface of the solid particle coated with a lipid layer provides an environment similar to that experienced by an insoluble or membrane protein in the cell membrane and as such, no chemical attachment/bonding is required, therefore there is a more natural environment that is analogous to the "natural" in vivo situation and therefore the target receptor will behave and function as in 7 vivo. It is expected that such a system comprising the carrier particles of the present - - invention will advantageously reduce the number of any false results in biochemical assays.

In a further embodiment of the invention, the carrier particle when it is a vesicle may be a vesicle into which appropriate and/or selected receptor(s) may be incorporated.

Preferably, in the instance where the carrier particle is a vesicle it may also include a further internal label or dye or any other agent which a user requires.

Preferably, the receptor target carried by the carrier particle is identifiable by the ratiometric proportion of the fluorophores attached thereto, incorporated therein or contained thereby.

This identification may be achieved by the detection and analysis of fluorescence signals emitted by the carrier particle upon exposure to suitable optical excitation.

Preferably, the receptor target carried by the carrier particle is capable or incapable of binding a given test ligand or number of test ligands.

Thus it will be appreciated that the carrier particles of the present invention, when used in an assay or diagnostic test will comprise a mixture of a set of particles capable of undergoing a binding event and a further set that are incapable of binding that ligand.

Preferably, the carrier particle is provided with more than one identical or different receptor target binding sites.

For example, and without limitation, the bound surface receptor target may be selected from the group comprising trans-membrane or G-protein coupled receptor proteins or peptides which may include ion channels, protein transport or other transport channels, or enzymes and so are especially suitable for detecting binding events to insoluble or membrane bound target receptors. It will be appreciated that any target receptor may be selected and that the foregoing is merely suitable samples from a non-exhaustive list that is not intended to limit the scope of the invention.

According to a second aspect of the invention there is provided a method of making a 8 carrier particle comprising: (i) providing a lipid vesicle or coating a solid microsphere in a lipid or mixture of lipids so as to provide, in both instances, a carrier particle comprising a lipid or lipophilic outer surface or shell; (ii) incorporating in or attaching to an outer shell of a carrier particle at least one target receptor; and (iii) impregnating or surface labelling the outer shell of the carrier particle or filling the core of the carrier particle when in the form of a lipid vesicle with a ratiometric mixture of fluorescent labelling agents.

It will be appreciated that step (iii) may be performed in advance of step (ii) and that strict adherence of stages is not intended to limit the scope of the invention.

Preferably, the second aspect of the invention further includes any one or more of the features of the first aspect of the invention.

Preferably, a mixture of carrier particles of either solid cored lipid coated microspheres or lipid vesicles with different target receptors and different fluorescent labelling agent ratios are prepared and mixed together in solution.

Preferably, where the carrier particles comprise lipid vesicles with a fluid filled interior they will be formed by extrusion through apertures of sizes ranging from lOnm to 50 pm or sonication of a solution of the lipid or mixture of lipids and fluorescent labelling agents.

Preferably, where the carrier particles comprise lipid vesicles with a fluid filled interior the target receptors will be incorporated in the lipid shell of said vesicle by controlled solubilisation of the vesicles with detergent followed by controlled removal of the detergent. It will be appreciated that this is only one of a number of possible strategies for insertion of the target receptor into the lipid vesicle.

Preferably, where the carrier particle has a gel or solid core the lipid shell will be coated onto the gel or solid core by adsorption or will be attached by appropriate chemical anchoring groups.

According to a third aspect of the invention there is provided a method of screening one or more test ligands in solution to identify which ligand(s) is/are capable of undergoing a binding event and the level of said binding event, with a mixture of carrier particles r. 10 comprising a lipid or lipophilic outer surface or shell and attached thereto or incorporated therein or contained within, at least two different fluorescent labelling agents present in a ratiometric amount so as to generate a number of species of carrier particles identifiable by their fluorescent spectral properties, each carrier particle further comprising at least one receptor target, being either attached to or incorporated in the outer surface or shell of said carrier particle, the binding event and level of binding being identifiable by either homogeneous readout or by sorting and subsequent analysis.

Reference herein to "level of binding event" also includes the degree of binding or the propensity to bind of the one or more test ligand with the at least one receptor target carried by the carrier particle.

Preferably multiple different species of receptor target, each carried on their own encoded carrier particles, and thus identifiable by their unique ratiometric fluorescent code, will be placed together in a fluid environment. One or more test ligands may then be added to this mixture of carrier particle borne receptor targets such the ligands may bind or not bind to the various receptor targets. The carrier particles may then be individually interrogated whilst still in solution to determine whether binding of the test ligand has taken place. The ratiometric encoding of the carrier particle enables the identity of the target receptor to be determined without the need to directly label the receptor. Alternatively, following exposure of the target receptors to the test ligands the ratiometric fluorescent encoding of the carrier particles may be used to sort and separate the different target receptor species. Once each target species is isolated the total amount of ligand bound to that species may be determined using one of a number of possible analytical techniques such as, but not limited to, mass spectrometry or optical spectroscopy. Thus it will be appreciated that the invention may be used as a homogeneous multiplex assay or diagnostic test in which the propensity of one or more test ligands to bind to multiple target receptors may be determined simultaneously.

Preferably, in the instance where the method involves homogeneous read out, detection of binding events is obtained from fluorescent correlation spectroscopy, fluorescence cross correlation spectroscopy, burst analysis or microscopic video rate fluorescence imaging.

Preferably, in the instance where the method involves sorting and subsequent analysis, the sorting. is achieved by fluorescence activated flow cytometry or microfluidics and preferably the subsequent analysis is achieved by a method selected from the group comprising mass spectrometry, HPLC,intrinsic fluorescence, Raman or non-linear Raman spectroscopy, absorption or other spectroscopy.

It will be appreciated that in the instance where the method involves sorting and subsequent analysis the method is able to differentiate the bound and unbound state of the receptor in the presence of a ligand which does not require the ligand to be labelled with a fluorescent labelling agent.

In this embodiment of the invention where the ligand is not tagged with a fluorescent labelling agent, but is present in for example a body fluid such as blood derived fluid or urine, the sorting and subsequent analysis method is of particular use in diagnosing the presence or absence of the particular ligand.

In the instance where the ligand is tagged with a fluorescent label the fourth aspect of the invention is particularly suitable.

According to a fourth aspect of the invention there is provided a method to simultaneously screen a binding event and level of binding of a test ligand tagged comprising a first fluorescent label with multiple target receptors comprising: (i) mixing in solution multiple species of carrier particles, the first species of carrier particles carrying a first receptor or set of receptors and labelled with a specified dye ratio of at least a second and third distinct fluorescent labelling agent compared to the tagged ligand and further species of carrier particles carrying different target receptors or sets of receptors and each labelled with a specified dye ratio of the at least second and third fluorescent labelling agents with that dye ratio being unique to each species of carrier particle; (ii) adding the test ligand or ligands tagged with a first fluorescent label to the mixture; (iii) passing the mixture through an excitation light source which may be a focused laser beam linked to a fluorescence spectrophotometer and; (iv) differentiating between a free ligand and a ligand which has undergone a binding event.

Preferably, the step comprising differentiation of the free ligand and one which has undergone a binding event employs a technique known as fluorescent coincidence analysis or fluorescence cross correlation analysis to detect coincident or non-coincident fluorescence and similarities in the temporal properties of the fluorescence from the ligand labelling agent and the carrier particle labelling agents so as to differentiate between a free ligand and a ligand which has undergone a binding event. Thus the method uses fluorescence emission from the ligand labelling agent Alternatively, preferably the step comprising differentiation of the free ligand and one which has undergone a binding event fluorescence correlation spectroscopy to determine the speed of diffusion of the ligand in solution and thus differentiate between a free ligand and one which has undergone a binding event.

In a yet further alternative embodiment, the step comprising differentiation of the free ligand and one which has undergone a binding event uses video rate microscopic fluorescent imaging of the encoded carrier particles and labelled ligands in solution with spectrally distinct detection channels to look for spatial coincidence and coordinated motion of ligand and carrier particle fluorescence to distinguish between free and bound ligands.

Preferably, the step comprising differentiation of the free ligand and one which has undergone a binding event simultaneously uses the ratio of intensity of the second and third labelling agents to identify the species of a given carrier particle and thus the identity of the target receptor or receptors.

Alternatively, the step comprising differentiation of the free ligand andone which has undergone a binding event simultaneously uses the intensity of the fluorescent emission from the labelling agents of test ligand molecules determined to have undergone a binding event to determine the number of test ligands bound to a given carrier particle.

Preferably, the fluorescence detection system has a number of detector channels commensurate with the number of different fluorescent labelling agents. In this respect it will be appreciated that the system typically has at least a three spectroscopically distinct detector channels; one to measure the photon burst from the first fluorescent labelling agent on the ligand and two further channels to measure the ratio of the second 12 and third distinct fluorescent labelling agents on or in the carrier particles. However, it may be designed for detecting further fluorescent labelling agents with different spectroscopic characteristics and so the number of detection channels may be increased.

Preferably, where fluorescence correlation spectroscopy is employed, the transit time through the excitation light beam of the free or unbound ligand tagged with the first fluorescent labelling agent is determined by, for example but not exclusively, calculation of the autocorrelation function of the fluorescence signal from the first labelling agent and fitting this function with an appropriate theoretical model Preferably, where fluorescence correlation spectroscopy is employed, a ligand which is bound to a target receptor on or in a carrier particle is identified by slower diffbsion in solution than an unbound, ligand due to the larger size. and therefore slower diffusion of the carrier particle/ligand complex when compared with that of the free ligand. A carrier particle/ligand complex typically has a diffusion rate at least one order of magnitude slower than that of a free ligand Preferably, where fluorescence coincidence analysis or cross-correlation analysis is employed a binding event is indicated by the simultaneous detection of the fluorescent signals from the first labelling agent identifying the ligand and the second and third labelling agents identifying the carrier particle, and where these signals display the same temporal characteristics indicating the same transit times through the excitation focal volume.

Preferably, the ratio of the second and third fluorescent labelling agents which encode the identity of the different species of carrier particle are measured by the fluorescence spectrophotometer coincidently with the fluorescence from the labelled test ligand so as to identify to which species of carrier particle the ligand has bound. It will be appreciated that the ratios of the fluorescence intensities from the second and third fluorescent labelling agents are unique identifiers for a carrier particle carrying a specified receptor(s) so that the target receptor to which the ligand has bound can be identified.

Preferably where video rate fluorescence microscopic imaging is employed, the assay solution is contained in a receptacle such that the distance through the solution is small in the dimension parallel to the optical axis of the imaging system. An area of the 13 sample is illuminated, or a point of light rapidly scanned over the sample solution, such that fluorescence from the various labefling agents is excited and images are obtained of the sample solution simultaneously at discrete spectral wavelengths corresponding to the emissions from the various labelling agents. Two or more such images are recorded in quick succession such that the motion of the various fluorescent particles may be observed. Labelled ligand molecules that have undergone a binding event may be identified by co-ordinated motion of their fluorescence signal with that of an individual carrier particle whereas unbound ligand molecules are characterised by more rapid, uncoordinated motion. The identity of the carrier particle, and thus the identity of the target receptor to which the ligand has bound may be determined from the ratio of intensities of the two or more fluorescence dyes used to encode the carrier particle.

Image analysis software may be used to extract this information from the recorded images and can simultaneously interrogate all the carrier particles within th f1d c.f cw for the presence of bound labelled ligands.

Preferably, the solution into which the different species of carrier particle and ligands are allowed to freely diffuse is of a physiological nature i.e. a buffered aqueous solution.

Preferably, the concentration of the carrier particles and ligands in the solution is kept low to ensure that, on average, only a single carrier particle or ligand at a time diffuses through the excitation volume.

Preferably, the total concentration of entities in the solution is in the range 1 pM to 1 OOnM and more preferably about 1pM - I nM.

Such a low concentration range is particularly advantageous so that very small amounts of ligands and target receptors may be screened obviating the need for any high scale production of either these or the carrier particles. Such a low concentration also has the particular advantage of allowing fluctuation spectroscopy such as fluorescence correlation spectroscopy (FCS) to be used to determine the transit time of the entities as they move through the excitation source.

A particular advantage of the methods of the present invention resides in obviating the need for construction of a 2D array, robotic spotting and microfluid handling techniques.

The methods of the present invention are ideally suited to high throughput screening of a variety of ligands/receptor interactions including ligands/insoluble membrane protein 14 receptors and provides a simple and rapid screening technique. Moreover, by using a mixture of carrier particles with lipid shells and other surfaces a single instrument/apparatus/system can be used for simultaneous screening of ligands against soluble protein, oligonucleotide and insoluble protein target receptors.

According to a fifth aspect of the invention there is provided use of ratiometrically labelled carrier particles in detecting a binding event between a ligand and at least one target receptor carried on or in the lipid or lipophilic surface of said carrier particle so as to diagnose the presence or absence of the ligand in a biological sample or to screen for candidate therapeutics.

Brief Description of the Figures

The invention will now be described by way of example o ly with reference to the following

brief description of the drawings wherein:

Figures 1 A-D shows a schematic representation of carrier particles of the present invention; Figures 2 A-C shows the carrier particle of Figure 1 B-D with their respective target receptors; Figure 3A shows a schematic representation of a binding assay with fluorescently labelled test ligands; Figure 3B shows a schematic representation of a solution based biological sandwich type assay; Figure 3C shows a schematic representation of a solution based competitive/displacement assay; Figures 4A-D shows a schematic representation of a method of sorting encoded carrier particles and subsequent detection of bound unlabelled test ligands; Figures 5A-D shows a schematic representation of a method of sorting encoded 35 carrier particles combining multiplex targets with multiplex test ligands; Figures 6A-D shows a schematic representation of a method of sorting encoded carrier particles combining more complex multiplex targets with multiplex test ligands ligands; Figure 7 shows a schematic representation of the method of the present invention of ratiometric multicolour fluorescent correlation screening (rm FCS); Figure 8A-G shows simulated data from the rm FCS of Figure 7.

Figure 9 shows fluorescence spectra recorded from four different batches of lipid vesicles, each prepared with different ratios of two differently fluorescently labelled lipids.

Figure 1OA shows autocorrelation function G (t) against t!rne for free dye and in Figure lOB autocorrelation function 0 (c) against approximately lOOnm diameter liposomes labelled with 0.001% of dyed lipid.

Figure 11 shows the results of measurements from different species of ratiometrically fluorescent encoded liposomes.

Materials and Methods Encoded Carrier Particle Preparation A range of methods to prepare liposomes are well known to the art and man-made' liposomes are widely used in applications ranging from drug delivery to cosmetics.

Detailed descriptions of some of these preparation methods are presented, for example, in reference 10 and further references contained therein. Possibly the most straightforward method to prepare unilamellar liposomes of well defined size is to pass a solution containing lipid molecules through a filter with known pore size. After repeated passes through the filter the lipids self-assemble into unilamellar vesicles whose diameter is defined by the filter pore size. Suitable filters with a wide range of well defined pore size are readily available from a number of vendors.

In order to produce the ratiometrically labelled liposomes employed in the invention described here a mixture of undyed lipid molecules and at least two different types of fluorescently labelled lipids are used to make the liposomes. Non-fluorescent lipids are readily available from a number of vendors and a range of fluorescently labelled lipids are available from vendors such as Invitrogen/Molecular Probes.

In order to produce different ratiometric fluorescent codes the ratio of the undyed lipid to fluorescently dyed lipid and the ratios of the different fluorescently labelled lipids to one another are varied in the lipid solution before extrusion through the filter. Using these methods ratiometrically labelled liposomes with sizes typically in the range 50 nm to 300 nm may be made.

Alternative methods of manufacture of unilamelar liposomes include ultrasonication, detergent removal and electroformation, details of which are readily available in reference 10 and the scientific literature.

Insertion of Membrane Protein Targets into Encoded Carrier Particles Various methods to insert (reconstitute') membrane proteins into liposomes are known to the art and descriptions of these methods are readily availble in the scientific literature; reference 11 reviews some of the main methods in some detail.

One example approach known to the art and described in detail in reference 11 uses detergents to solubilise preformed liposomes to varying degrees. The protein to be inserted is added to the liposome-detergent solution and the detergent is then gradually removed by, for example, dialysis. As the detergent is removed the liposomes reform and the membrane proteins insert into the lipid bilayer shell of these. The precise details of this process will depend somewhat on the exact nature of the membrane protein to be inserted.

Fluorescence Correlation Spectroscopy for Read Out of Encoded Liposome Assay with Fluorescent Test Ligands Fluorescence correlation spectroscopy is a well developed technology and a considerable body of literature exists regarding the theory and practical application of the technique. Example reviews are provided in references 12 and 13. Suitable instrumentation is commercially available or may be constructed by those knowledgeable in the art.

Fundamentally FCS measures the speed at which particles/molecules diffuse in solution 17 as a result of Brownian motion. The speed with which the solute particles diffuse at a given temperature depends principally on their size. Large particles diffuse more slowly than small particles and so by measuring the speed with which a given solute particle moves in solution its size may be determined. This may be used to distinguish between relatively small ligand molecules that are free in solution (and so moving fast) and ligand molecules that are bound to liposomes by a target receptor (and are therefore moving more slowly).

Experimentally FCS is commonly performed by focussing a laser beam into the sample solution with a high numerical aperture microscope objective. This laser excites fluorescence in suitably dyed molecules in the solution and this fluorescence is collected by the microscope objective. The collected fluorescence is passed through a small pinhole and then passed to an optical detector capable of counting single photons. The use of the pinhole ensures that only light from a very small volume of the sample centred on the focus of the exciting light reaches the detector (this is described as a confocal system). If a fluorescent molecule or particle passes through the focal volume then a burst of photons will be detected, If the particle is relatively large then the rcsulting burst will be long as it moves slowly across the focal volume whereas if the particle is sma the resulting burst of photons will be short as it quickly passes through the focal volume.

The invention described herein requires that multiple wavelength emissions from the focal volume are simultaneously detected. A minimum of two channels are required to allow read-out of the liposome encoding and another to detect the emission from fluorescently labelled ligands. This is readily achieved using suitable optical filters and multiple detectors, one for each wavelength required. Where more than one exciting wavelength is required, the output from two or more different types of laser may be arranged such that they are simultaneously focused to the same point in the sample solution.

In practice the temporal properties of the signal from the photodetectors are typically analysed by calculating the auto-correlation function of the signal and then fitting suitable models to this to extract the transit times of the fluorescent species through the excitation focal volume. Suitable electronics and software to perform this function are commercially available or may be constructed by those knowledgeable in the art.

The ratios of the intensities of the fluorescence from the different labelling agents used to encode the carrier particle may simultaneously be determined by comparing the 18 number of photons counted per unit time in each of the relevant detector channels as the particle transits the excitation focal volume. In this way the species of carrier particle may be identified and so the identity of the target receptor or receptors bourn on that carrier particle may be known.

Where the ligand fluorescence signal is identified as originating from bound tigands, the number of ligarids bound may be determined from the number of photons emitted per unit time from in the ligand labelling agent detection channel as they pass through the excitation focal volume.

It may thus be appreciated that the described instrumentation may then be used to simultaneously read' and distinguish between the ratiometrically labelled liposomes and to detect and distinguish between bound and unbound fluorescently labelled ligands and to determine how many ligands have bound to a given carrier particle.

Additionally, different sized liposomes may be distinguished from one another if required allowing the potential for size encoding of the carrier particles in addition to the ratiometric fluorescence encoding.

It will be appreciated that the described instrumentation may also be used to interrogate assays based on fluorescently encoded beads already known to the art and thus enables the readout of an assay composed of various different types of encoded carrier particle.

Coincidence or Cross-Correlation Spectroscopy for Read Out of Encoded Liposome Assay with Fluorescent Test Ligands The technique of fluorescence coincidence spectroscopy is known to the art. Alternative names for this and related techniques include dual colour fluorescence cross-correlation spectroscopy (DC-FCCS) and dual colour cross correlation analysis. Details of the technique may be found for example in references 14 and 15 and references therein.

Briefly, to perform DC-FCCS an experimental arrangement essentially the same as that for multi-channel FCS described herein is required. The autocorrelation functions for each of the two spectral channels is calculated as with the FCS described previously. In addition for DC-FCCS the cross-correlation function is obtained by correlating intensity fluctuations of both channels with each other. Essentially this cross correlation function indicates whether the two species of particle monitored by each of the fluorescence 19 channels are passing through the excitation focal volume together. In the case of the invention described herein this will reveal whether the labelled test ligand has bound to a target receptor bourn on a fluorescently encoded carrier particle. The ratios of the intensities of the fluorescence from the different labelling agents used to encode the carrier particle may simultaneously be determined by comparing the number of photons counted per unit time in their detection channels as the particle transits the excitation focal volume. In this way the species of carrier particle may be identified and so the identity of the target receptor or receptors bourn on that carrier particle may be known.

The number of photons emitted per unit time from ligand labelling agents that are cross- correlated with the emissions from the carrier particle indicates how many ligands have bound to the target receptors on a given carrier particle. DC-FCCS retains the benefits of FCS and so different sized carrier particles may also be distinguished with this technique.

Where there is a large difference in size between the carrier particle and the test ligand and at sufficiently low solute concentrations a simpler analysis of the data may optionally be used which may be described as ratiometric photon burst analysis (rmBA) in which the number of photons, duration and synchronicity of photons detected on the various detection channels are analysed directly without prior calculation of the autocorrelation function of the individual channels. In this binding of fluorescently labelled ligands to the liposomes are detected by looking for instances where the burst of photons in the liposome channel is the same length of the burst form the ligand channel.

Ratiometric Burst Analysis Read Out of Fluorescently Encoded Carrier Particles and Unlabelled Ligands This technique is similar to the ratiometric burst analysis read out described herein before for the case where the ligands are fluorescently labelled. In this instance, however, the ligands are not fluorescently tagged and are detected by some other optical means. The method selected to detect the presence of a ligand will depend on the photo-physical propertied of a given ligand but can be selected from a group comprising intrinsic fluorescence, Raman or nonlinear Raman, Raman optical activity, absorption or other spectroscopy. As with the read out mechanisms describedjreviouslv the identity of the target receptor is determined from the simultaneous measurement of the intensity ratio of the carrier particle's fluorescent labels. Bound Jigands are identified by the temporal coincidence of the tigand signal with carrier particle encoding signal.

Unbound ligands may be distinguished from bound ligands due to their shorter transit times through the focal volume and their lack of temporal coincidence with the carrier particle fluorescence signal.

Use of Flow Cytometer Type Systems to Read Out Assay Based on Encoded Liposome Carrier Particles with Fluorescently Labelled Test Ligands.

Proprietary bead based assay read out devices based on flow cytometer type instruments may be used in conjunction with the encoded carrier particles described herein. These instruments typically flow the sample solution down a confined channel such that the carrier particles pass through a small detection volume one at a time. Light from one or more lasers is focussed into this detection volume and any resulting fluorescence is collected and sent to a number of spectrally distinct detection channels.

The identity of the particles is determined from the ratio of the intensities of any labelling agents in the carrier particle while simultaneous detection of a signal from a fluorescently labelled ligand is taken as evidence that a binding event has taken place.

Use of Encoded Carrier Particles with Unlabelled Ligands: Displacement and Sandwich Type Assays Where it is not desirable or possible to fluorescently label the test ligand then either a displacement (competitive) or sandwich type approach to detecting ligand binding to the target receptors may be employed. Both displacement and sandwich approaches to detecting unlabelled ligand binding are well known and widely used in biological assays and may be adapted for use in the current invention.

In a displacement or competitive assay the target molecules carried on the encoded liposomes have suitable fluorescent ligands bound to them before exposure to the test ligands. Test ligands that have a strong affinity to a given target receptor will displace 2 1 the fluorescent ligands and bind to the receptor. This can be detected usinq the multi- channel FCS approach described above; where no binding of the test ligand has taken place the signal from an encoded liposome passing through the focal volume of the FCS instrument will consist of two or more fluorescent ratiometric encoding channels to identify the target molecule plus a large coincident signal from the bound fluorescent ligands; where the test ligand binds strongly to a given target molecule in the assay the fluorescent ligand will be displaced and the intensity of this channel will consequently be reduced or extinguished. As described previously the differences in diffusion speed as revealed by the FCS instrument may be used to distinguish between bound and displaced fluorescent ligands.

In an approach similar to a conventional sandwich type assay the test ligand(s) are added to the target molecules inserted into to their respective encoded carrier particles and given the opportunity to bind. Additional suitably engineered fluorescently labelled ligands which will bind to a different part of the test ligand to that involved in binding to the target molecule are added to the solution. The test ligands are therefore effectively fluorescently labelled and those that are bound to a target molecule may be distinguished from those that are free in solution using FCS as previously described.

Use of Encoded Carrier Particles with Unlabelled Ligands: Sorting of Target species and Subsequent Analysis of Bound Ligands.

Where the test ligands are unlabelled the ratiometnc labelling of the liposome carrier particles may be used to sort and separate the different target species and subsequent analysis may be used to detect which test ligands have bound to each target.

In this approach the test ligand(s) are added to a solution containing a multitude of test molecules in or on their respective encoded carrier particles. Sufficient time is then allowed to let binding take place and then the liposomes are sorted and separated according to their ratiometric fluorescent code. In this way each species of target molecule is isolated and any one of a range of analytical techniques known to the art may be used to detect and quantitate the presence of target ligand that has bound to that particular target receptor. Examples of such analytical techniques for the detection of the bound ligand include mass spectrometry, various forms of chromatography, optical absorption spectroscopy and Raman spectroscopy. Where the analytical technique allows discrimination between different test ligands then multiple test ligands may be used simultaneously in the assay.

The sorting arid separation of the encoded liposomes may be performed with a multiple optical channel fluorescence activated cell sorter' (FACS) which are available from a number of vendors. Alternatively a number of microfluidic devices which can perform this function have been described in detail in the scientific literature.

Detailed Description of the Invention

With reference to Figures 1A-D there is shown a carrier particle (1), comprising a number of lipid or lipophilic molecules (2) that form a shell and enclose a core (3). In the embodiment shown in Figure 1 A the core surrounded by the lipophilic shell is solid whereas in Figures 1 B, C and D the core is fluid-filled by, for example, an aqueous solution such as a buffer or by a hydrogel. Thus in all embodiments the carrier particle outer surface or shell provides a lipid or lipophilic environment similar to that experienced by a membrane protein in situ in the cell membrane. In this way the carrier particle surface will allow a target receptor such as a membrane protein or other insoluble protein to become attached or bound without chemical attachment or alteration, as a result the membrane protein remains substantially unaltered and retains functional properties as it would in its native environment.

Carrier particle (1) is ratiometrically labelled with multiple and distinct fluorescent labelling agents such as flourophores (4) and (5). The fluorescent labelling agents can be attached to the shell surface, reside in it or even be contained within the core (3) providing it is of the fluid-filled variety along with other entities such as a biosensitive dye. In the depiction of Figures IA-D of the carrier particles only two different types of flourophores are shown, however the carrier particle may be labelled with more than two types of fluorescent labels. Referring to Figure 1A where the carrier particle comprises a solid microsphere coated in a lipid or lipophilic shell, the ratio of the first fluorescent labelling agent (4) to the second fluorescent labelling agent (5) is 3:1. Similarly, the fluid filled carrier of Figure lB has a ratiometric value of 3:1, whereas the carrier particle of Figure 1 C has a ratiometnc value of 2:2 and Figure ID a value of 1:3.

The carrier particle also comprises a target receptor (6) that resides in, or on, the surface or shell of the carrier particle. In Figure 2A, the carrier particle has a ratiometric value of a first to second fluorescent labelling agent of 3:1 and so is the carrier particle depicted in Figure 1 B, this carrier particle carries target receptor (6a) and so any binding 23 event with target receptor (6a) can be tracked back or attributed to the encoded carrier particle of Figure 1 B. As regards the carrier particles of Figures 2B and 2C, these show the ratiometrically labelled carrier particles of Figures 1C and 1D carrying target receptors (6b) and (6c) respectively. As described above any binding event with a carrier particle of 2:2 or 1:3 ratiometric value can therefore be attributed to an interaction with target receptor (6b) or (6c) respectively.

In use, and in one method that is applicable to the detection of binding events (see Figure 3A), encoded carrier particles carrying target receptors (6a, 6b or 6c) are combined in a multiplex solution assay with fluorescently labelled test ligands (7) that carry a further fluorescent labelling agent that is distinct from either fluorescent labelling agents (4) and (5). Thus when the fluorescent read-outs are decoded for fluorescent labelling agents (4):(5):(7) the ratiometric value for carrier particles carrying target receptor (6a) will be 3:1:0, for the carrier particles carrying target receptor (6b) it will be 2:2:0 and for carrier particles carrying target receptor (6c) it will be 1:3:1, indicating that the third fluorescent labelling agent (7) is associated with the carrierparticle carrying the target receptor (6c). Such a read out indicates that the fluorescently labelled test ligand (7) binds to receptor (6c) but not to (6a) or (6b).

It will be appreciated that other methods for detecting binding events where the test ligand is not fluorescently labelled may also be employed to detect binding events. For example, in a solution based biological assay, with reference to Figure 3B, there is shown a mixture of encoded carrier particles as hereinbefore described which have been pre-mixed with a fluorescent ligand (9) that binds to a non-specific part of the unlabelled test ligand (8). The test ligand (8) and carrier particles are mixed in solution and the read out data decoded as before by the ratio of fluorescent labelling agents (4):(5):(9) so that the ratiometric value for carrier particles carrying target receptor (6a) will be 3:1:0, for the carrier particles carrying target receptor (6b) it will be 2:2:0 and for carrier particles carrying target receptor (6c) it will be 1:3:1. The decoded read out indicates that the unlabelled test ligand (8) with fluorescent ligand (9) binds to receptor (6c) but not to (6a) or (6b).

In a yet further method for detecting binding events, as shown in Figure 3C, ratiometrically labelled carrier particle carrying target receptors (6a), (6b) or (6c) are further labelled on each target receptor with a fluorescent ligand (10) that can be displaced by test ligand (8). Thus when test ligand (8) and carrier particles are mixed in 24 solution and the read out data decoded as before by the ratio of fluorescent labellina agents (4):(5):(1O), the ratiometric value for carrier particles carrying target receptor (6a) will be 3:1:1, for the carrier particles carrying target receptor (6b) it will be 2:2:1 and for carrier particles carrying target receptor (6c) it will be 1:3:0. The decoded read out indicates that the unlabelled test ligand (8) displaces fluorescent ligand (10) from the target receptor and so binds to receptor (6c) but not to (6a) or (6b).

Figures 4A-D shows a flow diagram of a method of sorting encoded carrier particles and subsequent detection of bound unlabelled test ligands. In Figure 4A ratiometrically labelled carrier particle carrying target receptors (6a), (6b) or (6c) are mixed with unlabelled test ligands (1 1), in Figure 4B the test ligand (11) binds to one of the target receptors carried by the carrier particle, in this depiction the test ligand binds to the carrier particle carrying target receptor (6c). Unbound test ligands are then removed or washed away and fluorescence colour-coding is used to sort the various carrier particles (Figure 4C). The bound test ligands are then dissociated from the carrier particles and the resultant products of Figure 4D analysed to detect the presence of test ligands, as will be apparent the test ligand is only identified from the dissociation products from carrier particles carrying target receptor (6c) hence the test ligand only binds to target receptor (6c) but not to receptors (6a) or (6b).

With reference to Figure 5A-D, there is shown a flow diagram of a method of sorting encoded carrier particles combining multiplex targets receptors with mutiplex test ligands and subsequent detection of which bound unlabelled test ligand binds to which receptor.

Ratiometrically labelled carrier particle carrying target receptors (6a), (6b) or (6c) are mixed with mixture of unlabelled test ligands (11) (12) and (13), in Figure 5B the test ligand (11) binds to the target receptors carried by the carrier particle (6c), test ligand (12) binds to the target receptors carried by the carrier particle (6b) and test ligand (13) binds to the target receptors carried by the carrier particle (6a). Unbound test ligands are then removed or washed away and fluorescence colour-coding is used to sort the various carrier particles (Figure 5C). The bound test ligands are then dissociated from the carrier particles and subjected to a separation process, the resultant products of Figure 5D are then analysed to identify which test ligand is associated with which carrier particle target receptor.

In Figure 6A-D, a further more complex scenario is depicted than that of either Figures 4 or 5. Following the same process as that described above, it can be deduced that the carrier particle carrying target receptor (6a) does not bind any of the test!igands

F

whereas the carrier particle carrying target receptor (6b) is capable of binding test ligands (11) and (13) and the carrier particle carrying target receptor (6c) is capable of binding all test ligands i.e. (11) (12) and (13).

With reference now to Figure 7, there is shown a specific embodiment of the invention using labelled test ligands in a fluorescent correlation spectroscopy method.

Ratiometrically labelled carrier particle (14) carries a target receptor (18) which is not capable of binding to a fluorescently labelled test ligand (16), whereas ratiometrically labelled carrier particle (15) carries a target receptor (19) which is capable of binding to a fluorescently labelled test ligand (16).

A range of carrier particles of either those depicted in Figure 1A or lB may be used, that is to say the carrier particle core may either be fluid filled or solid and each may carry at least one or more target receptors with different fluorescent labelling agent ratios or intensities, these are then mixed together in solution to which one or more fluorescently labelled ligands are added for the assay.

Two example detection schemes will be described: rmFCS as described hereinbefore and an alternative method suitable for both labelled and unlabelled ligands which will be referred to as ratiometrically labelled burst analysis or rmBA.

With reference to figure 7 there is shown a schematic representation of the rmFCS screening method. Labelled carriers (14 and 15) with receptors (18 and 19 respectively plus free ligands (16) labelled with a fluorescent labelling agent f3 (21a) are mixed together in solution. The concentration of the freely diffusing solutes is kept low to ensure that, on average, only one or a few carrier particles, at a time diffuses through the focussed laser (17). Ratios of fluorescent labelling agents or flourophores f1(20) and f2(21) indicate which of the receptors (in Figure 7 one that is not capable (18) or one that is capable (19) of binding to the ligand) has passed through the laser beam. The system has three detector channels, two to measure the ratio of f1 to f2 as the particles diffuse through the laser beam which identifies the receptor and the third channel to measure the photon burst from f3 (the fluorescent labelling agent on the ligand). The time taken for a carrier particle or free ligand to pass through the laser beam is measured directly from the length of the burst of fluorescence or by calculation of auto- or cross-correlation functions of one or more of the fluorescence signals from the three 26 detector channels and fitting of these functions to appropriate theoretical mpdels.The unbound ligand (16) has the fastest "transit time". The "transit time" is the time taken to traverse the shortest route through the beam (17) and can be calculated from a correlation analysis of the fluorescence signals. Particle (14) which is a carrier particle with a receptor (18) that does not bind the ligand will not produce a signal from f3 since there is no bound ligand, and particle (15) which is a carrier particle with a receptor (19) that does binds ligand (16) will show a signal from f3 with a transit time slower than the free ligand thus indicating that binding has occurred and the ratio of f1 to f2 signals indicates which receptor has bound the ligand.

Simulated data from the rmFCS detection scheme described above is depicted in Figures 8A-G in which the signals in each of the three fluorescence detection channels are shown for different possible combinations of particles that may pass through the focal volume. In the plots showing the readout from the three fluorescent channels x axis corresponds to time and the y axis corresponds to intensity of the fluorescence. Figure 8A shows the read out in all three channels from a solution containing only the labelled free ligand (16) in which short bursts of light are periodically observed in channel f3 as the labelled ligands move in and out of the focal volume. Figure 8B shows the read outs for all three channels from a solution containing a carrier particle that has a receptor (18) that does not bind ligand (16) and so is in the unbound state; in this longer bursts of fluorescence of equal intensity are observed in channels fi and f2 (22 and 23 respectively) as encoded carrier particles move relatively slowly in and out of the focal volume. Figure 8C shows the read out (22 and 24) of a carrier particle with a receptor (19) that is capable of binding ligand (16) but which is in an unbound state. In this instance the ratio of the intensity of the bursts in the fi and f2 channels are different to that observed in figure 8B and this may be used to identify the fact that different species of receptor are carried on the particles shown in Figures 8B and 8C. Figure 80 shows the readout of a combination of the scenario described in Figures 8A and 88 and Figure 8E shows the readout of a combination of the scenario described in Figures 8A and 8C.

Figure BE shows the readout for a carrier particle with a single receptor (19) which has bound to the ligand (16) in which the slower transit time of the bound Iigand through the focal volume, compared with that of the free ligand, can be observed from the signal in the f3 channel. Additionally it can be observed that the signal in the f3 channel is coincident with that in the fi and f2 channels indicating the ligand is passing through the focal volume bound to the carrier particle. Figure 8G shows the readout for a carrier particle with two receptors (19) each bound to ligands (16) plus a free ligand of Figure 27 8A. In this case the signal from the free ligand may be distinguished from that of the bound ligand by its different temporal characteristics. Also indicated in Figure 8G is the increase in intensity in the f3 channel when multiple ligands bind to a carrier particle illustrating how the number of ligands bound to a given carrier particle may be quantitatively determined from the intensity of the bursts of fluorescence in the f3 channel.

It will be appreciated from the foregoing that the carrier particles of the present invention may be labelled with more than two flourophores and that they may also carry multiple receptors that are identical or the or different.

It will also be appreciated from the forgoing that the rmFCS technique is also a suitable readout mechanism for existing homogeneous/multiplex assays which employ fluorescently encoded carrier particles.

The second example method of detection is rmBA.. In this method the detection of coincident signals of the same duration in three channels f1, f2 and the third channel f3 is used to indicate a binding event. Such a situation is illustrated in Figure 8G. In an alternative arrangement the carrier particle is fluorescently encoded as described above but the ligand is unlabelled and its presence in the focal volume is detected by an optical means selected from the group comprising intrinsic fluorescence, Raman or non-linear Raman, Raman optical activity, absorption or other spectroscopy. In this the fluorescent channel f3 is replaced by a channel d3 which contains the signal from the optical technique employed. Channels f1 and f2 provide the same ratiometric fluorescence labelling of the carrier particles as in the rmFCS method and therefore identify each carrier particle and their receptors. An unbound ligand will generate a signal in channel d3 but since its passage through the laser beam will be of a different duration than that of a carrier particle, the signals in d3 and f1 and f2 will not be of the same duration. The signal from a ligand bound to a carrier particle will produce bursts of signal from the three channels that are of the same duration since they diffuse together through the laser beam and thus the binding event and receptor can be identified by the rmBA method.

It will also be appreciated from the forgoing that the rmBA technique is also a suitable readout mechanism for existing homogeneous /multiplex assays which employ fluorescently encoded carrier particles.

In a further example of the utility of the fluorescently encoded liposomes in assaying the presence of non-fluorescently labelled ligands a multitude of target receptors are carried on the encoded carrier particles in solution as described previously. One or more test ligands are added to the solution and given the opportunity to bind to the target receptors. The different types of target receptor are then physically separated from one another using the ratiometric fluorescent encoding of the carrier particles to identify and distinguish between the different target receptors and thus enable sorting. This sorting may be achieved using devices known to the art such as flow cytometry cell sorters and microfluidic devices (refer to Figures 4, 5 and 6). Once the different target receptor species have been separated then these may be interrogated using analytical methods known to the art to detect the presence of test ligands which have bound to the target receptor(s). Suitable techniques to achieve this, include, but are not limited to mass spectroscopy, HPLC, optical spectroscopy, Raman spectroscopy.

The methods of the present invention advantageously completely obviate the need for any immobilisation in a 2D array by using multicolour ratiometric labelling of carrier particles. Interrogation of a biological assay constructed using these encoded carrier particles using fluorescent correlation spectroscopy or burst analysis confers greater sensitivity and instrumental simplicity than existing homogenous assays. Assays may be constructed by employing multiple (in the region of 102_ 1 0) species of carrier particle each carrying at least one target receptor, in solution with either fluorescently labelled ligands or ligands without extrinsic labels. Fluorescent labels on the carrier particles identify which target receptor(s) to which the ligand binds and the detection of transit times using correlation spectroscopy or burst analysis indicates the binding has taken place. As hereinbefore stated the methods are particularly suitable for screening interactions or binding events of ligands with insoluble proteins and especially membrane proteins which are problematic to screen using conventional 2D array chip techniques but are of particular importance in biological function, drug discovery and diagnosis.

EXAMPLE I

Experiments have been performed to demonstrate the ratiometric fluorescence labelling of lipid vesicles. Lipid vesicles were prepared using the following materials: Un-dyed lipid: 1,2-Dioleoyl-sn-Glycero-3Phosphocholine (DOPC) (Avanti Polar Lipids) Dyed lipid A: - $Bodipy 500/510 C5-HPC (Invitrogen) and Dyed Lipid B: Bodipy 558/568 C12 (lrivitrogen).

The two dyes used in this experiment are commonly referred to as a fluorescence resonance energy transfer (FRET) pair. This means that the following process can occur: Dyed lipid A absorbs light at 475 nm and then re-emits this at 516 nm; some of this 516 nm emission is absorbed by dyed lipid B which re-emits this at -572 nm. This process helps enable the use of a single excitation wavelength to excite two dyes that emit at different wavelengths. The use of a FRET pair is not, however, required to make the technique work and is not to be construed as a limitation of the methods of the invention.

The extrusion method of preparation of unilamellar vesicles was used and is well known to the art (reference 16). The three species of lipid were weighed in the desired ratios and dissolved in 1-2 mL of chloroform in glass vials. The DOPC was at a concentration of 10 mgfL while the dyed lipids were at concentrations in the range 10- 100pM range dependent on the labelling ratio of choice. The resulting solution was then evaporated using a dry nitrogen stream in a fume hood. This process resulted in a lipid film being formed around the inside of the glass vial. Following evaporation to remove the chloroform, hydration followed. 1-2 m L of hydration buffer (100 mM NaCI, 1 mM EDTA and 10 mM TES, pH 7.4) was added whilst agitating the vial over a period of 10-20 minutes. The hydration step resulted in large, multilamellar vesicles (LMV) which were then downsized to large, unilamellar vesicles (LUVET) using the extrusion technique. A proprietary extruder was used (Avanti Polar Lipids, lnc) in combination with a polycarbonate membrane of pore size 100 nm. The hydrated lipid mixtures were loaded into gas tight glass syringes (Hamilton Company) and then passed through the filter >15 times. The resulting -100 nm unilamellar vesicles in aqueous solution were then transferred to clean vials for fluorescence measurements.

Dynamic light scattering was used to measure the size of liposomes prepared in this way. These measurements confirmed a narrow distribution of particle size centred around 100 nm indicating that - 100 nm diameter lipid vesicles had been successfully formed.

Four example batches of vesicles were produced. Different proportions of dyed lipid type A to Dyed lipid type B in the lipid solution to be passed through the extruderwere used for each batch. In this way four different fluorescently encoded species of liposome were produced. The composition of each of the four different species is detailed in table 1 below.

Table 1 shows the varying proportions of the three different lipids used to make - 100 nm liposomes using extrusion technique. Also shown in table is the measured ratio of the fluorescence intensity for the four different liposome species produced.

Vesicle Concentratio Concentratio Concentratio Ratio Fluorescenc Species n n n Dye e # Undyed Lipid Dyed Dyed A:Dye B Intensity LipidA Lipid B Ratio _______ _____________ (em. 516 nm] [em. 572 nm] _______ 1 lOmg/mL 10jiM 10MM 1:1 3.2 2 10 mg/ mL 10 jM 20 jM 2:1 1.56 3 10 mg/ mL 20 zM 2 M 10:1 0.64 4 lOmg/mL 40M I jtM 20:1 J 0.15 The fluorescence emission spectra from each of the four species of vesicle, when excited at 475 nm, was recorded with a fluorimeter (Shimadzu RF-5301). The resulting spectra are presented in Figure 9. It can be seen from these spectra that the ratio of the emission at 516 nm to that at 572 nm varies depending on the relative proportions of the two types of dyed lipid used to make the liposomes.

We have demonstrated that by having a sensitive fluorescence detection system with two wavelength channels, in this example one set to detect 516 nm emission and the second to detect 572 nm emission, the ratio of the emission intensity of the two dyes may be measured and this then used to identify the species of liposome.

EXAMPLE 2

We have been able to show an experimental demonstration of the principal of discriminating free and bound fluorescently labelled ligand based on differences in their diffusion speed in solution as measured with fluorescence correlation spectroscopy.

Fluorescence autocorrelation spectroscopy (FCS) is a technique known to the art and allows the measurement of the speed of diffusion of fluorescent or fluorescently labelled species in solution. 31 30

In the present invention, fluorescently labelled ligands may either be free in solution or alternatively bound to a receptor in an encoded liposome and it is necessary to distinguish between these two states. The free ligand will have a small mass and hydrodynamic radius and thus will diffuse relatively fast in solution. The liposomes by contrast will diffuse much more slowly due to their much larger size and mass. Ligands bound to a liposome will diffuse at the same speed as the liposome and will therefore be observed to diffuse more slowly than free ligands.

FCS measurements were obtained from samples of freely diffusing dye molecules and -100 nm diameter lipid vesicles labelled with low concentrations of fluorescently dyed phosphotipid. The FCS instrument employed to make the measurements presented herein is described in detail in Handbook of Single Molecule, Fluorescence Spectroscopy by Christopher Gell, David Brockwell and Alastair Smith, Oxford University Press, 2006.

Figure 10 A shows representative plots of the autocorrelation function, G(r) of the recorded data against time, for free dye in solution and lipid vesicles at 0.001 % dyed phospholipid content. Figure 10 B shows - 100 nm diameter liposomes labelled with 0.001% of dyed lipid. The thin, noisy line is the recorded auto correlation function and the smoother, thicker line is a double exponential fit to the data. By fitting to the recorded data it is possible to determine the diffusion times of the free dye and the dye labelled liposomes through the focal volume of the FCS instrument.

These measurements indicate that the diffusion time for the free dye is 0.03 ms while for the liposomes it is - 5 ms. Thus, the free and bound dye may be distinguished from one another based on the large difference in their diffusion speed. The two channel FCS instrument may also be used to read the ratiometric fluorescence encoding on individual vesicles as they pass through the focal volume of the instrument. This was demonstrated as follows.

Liposomes of - 100 nm diameter were prepared with different ratios of labelled lipids A and B and placed in the FCS instrument. Figure 11 shows a graph with FRET efficiency (effectively the ratio of fluorescence intensity of dye B to that of dye A) on the X axis and number of occurrences during the measurement time on the Y axis. This measurement demonstrates that individual encoded vesicles can be distinguished based on the different measured ratios of the fluorescence intensity from the two labelling dyes incorporated in the liposome.

With reference to Figure 11 there is shown the results of measurements from different species of ratiometrically fluorescence encoded liposomes with a high sensitivity, 2 channel, confocal fluorescence instrument of the type suitable for FCS experiments. The histograms show the FRET efficiency (effectively the ratio of fluorescence intensity of the two labelling dyes) against the number of occurrences during the time period of the experiment. The discreet distributions in the histogram for the three example different ratios of dyed lipid indicates that the instrument is capable of distinguishing different species of encoded liposome from one another. Measurements from -100 nm liposomes with 1:1, 2:1 and 20:1 dyed lipid B (emission at 572 nm) to dyed lipid A (emission at - 516 nm) are shown. Gaussian curves have been fitted to each of the distributions.

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12. Fluorescence correlation spectroscopy J.D. Muller Chen,Y.; Gratton,E. Methods in Enzymology v. 361: Biophotonics p. 69 ed. G.Marriott, l.Parker (2003) 13. Fluorescence Correlation Spectroscopy N.L.Thompson In Topics in Fluorescence Spectroscopy 1: Techniques, p. 337-378, J.R. Lakowicz, ed. (1991) 14. "Confocal fluorescence coincidence analysis: An approach to ultra high- throughput screening" T. Winkler, U. Kettling, A. Koltermann, and M. Elgen PNAS v.96 (4), p.1375 (1999) 15. Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusionat analysis in solution P. Schwille., F.J. Meyeralmes, R.Rigler, Biophys. J., V. 72, 1878 (1997) 16. MacDonald, R. C., R. I. MacDonald, B.P. Menco, K. Takeshita, N.K. Subbarao, and L. R. Hu. (1991). Small-volume extrusion apparatus for preparation of large, unilamellar vesicles, Biochim Biophys Acta 1061: 297-303). H 36

Claims

Claims 1. A carrier particle comprising a lipid or lipophilic outer

surface or shell and attached thereto or incorporated therein or contained within, at least two different fluorescent labelling agents present in defined ratiometric amounts, the at least two different carrier particles being distinguishable by the different ratios of their labelling agents, the carrier particle further comprising at least one receptor target, being either attached to or incorporated in the outer surface or shell of said carrier particle.
2. A carrier particle according to claim 1 wherein the lipid or lipophilic outer surface or shell of the carrier particle comprises a lipid or mixture of lipids selected from the group comprising glyco-, sphingo-, phospho- lipid.
3. A carrier particle according to either claim 1 or 2 wherein the lipid or lipophilic outer surface or shell of the carrier particle also contain a moiety selected from the group comprising cholesterol, proteins, peptides or any other moiety that will reside in a lipid membrane.
4. A carrier particle according to any preceding claim wherein the at least two fluorescent labelling agents can each or separately be carried on the surface of the shell or in the shell itself or be enclosed within the shell.
5. A carrier particle according to any preceding claim wherein the ratiometric fluorescent labelling agent is a fluorophore or a fluorescent dye or an inorganic fluorescent entity.
6. A carrier particle according to claim 5 wherein the fluorophore or fluorescent dye or inorganic fluorescent entity is lipophilic or conjugated to a lipophilic agent and capable of integration into a lipid membrane or chemical attachment to the lipid or lipophilic surface or shell of the carrier particle.
7. A carrier particle according to any preceding claim wherein the intensity or level of labelling with the at least two fluorescent labelling agents is varied.
8. A carrier particle according to any preceding claim that is a microspherical particle or microsphere or any other shape that retains the functional capability of

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carrying at least one target receptor and at least two or more ratiometrically labelled fluorescent agents.
9. A carrier particle according to any preceding claim wherein the lipid or lipophilic shell contains within it or surrounds a solid or fluid or gel.
10. A carrier particle according to any preceding claim that is in the form of a vesicle.
11. A carrier particle according to any preceding claim that is in the form of a liposome.
12. A carrier particle according to any of claims 9 to 11 wherein the fluid filled interior of the lipid vesicle or liposome carrier particle comprises any one or more of the group comprising a buffer, an aqueous solution which optionally may contain a biochemically sensitive agent, a hydrophilic environment and a hydrogel.
13. A carrier particle according to any one of claims 1 to 9 wherein lipid or lipophilic shell is a monolayer or bilayer and it surrounds a solid particle.
14. A carrier particle according to claim 13 wherein the solid particle is made of latex or glass or a synthetic polymer material.
15. A carrier particle according to any preceding claim wherein the lipid or lipophilic shell is uni-lamellar or multi-lameflar.
16. A carrier particle according to any preceding claim having a diameter in the region of lOnm to SOpm.
17. A carrier particle according to claim 16 having a diameter in the region of 0.lpm tolOpm.
18. A carrier particle according to claim 17 having a diameter in the region of 0.4pm to 1 pm.
19. A plurality of carrier particles according to any preceding claim comprising a 2 mixture of: (I) particles with solid or fluid filled cores of the same or different diameters (ii) fluid filled particles of the same or different diameters or; (iii) solid core particles of the same or different diameters.
20. A carrier particle according to any preceding claim wherein the at least one target receptor carried by the carrier particle is identifiable by the ratiometric proportion of the fluorescent labelling agent attached thereto, incorporated therein or contained thereby.
21. A carrier particle according to any preceding claim wherein the at least one receptor target carried by the carrier particle is capable or incapable of binding a ligand.
22. A carrier particle according to any preceding claim wherein the carrier particle is provided with more than one identical or different target receptors.
23. A method of making a carrier particle comprising: (I) providing a lipid vesicle or coating a solid microsphere in a lipid or mixture of lipids so as to provide, in both instances, a carrier particle comprising a lipid or lipophilic outer surface or shell; (ii) incorporating in or attaching to an outer shell of a carrier particle at least one target receptor; and (iii) impregnating or surface labelling the outer shell of the carrier particle or filling the core of the carrier particle when in the form of a lipid vesicle with a ratiometric mixture of at least two fluorescent labelling agents.
24. A method according to claim 23 further including any one or more of the features of claims 1 to 22.
25. A method according to either claim 23 or 24 wherein a mixture of carrier particles of either solid cored lipid coated microspheres or lipid vesicles with different target receptors and/or different fluorescent labelling agent ratios are prepared and mixed together in solution.
26. A method according to any of claims 23 to 25 wherein in the instance where the carrier particles comprise lipid vesicles with a fluid filled interior they are formed by 3 extrusion through apertures of sizes ranging from lOnm to 50 pm or sonication of a solution of the lipid or mixture of lipids and fluorescent labelling agents plus the target receptors in solution.
27. A method according to any of claims 23 to 26 wherein in the instance that the carrier particles comprise lipid vesicles with a fluid filled interior the target receptors are incorporated in the lipid shell of said vesicle by controlled solubilisation of the vesicles with detergent followed by controlled removal of the detergent.
28. A method according to any of claims 23 to 27 wherein in the instance that the carrier particle has a gel or solid core the lipid shell is coated onto the gel or solid core by adsorption or is attached by appropriate chemical anchoring groups.
29. A method of screening one or more test ligands in a fluid environment to identify which ligand(s) is/are capable of undergoing a binding event and the level of said binding event, with a mixture of carrier particles comprising a lipid or lipophilic outer surface or shell and attached thereto or incorporated therein or contained within, at least two different fluorescent labelling agents present in a ratiometric amount so as to generate a number of species of carrier particles identifiable by their fluorescent spectral properties, each carrier particle further comprising at least one receptor target, being either attached to or incorporated in the outer surface or shell of said carrier particle, the binding event and level of binding being identifiable by either homogeneous readout or by sorting and subsequent analysis.
30. A method according to claim 29 wherein multiple different species of receptor target, each carried on their own encoded carrier particles are identifiable by their unique ratiometric fluorescent code.
31. A method according to either claim 29 or 30 wherein the one or more test ligands is added to a mixture receptor targets borne by carrier particles such the ligands may bind or not bind to the various receptor targets.
32. A method according to any of claims 29 to 31 wherein the carrier particles are individually interrogated whilst still in solution to determine whether binding of the test ligand has taken place.
33. A method according to any of claims 29 to 31 wherein following exposure of the target receptors to the test ligands the ratiometric fluorescent encoding of the carrier particles is used to sort and separate the different target receptor species.
34. A method according to claim 33 wherein once each target species is isolated the total amount of ligand bound to that species is determined using mass spectrometry or optical spectroscopy.
35. A method according to any one of claims 29 to 34 wherein, in the instance where the method involves homogeneous read out, detection of binding events is obtained from fluorescent correlation spectroscopy, fluorescence cross correlation spectroscopy, burst analysis or microscopic video rate fluorescence imaging.
36. A method according to any one of claims 29 to 34 wherein, in the instance where the method involves sorting and subsequent analysis, the sorting is achieved by fluorescence activated flow cytometry or microfluidics and subsequent analysis is achieved by a method selected from the group comprising mass spectrometry, HPLC, intrinsic fluorescence, Raman or non-linear Raman spectroscopy, absorption or other spectroscopy.
37. A method to simultaneously screen a binding event and level of said binding event of a test ligand tagged comprising a first fluorescent label with multiple target receptors comprising: (I) mixing in solution multiple species of carrier particles, the first species of carrier particles carrying a first receptor or set of receptors and labelled with a specified dye ratio of at least a second and third distinct fluorescent labelling agent compared to the tagged ligand and further species of carrier particles carrying different target receptors or sets of receptors and each labelled with a specified dye ratio of the at least second and third fluorescent labelling agents with that dye ratio being unique to each species of carrier particle: (ii) adding the test ligand or ligands tagged with a first fluorescent label to the mixture: (iii) passing the mixture through an excitation light source and; (iv) differentiating between a free ligand and a ligand which has undergone a binding event.
38. A method according to claim 37 wherein the step comprising differentiating between the free ligand and one which has undergone a binding event employs fluorescent coincidence analysis or fluorescence cross correlation analysis to detect coincident or non-coincident fluorescence and similarities in temporal properties of the fluorescence from the ligand labelling agent and the carrier particle labelling agents so as to differentiate between a free ligand and a ligand which has undergone a binding event.
39. A method according to claim 37 wherein the step comprising differentiating between the free ligand and one which has undergone a binding event employs fluorescence correlation spectroscopy to determine the speed of diffusion of the ligand in solution.
40. A method according to claim 37 wherein the step comprising differentiating between the free ligand and one which has undergone a binding event employs video rate microscopic fluorescent imaging of the encoded carrier particles and labelled ligands in solution with spectrally distinct detection channels to look for spatial coincidence and co-ordinated motion of ligand and carrier particle fluorescence to distinguish between free and bound ligands.
41. A method according to any one of claims 37 to 40 wherein the step comprising differentiating between the free ligand and one which has undergone a binding event employs simultaneously use of the ratio of intensity of the second and third labelling agents to identify the species of a given carrier particle and thus the identity of the target receptor or receptors.
42. A method according to any one of claims 37 to 40 wherein the step comprising differentiating between the free ligand and one which has undergone a binding event employs simultaneously use of the intensity of the fluorescent emission from the labelling agents of test ligand molecules determined to have undergone a binding event to determine the number of test ligands bound to a given carrier particle.
43. A method according to any one of claims 37 to 42 wherein the fluorescence detection system has a number of detector channels commensurate with the number of different fluorescent labelling agents.
44. A method according to claim 39 wherein in the instance that fluorescence correlation spectroscopy is employed, transit time through an excitation light beam of the free or unbound ligand tagged with the first fluorescent labelling agent is determined by autocorrelation function of the fluorescence signal from the first labelling agent and fitting this function with an appropriate theoretical model.
45. A method according to claim 39 wherein in the instance that fluorescence correlation spectroscopy is employed, a ligand which is bound to a target receptor on or in a carrier particle is identified by slower diffusion in solution than an unbound, ligand due to the larger size and therefore slower diffusion of the carrier particle/ligand complex when compared with that of the free ligand.
46. A method according to claim 38 wherein in the instance that fluorescence coincidence analysis or cross-correlation analysis is employed a binding event is indicated by simultaneous detection of fluorescent signals from the first labelling agent identifying the ligand and the second and third labelling agents identifying the carrier particle, and where these signals display the same temporal characteristics indicating the same transit times through the excitation focal volume.
47. A method according to claim 46 wherein the ratio of the second and third fluorescent labelling agents which encode the identity of the different species of carrier particle are measured by the fluorescence spectrophotometer coincidently with the fluorescence from the labelled test ligand so as to identify to which species of carrier particle the ligand has bound.
48. A method according to claim 40 wherein in the instance of video rate fluorescence microscopic imaging being employed, the assay solution is contained in a receptacle such that the distance through the solution is small in the dimension parallel to the optical axis of the imaging system.
49. A method according to claim 48 wherein an area of the sample is illuminated, or a point of light rapidly scanned over the sample solution, such that fluorescence from the various labelling agents is excited and images are obtained of the sample solution simultaneously at discrete spectral wavelengths corresponding to the emissions from the various labelling agents.
50. A method according to either claim 48 or 49 wherein two or more images are recorded in quick succession such that the motion of the various fluorescent particles are observed and labelled ligand molecules that have undergone a binding event are identified by co-ordinated motion of their fluorescence signal with that of an individual carrier particle whereas unbound ligand molecules are characterised by more rapid, uncoordinated motion.
51. A method according to any of claims 37 to 50 wherein the solution into which the different species of carrier particle and ligands are allowed to freely diffuse is a buffered aqueous solution.
52. A method according to any of claims 37 to 51 wherein the concentration of the carrier particles and ligands in the solution is kept low to ensure that, on average, only a single carrier particle or ligand at a time diffuses through the excitation volume.
53. A method according to claim 52 wherein the total concentration of entities in the solution is in the range 1pM to lOOnM.
54. A method according to claim 53 wherein the total concentration of entities in the solution is in the range of 1pM - 1 nM.
55. Use of ratiometrically labelled carrier particles in detecting a binding event and level of binding between a ligand and at least one target receptor carried on or in the lipid or lipophilic surface of said carrier particle so as to diagnose the presence or absence of the ligand in a biological sample or to screen for candidate therapeutics.