WO2007064297A1

WO2007064297A1 - Nano scale reaction system

Info

Publication number: WO2007064297A1
Application number: PCT/SE2006/050534
Authority: WO
Inventors: Karin Caldwell; Karin Fromell
Original assignee: Karin Caldwell; Karin Fromell
Priority date: 2005-12-01
Filing date: 2006-12-01
Publication date: 2007-06-07

Abstract

The invention relates to a reaction system for carrying out chemical reactions, comprising a hydrophobic surface and surfactant molecules attached to said surface. At least a fraction of said surfactant molecules are derivatized with two or more different types of functional groups having affinity for selected ligands. One or more different ligands are attached to said derivatized surfactant molecules via said functional groups. The average distance between ligands is in the nanomolar range. The invention can be used e.g. for a biosensor, bioassays or immunoassays, either on a flat surface or on particles such as nanoparticles.

Description

Nano scale reaction system

The present invention relates generally to the controlled immobilization of multiple types of active species (biomolecules) onto surfaces, in particular onto small particles, to render them multi-functional, thereby providing a reaction system for carrying out chemical reactions in a broad sense. The active species are provided in close proximity to each other to promote rapid mass transfer, for the purpose of enabling coupled reactions to be carried out on the surface at a higher rate than would otherwise be possible. Furthermore it relates to methods of making such multi-functionalized particles, and to such particles themselves.

Background of the Invention

Biological organisms are regularly executing series of coupled reactions whose outcomes depend on the efficient transformation of products from one reaction into reactants in the following step. This requires close proximity between the components, often catalysts, responsible for the consecutive reactions. It also demands a careful spatial organization to ensure high local concentrations of reactants at both donor and acceptor sites and thereby making the over-all reaction efficient. A typical example from biology is the mitochondrion, a small organelle that among other components houses the eighteen coupled electron transfer reactions which oxidize the proton to water during the metabolic process. These organelles differ in size from tissue to tissue, but typically range from a few hundred nm to several microns.

Sensitive diagnostic tools are often built around an analytical surface equipped with affinity ligands, such as antibodies or other especially designed structures that capture specific marker substances present in the minute quantities of blood, saliva, tears or other biological fluids that may be sampled from a patient. The binding event then has to generate a measurable signal that can be related to the concentration of marker present in the fluid. This coupling is nontrivial, and requires an effective communication between the binder and some signal generating system.

In the micro - nanomolar concentration range, the binding is often followed by optical detection. The captured marker is then frequently capped in a subsequent reaction with a "second antibody", i.e. an affinity ligand to which an enzyme has been conjugated. This enzyme is so chosen that it may convert a freely accessible substrate into a visible, fluorescent, or bioluminescent product; the amount of product then becomes a function of the amount of captured marker.

For diagnostic techniques to be used in "point-of-care testing" (POCT) it is typically required that the marker quantification be based on a signal that is reproducibly generated within a few minutes after sampling. In addition, the measuring platform has to be robust and able to withstand even harsh handling. This criterion excludes any technique sensitive to directional adjustment of an exciting light beam.

One detection mode of extreme sensitivity, in which the excitation is of chemical rather than optical nature, is based on bioluminescence. In this mode each specific chemical bond cleaved gives rise to one quantum of light, emitted spherically from the reaction site. This makes the bioluminescence measurement not only sensitive but also robust and generally free of background signal.

Summary of the Invention

An object o f the present invention is to provide means for enhancing the rate of reactions for a variety of biological, biochemical, chemical and other interactions (herein generally referred to as "chemical reactions"), thereby i.a. enabling detection of species that otherwise would be impossible or at least very difficult to detect. Synthesis of a variety of compounds that hitherto have been obtained at very low rates and yields will also be enabled.

In a first aspect of the invention there is provided a reaction system for carrying out chemical reactions (in a broad sense) as defined in claim 1.

In a second aspect there is provided a read-out system as define in claim 12, wherein a reaction system as claimed in claim 1 is attached to a read-out surface via a linker structure.

In a third aspect there is provided a method of making such a reaction system, said method being defined in claim 24.

In a fourth aspect there is provided a method of detecting unknown species in a medium, the method being defined in claim 25. In a fifth aspect there is provided a rapid and highly sensitive analytical sensor defined in claim 19. This is achieved by the ability to spatially arrange both capture and reporter groups in close proximity to one another on e.g. a nanoparticle surface.

Brief Description of the Drawings

Fig. 1 shows schematically the principle usable in the invention;

Fig. 2 illustrates multi-functionalized particles of a reaction system according to the invention;

Fig. 3 is a SEM image showing fully decorated particles in accordance with the invention;

Fig. 4 schematically illustrates a signal generating system for use in one embodiment of the invention;

Fig. 5a shows schematically an example of a signal generating construct; and

Fig. 5b shows the bioluminescence intensity produced by the multi-particle construct schematically illustrated in Fig. 5a

Detailed Description of Preferred Embodiments

For the purposes of the present invention the term "chemical reaction" shall be taken to encompass biological, biochemical, chemical and physico-chemical reactions.

The present invention is based on the insight that in order to enhance reaction kinetics for biological/ biochemical/ chemical system with low concentrations of reacting species, the reacting species need to be brought into close proximity of each other. This is virtually impossible in solution. A system in nature that has solved this problem is the mitochondrion, and thus, the inventors set out to provide a synthetic "device" mimicking the organization of mitochiondria. According to the present invention, in order to mimic the process efficiency present in biological tissues purified enzymes or other capture molecules known to participate in coupled reactions are attached to sub-micron sized polymeric particles. Through such an approach one would expect to gain the desired proximity between donor and acceptor in sequential reactions and locally reach the high reagent concentrations that promote fast reactions, even at low bulk concentrations. This situation is highly desirable, particularly in the designing of diagnostic tools when high sensitivity and rapid response are qualities in great demand. An illustration of the principle used in the invention is given in Fig. 1, where the lectin Concanavalin A (ConA) with high affinity for certain carbohydrates is attached to the surface of polystyrene

"nanoparticles" with a diameter of 240 nm and is allowed to bind the glycoprotein Ovalbumin. This particle is obviously comparable in size to the smallest mitochondria encountered in biology. The lectin attachment is accomplished via an adsorbed synthetic triblock surfactant named Pluronic F 108®. This surfactant has been end- group activated to allow the attachment of proteins and other reactive structures via its long and mobile poly- (ethylene oxide chains, PEO) as described in US-5, 516,703 and US-6,087,452. Different modes of end-group activation are utilized to steer the coupling of different structures to the surface. Since there is a large excess of PEO chains on the surface, and since such chains have a well established ability to suppress non-specific adsorption of proteins and other macromolecules, the surface in between the specifically attached proteins is well protected from undesired components. The Pluronic type of surfactant is a member of a class of surfactants referred to as poloxamers, which are a preferred class of surfactants for the purpose of the invention. Other types of polymers can also be employed, e.g. dextran provided they be derivatizecd so as to exhibit a hydrophobic part that can adhere to the surface. However, the efficiency will not be as high as with e.g. the Pluronic type of surfactants. As shown in the example given in Fig. 1, the local concentration of Con A on the particle surface can reach the remarkable level of 0.2 mM. When such particles are attached to a surface, as indicated in the figure, spots containing around 1 fmole of ConA have been shown to detect fluorescently labelled Ovalbumin in solutions with concentrations less than 1 nM.

In its most general form, the invention relates to a reaction system for carrying out chemical reactions comprising a hydrophobic surface. Surfactant molecules are attached to said surface and at least a fraction of the total number of said surfactant molecules have been derivatized with two or more different types of functional groups having affinity for selected ligands. There must always be a fraction that is not derivatized, so as not to bind any ligands, in order to provide some spacing between ligands. However, there is a proviso that each surfactant molecule is derivatized with only one type of functional group. One or more different ligands are attached to said derivatized surfactant molecules via said functional groups. The distribution of the ligands is such that the average distance between ligands is in the nanometer scale. In preferred embodiments the average distance between the ligands is < 100 nm, preferably <40 nm, more preferred < 15 nm, but >5 nm.

In one embodiment the reaction system according to the invention is confined to a generally flat surface.

In another embodiment the reaction system is confined to a particle having a hydrophobic surface, the particle preferably having a size in the range larger than 10 nm but smaller than 1000 nm, more preferably larger than 100 nm but smaller than 500 nm.

Preferably the particles are made of any of a polymeric material, a metal, a glass, a ceramic, as long as they exhibit the desired hydrophobic surface.

Surfactantas are suitably selected from poloxamers, dextran and other hydrophobized hydrophilic polymers.

The ligands for use in the invention can be selected from proteins, nucleic acids, carbohydrates and lipids.

In order to enable binding of certain desirable ligands, a part of the surfactant molecules can be charged with a metal ion. This will enable binding ligands having a histidine tag to the particles via said metal ion. In a preferred embodiment the ligand is lucif erase, and the metal ion is Ni²⁺.

In a further aspect of the invention there is provided a read-out assembly, wherein the particles are attached to a read-out surface via a linker structure. The linker structure can comprise complementary oligonucleotides, a first oligonucleotide present on the read-out surface, and a second oligonucleotide present on the particles of the reaction system, and the coupling is achieved by a hybridization between said complementary oligonucleotides. The linker structure can suitably comprise an oligo-guanine (dG) residue hybridized to an oligo-cytosine (dC) residue.

In one embodiment of the read-out assembly the particles are laid out on said read-out surface in discrete spots.

In a still further aspect of the invention there is provided a synthesis reactor comprising a reaction system as described above. Such a system can comprise any kind of hardware vessel that can house a reaction system such that reagents in solution can interact with the active members of the reaction system.

Also, in another aspect, there is provided an analytical device which comprises a reaction system as describe above, wherein one of the ligands is coupled to a species capable of generating a signal in response to reactions occurring at other ligands. In a particular embodiment the reaction system is confined to particles, the signal generating species is luciferase, and the reactions occuring at other ligand(s) generates ATP which can interact with luciferase and luciferin, thereby emitting detectable light.

Such devices can generelly be referred to as biosensors, a concept which also falls within the scope of the invention. In particular the biosensor comprises a reaction system confined to particles in the nanodiameter scale, and the particles are attached to other substantially larger particles, preferably an order of magnitude larger than the particles of the reaction system itself, forming aggregates. In particular the size of the aggregates is large enough to render them filterable.

An aspect of the invention is also a method of making a reaction system. It comprises incubating a hydrophobic surface with polymeric surfactant molecules, wherein at least a part of said polymeric surfactant molecules has at least two types of free reactive groups, but wherein each surfactant has only one type of free reactive group or none. A selected ligand having groups that can react with the free reactive groups is bound to the surfactant adsorbed to the particles.

Furthermore, the invention provides a method of analyzing species in solution, comprising contacting a sample containing a species of interest with an analytical device as claimed in claim 19, and detecting the signal generated from the reaction system.

The invention will now be described in more detail with reference to the figures.

Thus, the invention provides a hydrophobic surface, most frequently that of a nanoparticle (flat surfaces can also be used in accordance with the ionvention, but with reduced efficiency), which is coated with a mixture of differently derivatized polymeric surfactants composed of a hydrophobic block accomplishing the adsorption and one or more hydrophilic blocks to which a functional group of some kind is attached. Said functional group is incorporated to permit the subsequent specific attachment of a chosen ligand, chemically composed so as to specifically bind to that functional group. The differently derivatized surfactant molecules preferably all have the same polymeric base structure and their only difference lies in the chemical nature of the small functional group that is terminally attached to the hydrophilic block. Mixtures of such differently derivatized surfactants can be made to adsorb to hydrophobic surfaces such as those presented by polymeric nanoparticles, resulting in coated particles which present reactive structures in the same relative proportion as they had in the coating mixture. Each type of functional group will then be capable of binding a specific ligand molecule with matching chemical reactivity. The intended ligand molecules can either be attached one component at a time, or all together as a mixture in which case the specific chemistry of each will ensure coupling to the matching chemical groups on the surface. The former is a mode used for quantification of each component (see Example 1 and Figure 2) while the latter has the convenience of sending each ligand to its intended, specific chemical address in one reaction step. It is essential that the polymeric surfactant mediating the attachment of the, often structurally fragile, biomolecules contain hydrophilic blocks of moderately long chained poly- (ethylene oxide) or some similarly non-denaturing polymer. Through this arrangement an attached biomolecule will retain some mobility even while affixed to the particle surface. This is highly desirable from a reaction rate perspective. The non-denaturing polymer chain, in turn, protects the biomolecule from destructive interactions with the underlying hydrophobic surface so that the attached biomolecule retains its biological activity. One polymeric surfactant that has proven well suited for an attachment strategy of this kind is the triblock copolymer of poly- (ethylene oxide, PEO) and polypropylene oxide, PPO) with the overall chemical composition (PEO) i29(PPO)₅7(PEO) 129 and the trade name of Pluronic F 108®. This polymer, i.e. without the introduction of terminal reactive groups, is well known to prevent surfaces to which it is adsorbed from non-specific uptake of proteins. In a mixture of differently derivatized surfactants, as discussed here, one part of the surfactant molecules is generally left un-derivatized, such that the surface in between the attached biomolecules is shielded from non-specific uptake of proteins.

Among the functional groups that one attaches to a nanoparticle surface it is convenient to have one that is specifically intended to link the particle to a flat surface for positioning and detection purposes. In such a case, the flat surface might be coated with the same type of basic surfactant as is placed on the nanoparticle, with the exception that this flat surface for shielding purposes contains a coating that mostly consists of underivatized surfactant molecules although it has a minor admixture of surfactant capable of engaging in a particular linking chemistry, as illustrated in Fig. 1.

If a minute aliquot of a suspension of coated particles is deposited, e.g. with a so called "dot-blotter", onto a surface coated with underivatized Pluronic F 108 with at least 5% addition of surfactant capable of performing particle attachment via oligonucleotide hybridization, as in Fig.1 , a coupling occurs in a matter of less than 5 minutes. This attachment is robust and withstands vigorous flushing with water or aqueous buffers. The SEM image in Fig. 3 shows fully decorated particles, according to Fig. 2 and Example 2, after deposition on a suitably prepared surface according to the above. After the deposition had followed a five minute period of binding in turn followed by a careful flush-wash to remove all unattached particles. Particles functionalized to perform as miniaturized bioreactors can through this procedure be positioned at such locations where they can best be monitored and their reaction products quantified.

A specific purpose for using the type of multi-functionalized nanoparticles described here is in the context of a biosensor design, where the capture of marker molecules, the "analyte", has to be efficient even when present in high dilution. In order to quantify the captured molecule it must be capped with a second entity with high, specific affinity for the analyte. This entity, which can genetically be referred to as "second antibody" even though it might not contain an antibody in the immunological sense of the word, is linked to a signal producing enzyme as shown in Fig. 4. In the specific Example 2, the enzyme is a Pyruvate Kinase which produces ATP from ADP supplied by the reagent solution surrounding the complex. The ATP is not directly measurable, but through its very close proximity to a Luciferase enzyme molecule immobilized nearby, it can be quantitatively reacted with available luciferin and oxygen to produce oxyluciferin under the emission of light, as illustrated in Fig. 4. The quantum yield of this reaction is near unity, and the emitted light, which can be measured with a CCD camera, is proportional to the amount of ATP produced, in turn proportional to the amount of second antibody that has bound to the captured analyte.

The ability to close-pack functional structures on the particle surface is obviously limited by available space, and where possible it is desirable to work with small binder molecules, as in Fig. 5, rather than the large IgY antibodies in Fig. 2. The analysis detailed in Fig. 2 shows particles with a diameter of 240 nm to take up 145 IgY molecules, placing these capture-entities an average of 35 nm apart. In a parallel analysis of the close-packing accomplished with the small binders of Fig.5 one finds that the surface can accommodate about 2000 such structures, implying that they are placed less than 10 nm apart. Suitably, the average distance in general between ligands is < 100 nm, preferably <40 nm, more preferred < 15nm, or even IOnm or less, but >5 nm. The invention will now be illustrated in more detail by way of the following non- limiting examples. The examples relate to application of the inventive concept to particles. However, the basic principles are equally well adaptable to flat surfaces, as is realized and understood by the skilled man who would be able to implement the invention for flat surfaces without undue experimentation. Thus, the invention as defined in the claims are to be construed as encompassing all hydrophobic surfaces.

Examples

EXAMPLE 1

Immobilization of affinity ligand lectin Concanavalin A on PS particles

A 1 % (w/v) suspension of 240 nm diameter poly-styrene (PS) particles (obtained from Bangs Lab., Hamilton, Indiana, USA) was incubated with 10 g/ L of the surfactant F108-PDS (obtained from Allviva Inc. Lake Forest, CA, USA) (PDS = pyridyl-disulfide) in buffer solution for 24 hours at room temperature under constant end-over-end shaking. After adsorption unbound and loosely bound materials were removed by centrifugation in a table-top centrifuge (Eppendorff 5417 C) at 14,000 rpm for 20 min followed by removal of supernatant and resuspension of the pellet in buffer. This washing procedure was repeated three times. In order to bind the lectin Concanavalin A (ConA) to the free pyridyldisulfide groups on the Pluronic F 108 adsorbed to the particles, free thiol groups must first be introduced into the ConA molecules. This was done using the heterobifunctional reagent, N-Succinimidyl 3-(2-pyridyldithio) propionate (SPDP) [Carlsson, J., Drevin, H. & Axen, R. Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3-(2-pyridyldithio)propionate, a new heterobifunctional reagent. Biochem J. 173, 723-737 (1978)]. A 10 μl aliquot of 30 mM SPDP-reagent in ethanol was rapidly added to 1 mL of ConA in buffer solution (3.1 mg/mL). The reaction was proceeding for 15 min at room temperature with occasional stirring before excess reagent and low molecular weight products were removed by gelfiltration using a PD-10 column (GE Healthcare). The newly introduced 2-pyridyl disulfide was then cleaved off with DTT (Sigma) to give the thiolated ConA. After removal of excess DTT and pyridine-2-thione by gelfiltration with a NAP-10 column (GE Healthcare), the thiolated product was immediately transferred to the previously prepared suspension of Pluronic F108-PDS-dG coated particles. The coupling reaction was allowed to proceed for 20 minutes at room temperature. The surface concentration of adsorbed F 108 and attached ConA was analyzed using SdFFF(Sedimentation Field-Flow Fractionation.

EXAMPLE 2

Preparation of multifunctionalized particles

A 1-% (w/v) suspension of 240 nm PS particles was incubated with 40 % surfactant F108-PDS (10 g/L) and 60 % F108-NTA (NTA = Nitrilotriacetic acid) in MiIIiQ for 24 hours at room temperature under constant end-over-end shaking to adsorb the surfactant. After the adsorption was completed, unbound and loosely bound materials were removed by centrifugation in a table-top centrifuge at 14,000 rpm for 20 min followed by removal of supernatant and resuspension of the pellet in buffer. This washing procedure was repeated three times. 3 μL was taken out for analysis with SdFFF.

A small portion of thiolated 15-mers of guanine (dG) oligonucleotides (300 μL of 10 pmol/μL dG) were allowed to bind to some of the PDS-groups on the Pluronic coated PS particles for 20 hours at room temperature. The particles were then washed 3 times by means of centrifugation at 14000 rpm for 20 min. The supernatant was removed after each centrifugation and replaced with buffer solution. 3 μL was taken out for analysis with SdFFF.

To bind the antibody (IgY) to the free PDS (pyridyldisulfide) groups on the Pluronic F 108 adsorbed to the particles, free thiol groups must first be introduced into the IgY molecules as described in EXAMPLE 1 (with ConA) above. A 30 μl aliquot of 30 mM SPDP-reagent in ethanol was rapidly added to 1 mL of IgY in buffer solution (1.16 mg/mL). The reaction was proceeding for 30 min at room temperature with occasional stirring before excess reagent and low molecular weight products were removed by gelfiltration using a NAP-10 column. The newly introduced 2-pyridyl disulfide was then cleaved off with DTT to give the thiolated ConA. After removal of excess DTT and pyridine-2-thione by gelfiltration with a NAP-10 column, the thiolated product was immediately transferred to the previously prepared suspension of Pluronic F108-PDS-dG coated particles. The coupling reaction was allowed to proceed over night at room temperature, followed by washing 3 times by centrifugation at 14000 rpm for 20 minutes.

The particles were charged with Ni²⁺ by incubating in 50 mM NiCb over night at room temperature. The particles were then washed 3 times by centrifugation at 14000 rpm for 20 minutes and 250 μL of His-tagged Luciferase (0.5 mg/mL) was added to the particle suspension and incubated 1 h at room temperature, followed by washing as described above.

SdFFF was used to characterize the multifunctionalized particles. The core particles were sized in 0.1 % aqueous FL-70 detergent while the coated particles were analysed in 2 mM NH₄HCC<3. The field strength used was 1400 rpm, the relaxation time was calculated to 12 min and the carrier flow was maintained at 1.5 ml/ min throughout each experiment. The mass of the bare particle and the mass of the adsorbed respectively attached layer was determined according to Li, J. -T 8& Caldwell, K. D. Sedimentation field-flow fractionation in the determination of surface concentration of adsorbed materials. Langmuir. 7, 2034-2039 (1991). Mass determinations for the bare particles and their coating layers are summarized in Table 1.

Table 1

Size of bare particles: 239 nm

EXAMPLE 3

Attachment of fully decorated particles to the surface A planar surface was placed face down in a solution of Pluronic F108-PDS (10 g/L). The adsorption was allowed to proceed for 24 hours at room temperature followed by washing with buffer solution.

The Pluronic F108-PDS coated planar PS surface was placed in a 1.6 μM solution of thiolated 15-mers of cytosine (dC) oligonucleotides and incubated at room temperature for several hours, followed by extensive washing of the surface with buffer solution.

To attach the multifunctionalized particles to the surface a suspension of particles coated with IgY, luciferase and dG, described in example 2, was spotted out in droplets on the analytical surface preadsorbed with Pluronic F108-PDS and saturated with dC using a pipette. The complementary dC/dG oligonucleotides were allowed to hybridize in a humid environment at room temperature for 1 h followed by repeated rinsing of the surface with buffer solution to wash away all unbound and loosely bound particles.

The particle distribution was determined by Scanning Electron Microscopy (SEM) when the fluorescence measurements were completed. A FEG-SEM (LEO 1550) was used, and the sample was coated with a thin Au/Pd film prior to imaging at 3.5 kV. The SEM picture is shown in Figure 3.

EXAMPLE 4

Complete multiparticle arrangement in suspension shows activity

During adsorption a 1-% (w/v) suspension of 240 nm PS particles was incubated with 40 % F108-PDS (10 g/L) and 60 % F108-NTA in MiIIiQ for 24 hours at room temperature under constant end-over-end shaking. After adsorption unbound and loosely bound materials were removed by centrifugation in a table-top centrifuge at 14,000 rpm for 20 min followed by removal of supernatant and resuspension of the pellet in buffer. This washing procedure was repeated three times.

Thiolated synthetic binders (Patent application SE-0600794-2; Baltzer) with affinity for CRP (C-reactive protein) was added to the F108-coated particles in a 1: 1 ratio to the number of F108-PDS groups available on the particle surface and incubated for 1 h at room temperature. After washing (3 times at 14000 rpm for 20 min., the supernatant was removed and the pellet was resuspended in buffer solution) 100 μL CRP (2.3 mg/mL) was added to the particles and they were incubated over night. The particles were washed again by centrifugation (3 times) and Binder-Pyruvate kinase conjugates were added. The Binder-Pyruvate kinase conjugates were prepared by adding thiolated Binder and Pyruvate kinase equipped with a PDS-group (prepared by SPDP modification, see examples above) in a 2: 1 ratio. The molecules are allowed to conjugate for 30 min., and the conjugates were separated from unconjugated Binders using a NAP-10 column. The Binder-Pyruvate kinase were incubated with the particles over night and were then washed 3 times as described before.

The particles were charged with Ni²⁺ by incubating in 50 mM NiCb 1 h at room temperature, and washed 3 times by centrifugation at 14000 rpm for 20 minutes. 300 μL of His-tagged Luciferase (approx. 0.5 mg/mL) was added to the particle suspension and incubated 20 min. at 5°C, followed by washing 3 times by centrifugation at 14000 rpm for 20 min. at 5°C. The supernatant was removed and the particles were resuspended in a buffer solution containing 50 mM Glycine, ImM TRIS, 5mM MgSO₄ and 0,05% BSA.

Figure 5a shows schematically a signal generating construct. The pentameric CRP is recognized by a binder and generating ATP by a second binder conjugated to a kinase molecule.

To the particles equipped with Binder, CRP, Binder-Pyruvate Kinase, and Luciferase 10 μL 6 mM ADP, 10 μL 43 mM PEP and 10 μL 15 mM luciferin was added. The bioluminescense signal was detected using a CCD camera. The result is presented in Figure 5b, which shows the bioluminescence intensity produced by the multi-particle construct schematically illustrated in Fig. 5a.

Claims

CLAIMS:

1. A reaction system for carrying out chemical reactions, comprising:

a hydrophobic surface;

surfactant molecules attached to said surface, at least a fraction of said surfactant molecules being derivatized with two or more different types of functional groups having affinity for selected ligands, with the proviso that each surfactant molecule is derivatized with only one type of functional group;

one or more different ligands attached to said derivatized surfactant molecules via said functional groups, said ligands being reaction partners in the chemical reaction(s) to be carried out, wherein

the average distance between ligands is in the nanometer scale.

2. The reaction system as claimed in claim 1, wherein the average distance between the ligands is < 100 nm, preferably <40 nm, more preferred < 15 nm, but >5 nm.

3. The reaction system as claimed in claim 1, wherein the reaction system is confined to a generally flat surface.

4. The reaction system as claimed in claim 1, wherein the reaction system is confined to a particle having a hydrophobic surface, the particle preferably having a size in the range 10-1000 nm, more preferably 100-500 nm.

5. The reaction system as claimed in claim 4, wherein the particles are made of any of a polymeric material, a metal, a glass, ceramics.

6. The reaction system as claimed in any preceding claim, wherein the surfactants are selected from poloxamers and dextrans, and other hydrophobized hydrophilic polymers.

7. The reaction system as claimed in any preceding claim, wherein ligands are selected from proteins, nucleic acid, carbohydrates, lipids.

8. The reaction system as claimed in any preceding claim, wherein a part of the surfactant molecules are charged with a metal ion, and ligand having a histidine tag is coupled to the particles via said metal ion.

9. The reaction system as claimed in claim 8, wherein the ligand is lucif erase and the metal ion is Ni²⁺.

10. A read-out assembly, comprising a reaction system as claimed in claim 3, wherein the particles are attached to a surface via a linker structure.

11. The read-out assembly as claimed in claim 10, wherein the surface is on a particle which is substantially larger, preferably an order of magnitude larger than the particles of the reaction system itself.

12. The assembly as claimed in claim 11, wherein the linker structure comprises complementary oligonucletudes, a first oligonucleotid present on the surface, a second oligonucleotide present on the particles of the reaction system.

13. The assembly as claimed in claim 12, wherein the linker structure comprises an oligo-guanine (dG) residue hybridized to an oligo-cytosine (dC) residue.

14. The assembly as claimed in any of claims 10-13, wherein the particles are laid out on said surface in discrete spots.

15. A synthesis reactor comprising a reaction system as claimed in claim

1.

16. An analytical device, comprising a reaction system as claimed in claim

1 , wherein one of the ligands is coupled to a species capable of generating a signal in response to reactions occurring at other ligands.

17. The device as claimed in claim 16, wherein the reaction system is confined to particles, the signal generating species is luciferase, and the reaction occuring at other ligand(s) generates ATP which can interact with luciferase and luciferin, thereby emitting detectable light.

18. A biosensor comprising a reaction system as claimed in claim 1.

19. The biosensor as claimed in claim 18, wherein the reaction system is confined to particles in the nanodiameter scale, and the particles are attached to other substantially larger particles, forming aggregates.

20. The biosensor as claimed in claim 19, wherein the aggregates are of a size that renders them filterable.

21. A method of making a reaction system as claimed in claim 1 , comprising the steps:

incubating a hydrophobic surface with polymeric surfactant molecules, wherein at least a part of said polymeric surfactant molecules has at least two types of free reactive groups, but wherein each surfactant has only one free reactive group or none;

providing at least two selected ligands having groups that can react with the free reactive groups on the surfactant adsorbed to the particles; and

binding said ligands to the surfactants..

22. A method of analyzing species in solution, comprising contacting a sample containing a species of interest with an analytical device as claimed in claim 16, and detecting the signal generated from the reaction system.