GB2445579A

GB2445579A - An encoded microsphere

Info

Publication number: GB2445579A
Application number: GB0700531A
Authority: GB
Inventors: Robert Wilson
Original assignee: UK Secretary of State for Defence
Current assignee: UK Secretary of State for Defence
Priority date: 2007-01-11
Filing date: 2007-01-11
Publication date: 2008-07-16
Also published as: WO2008084247A1; GB0700531D0

Abstract

A method for encoding a microsphere comprising the steps of i) providing a layer of a polyionic polymer to the microsphere 11, ii) coating the layer with quantum dots iii) optionally, repeating steps i) and ii), iii) providing one or more protective layers of a transparent polyionic polymer 12a, 12b, 12c and iv) providing to the microsphere so obtained an overcoat of a transparent material 12d which is or can be adapted for the attachment of a molecule or moiety capable of recognising a target molecule 12e. The beads may be paramagnetic, latex or silica spheres or beads, the polyionic polymer may be polyallylamine or polystyrene sulphonate, the quantunm dots may be CdSe/ZnS nanocrystals. Each layer may comprise only one type of quantum dot, or different dot types.

Description

AN ENCODED MICROSPFLERE

The present invention is directed to a method for encoding a microsphere, an encoded microsphere and uscs thereof. It is particularly, although not exclusively, concerned with use of the encoded microsphere for multiplexed assays, especially in relation to explosive mixtures.

Modern analytical methods are increasingly characterised by a requirement for screening of large compound libraries. Although traditional planar (two-dimensional) arrays are ideal for this purpose, they are accompanied by problems associated with diffusion and reproducibility which make them unsuitable for quantitative assay.

A suspension (three-dimensional) array can avoid many of these problems and offer reliable quantitative assay where specifically functionalised substrates can be manufactured in large quantity with high reproducibility. However, the freedom of movement available to substrates in suspension means that they must contain some kind of code which enables them to be uniquely identified.

Commercial substrates for suspension array are based on polystyrene microspheres in which one or more fluorescent dyes have been trapped by a swell-shrink cycle. * ** * * S * ** *.**

Their use for multiplexed assays is, however, limited by poor reproducibility in *SS manufacture where high levels of encoding are required, the need for solvent compatibility in the dyes and the avoidance of overlapping excitation or emission spectra and by the cost of decoding instruments requiring multiple excitation sources *S. *

The present invention generally aims to provide a method for the production of encoded microspheres which overcomes the above-mentioned limitations.

Quantum dots (QDs) are known to give rise to size-dependent photoluminescent emission spectra of narrow bandwidth (20 to 30 nm) and have been used to label a wide variety of solid substrates.

For example, polystyrene microspheres including a paramagnetic material can be labelled by trapping quantum dots (S. Mulvaney Ct al., BioTechniques, 2004, 36, 602- 607). Encoded non-paramagnetic polystyrene microspheres can be similarly prepared (X. Gao and S. Nie, Anal. Chem., 2004, 76, 2406 -2410; M. Han, et a!., Nature, 2001, 631-635) A quantum dot labelled polystyrene microsphere has been prepared using layer-by- layer (LbL) chemistry (D. Wang et a!., Nano Lett., 2002, 2(8), 857-861). This self-assembly chemistry has also been used for quantum dot labelling of negatively charged fernc oxide nanoparticles (X Hong et al., Chem. Mater., 2004, 16, 4022-4027).

*:*::* 20 It has now been found that LbL chemistry can provide microspheres encoded by * .** I.... quantum dots on a large scale and with good reproducibility. *SS.

S

Accordingly, in a first aspect, the present invention provides a method for encoding a microsphere comprising the steps of i) providing a layer of a polyionic polymer to the a S..

microsphere, ii) coating the layer with quantum dots and, optionally, repeating these steps, iii) providing one or more protective layers of a transparent polyionic polymer and iv) providing to the microsphere so obtained an overcoat of a transparent material which includes a moiety capable of recognising a target molecule or is, or can be, adapted for the attachment of a molecule capable of recognising a target molecule.

As used herein the term "quantum dot" will be understood to refer to a particle of a semiconductor material having a dimension smaller than the exciton Bohr radius corresponding to the bulk material.

References to different quantum dots herein are references to the same andlor other such materials which have particle dimension such that they emit light at a predetermined wavelength of incident light which can be resolved from light emitted by any other. References herein to the same quantum dots will be construed accordingly.

As used herein, the term "microsphere" will be understood to refer to a particle, particularly, although not essentially, to a particle of circular cross-section, which has largest dimension or mean diameter ranging from 1 to 10 jim.

... 20 The term "transparent" as used in relation to a polyionic polymer or a material will be I...

understood to mean that the polymer or material permits the propagation of light capable of exciting the quantum dots as well as the propagation of light emitted from them. The transparent polymer or material may, in particular, permit the propagation Si: : of wavelengths of light ranging from 250 to 1400 n-ni.

S

In one embodiment, the microsphere is functionalised by an uncharged organic moiety capable of forming a covalent bond with a polyionic polymer.

In this embodiment, the method provides a base, polyionic polymer layer which is covalently bound to the microsphere. The covalently bound polymer layer is suitable for direct coating with quantum dots by step ii) and the method avoids the need for one or more priming layers The method may nonetheless include the preliminary step of providing one or more priming layers to the microsphere. The preliminary step may use one or more polyionic polymers such that the final priming layer is of opposite charge to the polyionic polymer of step i).

The preliminary step may also result in a covalently bound base polymer layer -but embodiments in which the polymer is provided by an electrostatic interaction with the microsphere are also possible The method may use polyionic polymers of high molecular weight ( 10 kDa) for these steps. It may, in particular, use a high molecular weight polyamine (? 10 kDa) such as poly(allylamine), poly(ethylcneimine), poly(lysinc) or chitin andlor negatively * **.

charged polymers such as p01 y(sod turn 4-styrenesulphonate). *,**

I S...

S *

* Where the microsphere is charged (for example, by SO3) the method may S...

: additionally use high molecular weight (? 10 kDa) polyelectrolytic salts such as those *5I * 25 based on poly(diallyldimethylammoniurn) or poly(4-vinylpryridine) for these steps.

Other suitable polyionic polymers will be known to those skilled in the art. It will be understood, however, that the method need not use a transparent polyionic polymer for these steps.

However, where the method repeats step i) a transparent polyionic polymer is always required. The method may use any one of the aforementioned polymers for repeat step i).

The microsphere may comprise any suitable material, for example, a functionalised latex or silica microsphere. Advantageously, the microsphere is paramagnetic -so as to permit convenient handling by magnetic precipitation during the separation and re-suspension steps accompanying the method.

The microsphere may, in particular, comprise an epoxy-functionalised, paramagnetic or non-paramagnetic, polystyrene microsphere (-4.5 pm) -available from Dynal (UK) and Spherotech (US) respectively.

The method may use quantum dots which comprise compounds of Group Il-Vi elements, for example CdSe, or Group 111-V elements, for example InP. It may use quantum dots having an overcoat of a material of larger band gap but limited mismatch in crystal structure (for example, ZnS) -in order to increase S...

photoluminescent quantum yield. S...

S

S S..

S S...

S S S S. S

It has been found that where, for example, the method uses an excess of quantum dots for step ii), the amount of quantum dots deposited to a polyionic layer is determined largely by surface area.

The method, therefore, reproducibly loads a precise amount of quantum dots to the microsphere.

Furthermore, the intensity of the photoemission spectrum of a microsphere on which steps i) and ii) have been repeated once is roughly twice the Intensity of that of a microsphere on which these steps have been performed only once.

The method may, therefore, repeat these steps a predetermined number of times whereby to load the microsphere with a desired amount of one or more quantum dots.

It will also be understood that the method encodes the microsphere by the wavelength andlor intensity of its photoemission spectrum on excitation at a predetermined wavelength of incident light.

The wavelength of incident light is chosen to be lower than the wavelength of the first exciton peak of the quantum dots. The incident light may, for example, comprise a wavelength ranging from 350 to 400 nm. S.S * S...

S S...

* The method may use an excess of quantum dots for all steps ii) and repeat steps i) and

S

ii) from I to 20 times (for example, at least once, twice or three times) using the same S* and/or different quantum dots to those used in any one preceding step ii).

The method may, in particular, repeat these steps using different quantum dots for each step ii) to those used in any preceding step ii).

The amount of quantum dot encoding the rrncrosphere is to a lesser extent also controlled by the number of priming layers andlor the inclusion of one or more intervening layers.

In one embodiment, therefore, the method comprises the additional step of providing one or more intervening layers of a transparent polyionic polymer following at least one step ii).

In this embodiment, the method may use the high molecular weight polymers mentioned in relation to repeat step i) for the additional step.

The number of times steps I) and ii) can be repeated with or without the additional step appears limited only by practical considerations of time and economy.

In this regard, a paramagnetic microsphere is particularly advantageous -in that it permits rapid separation of the microsphere from solution by magnetic precipitation. * **

The method may, therefore, avoid time consuming and complicated separation **** techniques such as filtration and centrifugation. ***.

S ** S

S

In one embodiment, steps i) and ii) of the method are performed in a protic solvent *SSS * S * and step ii) uses an excess of quantum dots (opposite in charge to the polymer). S..

in this embodiment, the quantum dots may be capped by a negatively charged moiety -for example, thioalkyl carboxylate. Such quantum dots can be obtained by treatment of commercially available tri-n-octylphosphine oxide (TOPO)-or tn-n-octylphosphine (TOP)-capped quantum dots with mercaptoacetic acid.

The method may provide for encoding by consecutive layers of polymer coated in the same quantum dots. For example, the method may comprise five consecutive steps ii) using green quantum dots followed by five consecutive steps ii) using red quantum dots.

In another embodiment, step 1) of the method is performed in a protic solvent and step ii) is performed in an aprotic solvent using an excess of quantum dots.

In this embodiment, the method may use TOPO-or TOP-capped quantum dots.

However, quantum dots capped by other hydrophobic ligand may be suitable -especially if they are monovalent and/or can be displaced by, or interact with, the polyionic polymers used for step i).

In this embodiment, it is essential, in order to avoid irreversible aggregation on * 20 transfer of the microsphere between protic (for example, water) and aprotic solvent that step i) is followed by a drying step and that step ii) is followed by a wetting step. S... S... *S..

The drying step may comprise washing with a suitable organic solvent -but other means for removing the protic solvent may be employed. The wetting step may comprise washing with an organic solvent whereby to substantially remove the aprotic solvent.

The aprotic solvent may, in particular, comprise chloroform -in which case both the drying and the wetting step may comprise washing with methanol.

Those skilled in the art will appreciate that, in this embodiment, the assembly of quantum dot to polymer covered microsphere can not be attributed to the electrostatic interactions which are thought to underpin conventional LbL chemistry.

The mechanism remains unclear, but the method may perhaps be described as "amphiphilic" in the sense that microspheres are exposed alternately to protic and aprotic solution.

A single coating of quantum dots is sufficient for high intensity photoemission spectra according to this embodiment -especially where the polyionic polymer used for steps i) is poly(ethyleneimine).

The method may perform all steps ii) in an aprotic solvent. Alternatively, it may perform at least one step ii) in protic solvent and at least one step ii) in protic solvent. * S

* *. S In one embodiment, step iii) of the method comprises providing one or more layers of S...

a transparent polyionic polymer. It may, in particular, comprise repeating step i) or S..

S

repeating the preliminary or additional step. The method may use any of the above- :. 25 mentioned polymers for this step.

The protective layer encapsulates the QD assembly and provides an outer layer to the microsphere for the attachment of a transparent polymer or for coating with transparent nanoparticles.

In one embodiment, step iv) of the method comprises coating the protective layer with transparent nanoparticles.

Step iv) may, in particular use silica or germanium oxide nanoparticles -but other transparent nanoparticles, comprising for example chalcogenidcs, are envisaged.

Negatively charged silica nanoparticles are available under the trade name Ludox (Grace & Co, USA).

In this embodiment, step iv) may comprise the additional step of providing one or more layers of a low molecular weight (< 10 kDa) transparent polyionic polymer, such as those mentioned above, to the glass coated microsphere and coating the polymer with transparent nanoparticles.

The resultant (outer) vitreous coating can be functionalised by a convenient and short protocol which permits the covalent attachment of a wide variety of molecules. * ** * * .

In particular, silanization of the vitreous coating and chemical modification of the * S..

silamzed surface whereby to covalently attach or to permit covalent attachment of a *.** S...

* molecule capable of recognising the target molecule. * S.. * S*

S

S a.. a

In this crnbodiment, therefore, the method may include the further step of functionalising the vitreous coating and covalently attaching to the functionalised microsphere a molecule capable of recognising the target molecule.

In another embodiment, step iv) of the method may comprise providing a layer of a transparent polymer to the (outer) protective layer which polymer includes a moiety capable of recognising the target molecule. The polymer may electrostatically anchor or covalently attach to the protective layer.

The method is not limited by a requirement for any one type of target molecule -suitable target molecules comprise chemical compounds, antibodies, anti-antibodies, receptors or nucleic acids sequences.

The molecule or moiety capable of recognising the target molecule may, therefore, comprise any conventional probe -such as antibody, aptamer, hapten, oligonucleotide sequence or a ligand recognising a particular receptor.

In one embodiment, step iv) of the method uses a transparent polymer (for example. a dextran) comprising an analogue or hapten for one of the explosives RDX, PETN or TNT (see G.M. Blackburn et al., J. Chem. Soc., Perkin Trans. 1, 2000, 225-230; R. :.:: Wilson et al., Anal. Chem., 2003, 75, 4244-4249). * S.. I... S...

* in a second aspect, the present invention provides for an encoded microsphere S..

I

obtainable according to the first aspect. I... * I * SI.

S

It will be appreciated, therefore, that the present invention provides an encoded microsphere comprising a core-shell structure in which the core is comprised by the microsphere and the shell by one or more quantum dot coated polyionic polymer layers.

In one embodiment, the shell comprises a first layer of quantum dot coated polymer which i.s covalently bound to the core microsphere.

In another embodiment, the shell comprises a base, priming layer of polyionic polymer which is covalently bound to the core microsphere. In this embodiment, the shell may comprise one or more additional priming layers of polyionic polymer provided to the base priming layer.

The priming layer(s) and the first layer of quantum dot coated polymer need not comprise a transparent polymer -but the second (and any subsequent) polymer layer should comprise a transparent polyionic polymer.

The priming layer(s) and the first quantum dot coated polymer layer may, in particular, comprise high molecular weight ( 10 kDa) polyamines -for example, poly(allylamine), poly(ethyleneimine), poly(lysine) and chitin and poly(sodium 4- : .:: styrenesuiphonate). S..

* * S. S...

The second (and any subsequent) transparent polyionic layer may he comprised from the above-mentioned polymers as well as polyionic polymers based on quaternary *: 25 salts -such as poly(diallyldimethylammonium) and poly(4-vinylpyridine).

The shell may comprise any arrangement of quantum dot coated polymer layers. It may, in particular, comprise at least two, three, four or five overlying polymer layers each coated with one type of quantum dots.

The shell may alternatively or additionally comprise at least two, three, four or five overlying polymer layers each coated with different quantum dots from one another.

It may, in particular, comprise multiple layers of polymer coated with one type of quantum dots in which each layer is separated from the other by one or more layers of a polymer coated with a different type of quantum dots.

The shell may also comprise one or more intervening layers of a transparent polyionic polymer which separate one or more quantum dot coated polymer layers. The polyionic polymer of the intervening layer(s) may comprise one or other of the above-mentioned polymers.

The shell includes one or more protective layers of a transparent polyionic polymer which encapsulates the QD assembly. The protective layer(s) may, in particular, comprise any of the high molecular weight polymers mentioned in relation to repeat *:*::* 20 step i). a... * S S...

The shell includes an overcoat of a transparent material which includes a moiety S...

* capable of recognising a target molecule or is or can be adapted to recognise a target S..

S

molecule. *55S * S S

S * 25 *5S

S

The overcoat may, in particular, comprise a vitreous coating formed by treating the protective polymer layer with silica or germanium oxide nanoparticles. It may comprise additional layers of a low molecular weight (< 10 kDa) transparent polyionic polymer, for example polyamine, each of which is coated with silica or germanium oxide nanoparticles.

In one embodiment, the vitreous coating is functionalised by covalent attachment of a molecule capable of recognising the target molecule.

Alternatively, the overcoat may comprise a layer of a transparent polymer including a moiety capable of recognising the target molecule which is electrostatically anchored or covalently attached to the (outer) protective layer.

In one embodiment, the overcoat comprises a polymer (for example, a dextran) including an analogue or hapten for one of the explosives RDX, PETN or TNT (see G.M. Blackburn et al., J. Chem. Soc., Perkin Trans. I, 2000, 225-230; R. Wilson et a!., Anal. Chem., 2003, 75, 4244-4249).

The core microsphere may comprise any suitable material, for example functionalised *:::* 20 latex or silica. Advantageously, the microsphere is paramagnetic.

S * S *a..

* In a third aspect, the present invention provides for a library comprising a plurality of S...

encoded microspheres according to the second aspect of the invention. S... S*

S S..

The library may, in particular, be obtained according to the first aspect of the invention. The method is used to produce a plurality of microspheres each encoded by the wavelength andlor intensity of its photoemission spectrum on excitation at a predetermined wavelength of incident light.

The library may, in particular, comprise encoded microspheres including a protective vitreous overcoat -for functionalising by the end user. Alternatively, the library may comprise encoded microspheres which have already been adapted for a specific use.

In any case, the library may comprise microspheres encoded by different colours or by the same colour or colours at different intensities. Thus the encoding may rely on relative as well as absolute intensities.

In a fourth aspect, the present invention provides for use of the encoded microsphere or the library for multiplexed assays, multiplexed screening or for combinatorial synthesis.

Such use may provide for the detection of a variety of materials including drug, pesticide, explosive and biological materials. * **

For example, a competitive (reagent limited) immunoassay comprises exposing a sample of suspected explosive material to a mixture comprising two or more suitable S... *.

antibodies and two or more encoded microspheres, each of which codes for a different

S

hapten for one or other of the explosives RDX, PETN or TNT. **.. * SS *. S * 25

Suitable antibodies comprise antibodies for RDX, PETN or TNT and may, for example, carry a label or a moiety to which a label can be attached following the exposure. In the example below, biotinylated antibodies for RDX, PETN or TNT are labelled after the exposure by sequential treatment with an excess of polystreptavidin and AlexaFluor 660 biotinylated dextran.

The use may also provide for the detection of bacteria, spores and viruses, especially pathogenic types, by targeting certain antigen or nucleic acids sequences.

The multiplexed assays may comprise, for example, a sandwich (reagent excess) assay. It may, in particular, comprise exposing a suitable sample of the suspected material to a mixture comprising two or more suitable (detector) antibodies and two or more encoded microspheres, each of which codes for a (capture) antibody for a different target molecule.

The detector and capture antibodies may comprise antibodies for the material raised in the same animal species (X) -except where it is desired to label the detector antibodies by anti-species antibody. in that case, the capture antibodies and the anti-species antibody must be raised in a different animal species (Y). * ** * S *

Alternatively, the assay can comprise exposing a suitable sample of the suspected **** material to two or more encoded microspheres, each of which is conjugated to an S..

S S...

* oligonucleotide capable of hybridising to a different target oligonucleotide. * * *.. * S S a. * a

The assay may, in particular, target a RNA or DNA sequence -and if appropriate include a preliminary amplification (PCR or ligase chain reaction) step. Suitably, the label may be incorporated during the amplification step. Alternatively, it may be incorporated via a detector oligonucleotide or simply bind to the hybridised molecule The label may comprise a fluorescent label -but other types of label, are also contemplated. The fluorescent label should emit at a wavelength of light which is resolvable from the light emitted by the various quantum dots at the predetermined wavelength of incident light. Preferably, the fluorescent label emits in the red region.

It may, for example, comprise AlexaFluor 660 or Cy-5.

It will be appreciated that the use relies on reading and decoding the photoemission spectrum of an encoded microsphere on excitation at the predetermined wavelength.

The detection instrument may, in particular, comprise one or more optical detectors arranged in combination with a number of filters for fixed imaging or high throughput detection.

In one embodiment, the detection instrument comprises a flow cytometer. In any case, the detection instrument may be associated with a computer programme that * .* decodes the encoded microsphere. *.** * *

The library or encoded microspheres may be included in a test strip (lateral flow) *** device allowing use with, for example, a fixed imager, in the field andlor by low **.* * . * trained personnel. **S

S

The present invention provides a reliable method for large scale production of encoded microsphere with good reproducibility The method permits very precise loading with quantum dots of one or colours and in controlled amounts.

Those skilled in the art will appreciate, therefore, that the method offers high levels of encoding and multiplexed assays for many more analytes than is possible with dye encoded microspheres.

Although the examples below are concerned with two or three different colours it will be appreciated that the encoded microsphere may be loaded with additional colours of quantum dot and that each colour may be loaded to a specific intensity, In theory, therefore, the number of unique codes N that may be obtained is given by the formula N = Cv -I where C is the number of colours and v is the number of intensities. Loading with Just five different colours of quantum dot in five different amounts may provide 3124 resolvable codes -many more in practice than with fluorescent dyes.

Since each quantum dot may be excited at the same (predetermined) wavelength of * 20 incident light use of the encoded microsphere is not limited by a requirement for decoding instruments having a large number of excitation sources. **..

S **..

* A paramagnetic core provides for easy and rapid separation of the microsphere from *SS

S

aqueous and aprotic solution. It avoids the need for costly and time consuming * **. * S * S. *

S ***

S

filtration and/or centrifugation steps and may permit automation of the method as well as automation in use.

Another advantage of the method is that it can avoid the need to prepare and/or use hazardous mercapto-capped quantum dots.

The method also enables production of an encoded microsphere including a vitreous protective layer which can be functionalised according to short and convenient protocols -so permitting versatile use.

The method provides an encoded microsphere which may be rapidly adapted to different target molecules for multiplexed assays, multiplexed screening or combinatorial synthesis.

The present invention will now be described having regard to the following embodiments and with reference to the following examples and drawings in which Figure 1 is a scheme showing an encoded microsphere according to a first embodiment of the present invention; Figure 2 is a graph plotting zeta () potentials against number of layers and coatings obtained by microelectrophoresis during the production of the encoded microsphere of Figure 1 according to a first embodiment of the method of the present S...

invention; *..S Figure 3 a) shows UV/visible absorption spectra obtained by LbL assembly of quantum dots on a quartz cuvette in accordance with the first embodiment of the * 25 method of the present invention; Figures 3 b) and c) shows graphs plotting UV/visible absorbance obtained by LbL assembly of quantum dots on a quartz cuvette and intensity of photoemission spectrum after LbL assembly on microspheres against the number of coatings of quantum dots in accordance with the first embodiment of the method of thc present invention; Figure 4 shows photoemission spectra of a library of encoded microspheres produced according to the first embodiment of the method of the present invention; Figure 5 shows a UV/visible difference absorption spectrum reporting the attachment of a haptenylated dextran to an LbL assembly of quantum dots on a quartz cuvette in accordance with the first embodiment of the method of the present invention; Figure 6 a) to d) shows atomic force microscopy (AFM) images obtained during LbL assembly of quantum dots on a glass microslide in accordance with the first embodiment of the method of the present invention; Figures 7 a) to c) shows transmission electron microscopy (TEM)images obtained during the production of the encoded microsphere of Figure 1 in accordance with the first embodiment of the method of the present invention; Figure 8 is a scheme showing the production of an encoded microsphere according to a second embodiment of the method of the present invention; Figure 9 is a graph plotting -potentials against number of layers and coatings obtained by microelectrophoresis during production of an encoded microsphere in accordance with the second embodiment of the method of the present invention; isa.

* Figures 10 a) and b) shows UV/visible absorption spectra and photoemission a.' spectra obtained by LbL assembly of quantum dots on a glass cuvette in accordance iS*s 5* with the second embodiment of the method of the present invention, S..

Figure 10 C) shows photoemission spectra of a library of' encoded microspheres produced in accordance with the second embodiment of the method of the present invention; Figures 11 a) and c) are respectively scanning electron microscopy (SEM) and AFM images obtained during LbL assembly of quantum dots on a glass microslide in accordance with the second embodiment of the method of the present invention; Figure 11 b) shows high magnification SEM images obtained during the production of an encoded microsphere according to the second embodiment of the present invention; Figure 12 is a graph plotting the intensities of photoemission spectra obtained in microspheres encoded with 1 to 5 coatings of the same quantum dot; and Figures 13 a) and h) are schemes illustrating use of a library of encoded microspheres according to the present invention; Referring now to Figure 1, an encoded microsphere according to the present invention, generally designated 10, comprises a core-shell structure, in which a paramagnetic polystyrene microsphere 11 is surrounded by a shell 12 comprising a series a), b), c) of five concentric layers of a transparent polyionic polymer coated with the same quantum dots (green (G), red (R) or yellow (Y)). The shell includes protective layers of transparent polyionic polymers 12 d) and an outer layer of a haptenylated dextran 12 e). si.

* The encoded microsphere 10 was produced in accordance with a first embodiment of *1s

I

the method of the present invention -all steps in aqueous solution: * a.. * I * S. * * 25 a..

S

Example I

First Layer (step i) Paramagnetic, epoxy-functionalised microspheres (200 jil; 4 x 108 microsphere/mI water; mean diameter 4.5 jim; C.V. <5%; Dynal, UK) were washed (5 x 1 ml) and re-suspended in water (0.5 ml). To the vortexed suspension was added a solution of polyallylamine hydrochloride (PAH; Sigma; MW 70 kDa; 100 rnglml; pH 8.0) which was prepared by dissolving in 1 M NaCI solution and diluting (1: 1) with a solution of 0.1 M sodium tetraborate. After slow tilt rotation (Dynal, MX2 Sample Mixer) overnight, the polyallylamine (PAA) covered microspheres were washed sequentially with water (4 x 1 ml), 0.1 M sodium borate solution (pH 9.5; 4 x 1 ml) and 0.1 sodium acetate solution (pH 4.5; 4 x 1 ml). The latter two steps were repeated and the PAA covered microspheres finally washed with water (6 x I ml).

Refernng now to Figure 2, microelectrophoresis measurements (Brookhaven ZetaPlus potential analyser) made in air-equilibrated HPLC grade water (pH 6.5; 0.005 w% microspheres) show a change in -potential of the microspheres. The change, from - 10.9 mV to + 40.12 mV confirms the deposition of a PAA layer to the microsphere.

Brightfield images (not shown) show that there was no cross-linking of microspheres.

*:*::* 20 TEM images (JEOL TEM 2000 FX microscope operating at 200 V; not shown) show a thin halo surrounding the PAA polymer microsphere which was not present in * oss similar images of the epoxy-functionalised microsphere. *5**

S

I I... * IS S* *

S

Negatively Charged Quantum Dot TOPO coated CdSe/ZnS core shell quantum dots (Evident Technologies, USA, 100 il in toluene) were centrifugally precipitated with methanol (4 x 1 ml) at 9000g (15 mm.) in a sealable polypropylene vial. The pellet was suspended in chloroform (50 il) and to the suspension was added thioglycolic (mercaptoacetic) acid (MA; 25 p1; Sigma) followed by a solution (25% in methanol) of tetramethylammonium hydroxide (TMA; 25 p1; Sigma). The vial was sealed and the mixture sonicated (1 mm.) before warming in a water bath (60 C; lh). After centrifuge at 9000g (10 mm.), the supernatant was removed and the pellet centrifugally precipitated with methanol (3 x 1 ml) at 9000 g (15 mm.). The washed pellet of MA-functionalised, quantum dots was suspended in 10 mM sodium bicarbonate solution (I ml) and stored in the dark ready for use.

First Coating (step ii) To a suspension of PAA microspheres (20 p1 equivalent to 60 pg) in water (1 ml) is added an excess of MA functionalised, green quantum dots (50 p1) and the mixture (pH 7.0) slow tilt rotated (15 mm.). The coated microspheres were magnetically precipitated (Dynal MPC-S Sample Concentrator) and washed with water (4 x 1 ml; HPLC grade, pH 6.5).

*:*: Microelectrophoresis measurements (Figure 2) confirm the deposition of the green I.. S quantum dots to the PAA layer. The -potential of the microspheres became less positive but not negative (+ 9.93 mV) -suggesting either that the microspheres is not completely covered by quantum dots or that the PAA layer partly envelops them. S...

S S. * *S.

S

Epifluorescence imaging (Leica DMBL fluorescence microscope with SPOT 2 camera (using lOOx objective lens at a magnification of l000x) from SPOT Diagnostics, USA) shows that the microspheres were uniformly photoluminescent.

Second layer (step i) The coated microsphercs were re-suspended in a solution of branched polyethylene-imine (PEI; MW 750 kDa; Sigma; 1 mg/mI) in 0.5 M sodium chloride (pH 8.0) and rotated (15 mm.). The PEI covered microspheres were magnetically precipitated and washed with water (4 x I ml).

The PEI covered microsphere shows greater photolurninescence than the coated microsphere -which is surprising given that UV/visible measurements of these steps applied to a quartz cuvette show that about 30% of the quantum dots are displaced by PEI.

Refernng now to Figure 6, AFM images (Thermomicroscopic Explorer AFM; in tapping mode; scan rate 5.23 lims-l; SPMLAB Version 5.01 software from Windsor Scientific, UK using NanoSensors PPP-NCHR cantilever 125 im long, tip radius < nm, 42 N/rn spring constant, 33 kHz resonance frequency) of these steps on epoxy-functionalised glass niicroslides (a); Genetix, UK) show that the surface of the PEI polymer slide (d) is smoother than the surface of the coated PAA slide (c) and that the latter is more uneven than the surface of the PAA polymer slide (b). *.S. S...

S S... * . S *S S

S S..

S

Second coating (step ii) To a suspension of (PEI) polymer microspheres in water (1 ml) is added MA functionalised, green quantum dot and the mixture slow tilt rotated (15 mm). The coated microspheres were magnetically precipitated and washed with water (4 x I ml; HPLC grade, pH 6.5).

Subsequent layers and coatings Steps I) and ii) were repeated three times to give (5G) niicrospheres -five polymer layers each coated with green quantum dot.

Different quantum dots Steps i) and ii) were repeated a further five times with MA-functionalised red quantum dot to give (5G-5R) microspheres.

Microelectrophoresis measurements (Figure 2) confirm the successive deposition of PEI layer and quantum dot coating. As may be seen, the -potential of the microspheres oscillated between + 38.65 mV and + 10.46 mV.

Figure 3 a) shows the UV/visible spectra obtained when these steps (5G-5R) are applied to a quartz cuvette. Figures 3 b) and c) show a linear increase (upper part) in the absorbance of the first exciton peak of quantum dots with number of coatings on *S..

the cuvette and that the Intensity of luminescence (lower part) of microspheres similarly increased in line with the number of coatings (1-5-G; 1-5-R) on the I...

microsphere. S... S. *

S

Steps i) and ii) were repeated a further five times with MA-functionalised yellow quantum dot to give (5G-5R-5Y) microspheres.

The pattern of rougher and smoother surfaces continues as further PEI layers and quantum dot coatings are deposited. Although the surface of the microspheres becomes more uneven as the total number of PEI layers and quantum dot coatings increases, Figure 7 shows that the surface (C) of encoded microspheres with 15 PEI layer/coating is only slightly more uneven than the surface of encoded microspheres with 5 (A) and 10 (B) layers/coatings.

Confocal imaging (Zeiss LSM 510 laser scaiming confocal microscope; Zeiss META detector) of (5G-5R-5Y) individual microspheres (not shown) confirm the core-shell structure.

Referring now to Figure 3 c) the photoemission spectra of a library of encoded microspheres is shown. The library comprises 5G, 5G-5R and 50-5R-5Y encoded microspheres. As may be seen, the intensity of luminescence from the green quantum dots decreases when the microsphere also includes quantum dot that emits at longer wavelength The effect may be due to radiative andlor non-radiative energy transfer.

* :* ::* Protective Layers (Ste p iii) * ** * Step i) was used to obtain PEI covered (5G-5R-5Y) encoded microspheres. These microspheres (60 pg) were slow tilt rotated (15 mm.) with 0.5M NaC1 solution containing poly(sodium 4-styrenesulphonate) (PSS: MW 70 kDa; Sigma; 1 mg/mI). * S.. * S* *S * *S.

The PSS covered microspheres were magnetically precipitated and washed with water (4 x 1 ml). These steps were repeated to give (PELIPSS) 3 covered microspheres The inclusion of the protective layers is confirmed by microelectrophoresis measurements (Figure 2; for 5G-5R) which showed that the -potential of the microspheres became negative following treatment with PSS and then positive following treatment with PEI.

PETN-PDP-Dextran and RDX-PI)P-D ext ran To a solution (100 ul) of carhoxylated hapten for PETN (or RDX; prepared according to G.M. Blackburn et al., J. Chem. Soc, Perkin Trans. I, 2000, 225-230) in aceto-nitrile (50 mM) was added a solution (1 ml) of 0.2 M N-hydroxysulphosuccinimide, sodium salt (NHSS, Sigma) in 0.1 M sodium phosphate (pH 7.4) and a solution (2 ml) of N'-(3-dimethylammopropyl)-N-ethylcarbodiimide (EDC; Sigma) in 0.1 M sodium phosphate solution. The resultant mixture was stirred and a solution (1 ml) of aminodextran (70 kDa, Molecular Probes, USA; 22 primary amine/molecule; 10 mg/mi) in 0.1 M sodium phosphate added. After further stirring (2 h) a solution (400 .iI) of 2 mM 3-(2-pyridydithio)propionic acid succinimidyl ester (SPDP; Sigma) in ethanol was added and the stirring continued (1 h). The solution of haptenylated PETN dextran (haptenylated RDX-PDP-dextran) was dialysed (48 h) against water (3 x 11) at 4 C in darkness. * S

* S. S *5s5 TNT-PDP-D ext ran To a solution (4 ml) of PBS containing aminodextran (4 mg, 70 kDa) was added drop *: 25 wise with stirring a solution (100 Ml) of dimethylformamide (DMF) containing DNT-

S *S. *

NUS (0.25 mg preparcd according to G.H. Keller et al., Anal. Biochem., 1998, , 441). After stirring (2 h) a solution (400 p1) of 2 mM SPDP in ethanol was added and the stirnng continued (1 h). The solution of TNT haptenylated dextran was dialysed (48 h) against water (3 x 11) at 4 C in darkness.

Haptenylated Microspheres (step iv) PEI (5G-5R-5Y) encoded microspheres (60 pg) including protective (PElJPSS) layers obtained above were re-suspended in a solution of 0.1 M sodium bicarbonate.

To this solution was added a solution of SPDP (0.4 mg) in dimethylformamide (DMF) and the mixture slow tilt rotated (30 mm.). The addition was repeated and after further rotation (30 mm.) the microspheres were washed with 0.lM sodium bicarbonate solution (4 x 1 ml) and phosphate buffer solution (PBS; 15 mM sodium phosphate, 0.15 M NaCI; pH 7.4; 4 x 1 ml). The microspheres were re-suspended in a solution of DTT (10 mM) in 0.1 M sodium bicarbonate solution and slow rotated (15 mm.).

After magnetic precipitation, the microspheres were immediately re-suspended in buffer solution (0.33 ml; 3 x PBS; 45 mM sodium phosphate, 0.45 NaCl; pH 7.4). To the suspension was added a solution (0.66 ml) of haptenylated PDP-dextran in water and the mixture slow tilt rotated at 4 C overnight. The haptenylated microspheres were washed with PBS (4 x 1 ml) and water (4 x 1 ml) and stored in water at 4 C in * ** * darkness. * *.. ** I *S*. S * * I. S

S 55.

Refernng now to Figure 2, microelectrophoresis measurements confirm attachment of the haptenylated-PDP-dextran. The -potentia1 of the microspheres becomes negative (-20.95 mV).

Referring now to Figure 4, the difference UV/visible spectrum obtained by subtracting the underlying spectrum of the LbL assembly from the spectrum acquired for the haptenylated LbL assembly on a quartz cuvette shows a peak at 362 nm corresponding to DNP-PD P-dextran.

The stability of the haptenylated layer appears good -shaking with a solution of bovine serum albumin (BSA) in PBS (16 h) resulted in no recognisable change in this spectrum.

Referring now to Figure 13 a), a competitive assay for TNT employed a suspension array of encoded microspheres produced according to Example I.

Example 2

AlexaFluor 660 Biotinylated-dextran -obtained under conditions of low light as follows: To a stirred solution (2.5 ml) of aminodextran (500 kDa; 98 primary amine per molecule; Molecular Probes, USA; 5 mg) in PBS was added a solution (10 p1) of AlexaFluor 660 carboxylic acid * SI* S..... succinimidyl ester (Alexa-NHS; Molecular Probes, USA; 200ug) in DMF. The ** addition was repeated (four times) at intervals (30 mm.). The stirring was continued (1 h) and a solution (10 p1) of biotinamidocaproate succinimidyl ester (biotin-NHS; *: 25 Sigma; 200 pg) in DMF was added. The addition was repeated (four times) at SI.

I

intervals (30 mm) After further stifling (1 h) the solution was dialysed against water (4 x 11) at 4 C.

Competitive Assay A sample solution was prepared by dissolving TNT in acetonitrile and diluting to suitable concentration with PBS solution containing BSA (PBS-BSA; 10 mg/mI).

The suspension array (as follows 5G-PETN, 5G-5R-TNT and 5G-5R-5Y-RDX) was suspended in the sample solution (0.5 ml) and the mixture diluted (1:2) with a solution of hiotinylated antibodies to PETN, TNT and RDX in PBS-BSA to 25 nM each antibody.

The suspension array was slow tilt rotated (30 mm.), washed with PBS and re-suspended in PBS-BSA containing an excess of polystreptavidin (DakoCytomation, DK, supplied as 5.9 jiM dextran solution, mean 19 polystreptavidin molecules per molecule dextran). After further rotation (15 mm.), the array was washed with PBS and re-suspended in PBS-BSA containing an excess of AlexaFluor 660 biotinylated dextran. After further rotation (15 mm) the array was washed with PBS (3 x I ml) and resuspended in PBS (25 j.il) for epifluorescence imaging.

The biotinylated antibodies to PETN and RDX bind to the corresponding . microspheres -but antibodies to TNT do not bind. Consequently, there is no binding * S.

S

of polystreptavidin to the microsphere encoding TNT and no labelling by binding to biotinylated AlexaFluor 660. The microspheres specific for TNT are not visible through the AlexaFluor window (far right of Table I). S... *S S

S * S*

S

Example 3

Multiplexed Assays A competitive assay similar to Example 2 was used for sample solutions that contained PETN and RDX in ratios found in Serntex A and Semtex H. The assay reports the presence of these explosive materials at concentrations of PETN and RDX as low as 1000 ppb and 50 ppb respectively.

A second embodiment of the method of the present invention is shown in Figure 8 -step i) is performed in aqueous solution and steps ii) arc performed in chloroform (l-lCCl)

Example 4

First Layer (step i) To the vortexed suspension of washed, paramagnetic epoxy-functionalised microspheres (6 mg) in water (500 p1) was added a solution (500 p1) of branched chain PEI (MW 750 kDa, 100 mg/mI) of 1 M NaC1. After slow tilt rotation overnight, the (PEI) polymer microspheres were washed sequentially with 0.5 M NaCI solution (4 x 1 ml), 0.1 M sodium borate solution (pH 9.5; 4 x 1 ml), 0.1M sodium acetate solution (pH 4.5. 4 x 1 ml) and 0.5 M sodium chloride solution (6 x 1 ml). The PEI covered microspheres were re-suspended in water and stored at 4 C. * S. * . . * *. **S.

S..'.. Microelectrophoresis measurements (Figure 9) confirm the deposition of PEI layer -the negative -potential of the epoxy-functionalised microspheres becomes positive.

S *. * S. S. * S..

S

First Coating (step ii) PEI polymer microspheres were magnetically precipitated from a suspension (60 pg) in water (20 p1) and washed with HPLC grade water (pH 6.5; 4 x I ml) and then methanol (4 x 1 ml). The microspheres were re-suspended in chloroform (1 ml) and to the suspension was added an excess of TOPO-capped green quantum dot. The mixture was slow tilt rotated (lh) The coated microspheres were magnetically precipitated and washed with chloroform (1 ml), methanol (4 x I ml) and then water (1 ml).

Microelectrophoresis measurements (Figure 9) confirm the deposition of the quantum dot coating -the -potential of the microspheres again becomes less positive.

Intervening layers The coated microspheres were slow tilt rotated (1 h) with a solution (1 ml, pH 8.0) of 0.5 M NaCI containing PEI (1 mg/mI). The PEI covered microspheres were magnetically precipitated, washed with water (4 x I ml) and then slow tilt rotated (lh) with a solution (1 ml, pH 6.0) of 0.5 M NaCI containing PSS (1 mg/mI).

The inclusion of the intervening layers is confirmed by microelectrophoresis measurements (Figure 9) -the Ed-potential of the microspheres becomes negative following treatment with PSS and then positive following treatment with PEI. * S. *** S * S * *** *S*.

S **S. * S. * 5.. * SS S. S S.

S

Second layer (step i) After magnetic precipitation, the PSS covered microspheres were washed with water (4 x I ml) and slow tilt rotated (1 h) with a solution (1 ml, pH 8.0) of 0.5 M NaCI containing PEI (1 mg/mI).

Second coating (step ii) The PEI covered microspheres were washed with HPLC grade water (4 x I ml) and methanol (4 x 1 ml) and re-suspended in chloroform (1 ml). To the suspension was added an excess of TOPO-capped quantum dot and the mixture was slow tilt rotated (lh). The coated microspheres were magnetically precipitated and washed with chloroform (1 ml), methanol (4 x 1 ml) and then water (1 ml).

Subsequent layers and coatings PEI covered (1G) microspheres were obtained and coated with TOPO-capped red quantum dots according to the steps described above. The PEI (1G-IR) microspheres so obtained were similarly coated with TOPO-capped yellow quantum dots to give PEI covered (1G-1R-1Y) microspheres.

The successive deposition of PE! layer and quantum dot coating is confirmed by microelectrophoresis measurements (Figure 9) -again an oscillation in positive values of -potential again is seen. S... * S *.a.

Figure 10 a) shows the IJV/visible spectra obtained when these steps (5G-5R) are *.** applied to a quartz cuvette. Again a linear increase in the absorbance of the first cxciton peak when multiple coatings of the same quantum dot are deposited is found. S..

S

Vitreous Overcoat (step iv) To a suspension of (IG-1R-IY) encoded (PEI!PSSIPEI) microspheres (60 1g) in water (1 ml) was added an excess of silica nanoparticle (Ludox TM-40; SiNP) and the mixture (pH 9.5) slow tilt rotated (1 h). The SiNP covered microspheres were magnetically precipitated and washed with water (4 x I ml). The microspheres were slow rotated with a 0.5 M NaCI solution (1 ml, pFI 8.0) containing PEI (MW 10 kDa; 1 mg/mI).

The coating step was repeated together with the step providing PEI to give (IG-1R-IY) encoded microspheres having an overcoat of SiNP/PEIISiNP/PEIISiNP layers.

The successive deposition of PEI layer and SiNP coating is confirmed by microelectrophoresis measurements (Figure 2) -the -potential of the microspheres oscillates between positive and negative values.

UV/visible measurements show that the overcoat does not increase the absorbance of the microspheres in the range 200 -700 nm or decrease the intensity of the photoemission spectrum.

Referring now to Figure 11 SEM a) and b) and AFM c) images of surfaces obtained following assembly of three coatings of quantum dots (III) and a SiNP protective * I. I...

coating (IV) on epoxy-functionalised microspheres (b) and on epoxy-functionalised *** glass microslides (a, c) show that the surfaces are not noticeably rough compared to the surfaces of the starting microspheres and slides (I). Indeed, the surface of SiNP * **. * * * *. *

S S..

S

covered microspheres (b-IV) appears smoother than the surface of the epoxy-functionalised microsphere.

As may be seen, the size of the microspheres after the application of the overcoat (b-IV) is similar to the size of the unloaded microspheres (h-I).

The core-shell structure is again confirmed by confocal imaging of individual microspheres (not shown).

Refernng now to Figures 10 b) and c) the photoemission spectra obtained from a library of 1G (I), 1G-1R (II) and lG-IR-lY (III) encoded microspheres again show that the intensity of luminescence from green quantum dots decreases when the microsphere includes quantum dots emitting at longer wavelength (red). The effect may be due to radiative andlor non-radiative energy transfer.

Referring now to Figure 12. the photoluminescence intensities of microspheres including 1 to 5 layers of the same quantum dot show four distinctly resolvable groups of microsphere.

PDP-functionalised Albumin To a stirred a 0.1 M sodium bicarbonate solution (1 ml; pH 8.6) containing albumin * ** p* (10 mg; albumin (chicken egg white; OVA); bovine serum albumin (BSA), human serum albumin (HSA); Sigma) was added drop wise a solution (100 uI) of SPDP (0. 2 *.( S mg) in DMF. After further stirring (1 h) the PDP-albumin was purified by gel *: 25 exclusion chromatography (Sephadex G25).

S *S.

S

The molar ratio of PDP to albumin was determined as 2:1 by UV absorbance measurements (PDP at 343 nm after reduction with dithiothreitol (DTT); albumin at 280 nm corrected for PDP).

Albumin Microspheres (5G-5R-5Y) Encoded microspheres (60 j.tg) including the protective SINP/PEIJSiNP/PEL'SiNP layers mentioned above were washed with ethanol (4 x 1 ml) and slow tilt rotated (overnight) with a mixture (1 ml) of (95/3/2) ethanol/water/aminopropyltriethoxysilane (APTS). The microspheres were washed with ethanol (4 x I ml) and re-suspended in 0.1 M sodium bicarbonate solution (0.5 ml). The microspheres were added to a solution (0.5 ml) of 0.1 M sodium bicarbonate solution containing 2-iminothiolanc, hydrochloride salt (1 mg) and the mixture slow tilt rotated (30 mm.).

After washing with 0.1 M sodium bicarbonate solution (4 x 1 ml), the microspheres were re-suspended in PBS (1 ml) containing PDP-albumin (0.5 mg). The mixture was slow tilt rotated (overnight) and the microspheres washed with PBS (4 x 1 ml). The *:*::* microspheres were then slow tilt rotated (1 h) with blocking solution (PBS containing gelatin (cold water fish skin; 10 mg/mI; 1 ml) and washed with PBS (4 x 1 ml) and * *** stored in darkness.

I I.. *

*:::1: Referring now to Figure 13 b), a competitive assay for BSA employed a suspension * array of encoded microspheres produced according to Example 4:

Example 5

Competitive Assay A suspension array was prepared by mixing equal amounts of Example 3 encoded microspheres as follows 3G-OVA, 2G-IR-BSA and 5G-5R-5Y-HSA in antibody diluent. A sample solution (10 gIml) was prepared by dissolving BSA albumin in antibody diluent containing anti-albumin (anti-OVA, anti-BSA (mouse ascites fluid), anti-HSA; Sigma, IgG 5 pg/mI).

The suspension array was incubated with the sample solution (1:1 v/v) and the mixture slow tilt rotated (15 mm.). The microspheres were magnetically precipitated and washed with PBS containing 0.05% Tween-20 (1 x 1 ml).

The microspheres were then slow tilt rotated in antibody diluent containing Cy-5-labelled antimouse antibodies (AbCam, UK); IgG 10 pg/mI), washed with PBS containing Tween and imaged with an epifluorescence microscope.

The table shows that no anti-BSA antibody binds to the 2G-IR encoded microsphere *:*::* because they are bound to BSA in solution. Consequently, when the array is *. incubated with antibody specific to the antibodies there is no binding to the 2G-1R :" 20 encoded microspheres. Microspheres specific for BSA are not visible when imaged through the Cy5 window (far right of Table II). * *** * * S S. S..

Claims

CLAIrvIS 1. A method for encoding a microsphere comprising the steps of

i) providing a layer of a polyionic polymer to the microsphere, ii) coating the layer with quantum dots and, optionally, repeating these steps iii) providing one or more protective layers of a transparent polyionic polymer and iv) providing to the microsphere so obtained an overcoat of a transparent material which includes a moiety capable of recognising a target molecule or is, or can be, adapted for the attachment of a molecule capable of recognising a target molecule.
2. A method according to Claim 1, in which the microsphere is paramagnetic.
3. A method according to Claim 1 or Claim 2, in which the microsphere comprises a latex or silica microsphere.
4. A method according to Claim 3, in which the microsphere forms a covalent bond with the polyionic polymer.
5. A method according to any preceding Claim, in which the polyionic polymer of step i) is transparent.

*... 20 I...
6. A method according to any preceding Claim, in which the transparent polyionic polymer comprises a high molecular weight polyamine. S..

S
7. A method according to Claim 6, in which the polyamine comprises poly(allylarnine), poly(ethyleneimine), poly(lysine) or chitin.
8. A method according to any preceding Claim, in which step i) is carned out in a prone solution.
9. A method according to any of preceding Claim, in which step ii) is carried out in aprotic solution.
10. A method according to Claim 9, in which step i) is followed by a drying step.
11. A method according to Claim 10, in which step ii) is followed by a wetting step.
12. A method according to Claim 11, in which the drying step and the washing step comprise washing with methanol.
13. A method according to any preceding Claim, in which step ii) is repeated with the same quantum dots.

*:*::*
14. A method according to any preceding Claim, in which step ii) is repeated from 1 to 20 times. * ***

*.
15. A method according to any preceding Claim, in which step ii) is repeated with different quantum dots as those used for any preceding step. * ***

S
16 A method according to any preceding Claim, comprising the preliminary step of providing one or more priming layers of a polyionic polymer to the microsphere.
17. A method according to any preceding Claim, comprising thc stcp of providing one or more intervening layers of a polyionic polymer following step ii).
18. A method according to any preceding Claim, in which step iv) comprises coating the polyionic polymer of the outer protective layer with silica or germanium oxide narioparticles.
19. A method according to any preceding Claim, in which step iv) comprises providing an outer transparent polymer layer including a moiety capable of recognising a target molecule.
20. A method according to Claim 18, comprising the further step of silanizing the overcoat and directly or indirectly attaching a molecule capable of recognising a target molecule.
21. An encoded microsphere. obtainable by the method of any preceding Claim.

*:*::*
22. An encoded microsphere according to Claim 21, comprising from 1 to 20 different quantum dots. * *.*

*.
23. A library comprising one or more encoded microspheres according to Claim 21 or Claim 22. * * . *.S
24. Use of the library of Claim 22, for multiplexed assays, multiplexed screening or combinatorial chemistry.
25. Use according to Claim 24, for the detection of explosive materials.
26. Use according to CLaim 24 or Claim 25, in combination with a lateral flow device. * ** * * * * ** *** S * * S... S... *S. S

SS *SSS S5 S