WO2003091731A1

WO2003091731A1 - System and method for multiparameter analysis of analytes

Info

Publication number: WO2003091731A1
Application number: PCT/GB2003/001725
Authority: WO
Inventors: Christian Bunke; Caroline Garey; Jodie Hadley
Original assignee: Smartbead Technologies Limited
Priority date: 2002-04-24
Filing date: 2003-04-23
Publication date: 2003-11-06
Also published as: AU2003224297A1; GB2387903A; GB0209319D0

Abstract

There is described a system for multiparameter analysis of analytes. The system comprises: 1) a solid substrate (10) including a main surface (12) extending substantially in a two dimensional plane; 2) at least one primary analyte (17) bound to the main surface (12) of the substrate (10), the main surface (12) being capable in use of accommodating a liquid (11) including at least one secondary analyte (16); and 3) measuring apparatus (27, 29, 30) arranged in visual communication with the main surface (12) of the solid substrate (10) for monitoring the surface (12). The system is distinguished in that: 4) at least one support (1) suspended in use in the liquid (11); 5) the support (1) comprises a sequential identification (2) for identification thereof; 6) at least one secondary analyte (16) is attached to the support (1); and 7) the measuring apparatus (27, 29, 30) is arranged to detect post-reaction any interaction between one or more primary analytes (17) and one or more secondary analytes (16) based on the identification and final position of the support (1) on the substrate (10). There is also described a method of multiparameter analysis of analytes using the system.

Description

SYSTEM AND METHOD FOR MULTIPARAMETER ANALYSIS OF ANALYTES

Field of the Invention

The present invention relates to a system for multiparameter analysis of analytes; moreover, the invention also concerns a method of performing such multiparameter analysis of analytes.

Background to the Invention

During recent years, there has arisen a considerable interest in techniques and associated systems for determining large number of analyte characteristics through parallel testing. Earlier, tests for detecting analyte characteristics were performed manually in a sequential manner in laboratories. Later, technological developments relating to analyte characterisation evolved towards greater automation with associated higher detection throughput. Such technological developments have been prompted by, for example, the mapping of the human genome (e.g. SNP analysis, gene expression, drug targeting, proteomics), increased need for disease monitoring (e.g. foot and mouth, and BSE for animals), and testing for drug abuse (e.g. performance enhancing drugs). There is currently a need for massively parallel high throughput testing in industries performing analysis of analytes during research and development. Non-exhaustive examples of such industries are the biotechnology, pharmaceutical, diagnostics, veterinary, petroleum, pulp and paper, food and beverage, and chemical industries. This need for high throughput methods has resulted in many different technologies and associated methods of determining analyte characteristics becoming commercially available.

There are several known experimental techniques for detemiining analyte characteristics. These techniques involve a plurality of constituent experiments which are individually labelled; when the experiments have been completed, they can be read using their associated labels for identification. Some examples of labels used at present include: (a) the position of each experiment on the surface of a test integrated circuit substrate, also known as an array, DNA array, array chip or microarray;

(b) the position of each experiment in a microtitre plate or in a tube;

(c) the position of each experiment on the surface of a membrane;

(d) a chemically attached label, such as a radionucleotide or fluorescent probe; and

(e) solution based particle arrays where each array is identified by a code, which is unique for each type of array, allowing simultaneous testing of many different arrays in one vessel.

In a United States patent no. US 6, 027, 880, a microarray is described. The microarray concerns an integrated circuit whose two dimensional surface is partitioned into a plurality of spatially disposed sites, each site corresponding to an individual experiment. Each individual experiment is provided with one or more corresponding analyte e.g. nucleotides thereat. Each site is effectively labelled by virtue of its spatial position on the surface of the integrated circuit. This use of microarray technology has become the most common approach of testing large number of oligonucleotides against whole genome samples. A major drawback with microarrays is that they have limitations on the number of probes (query molecules), which can be deposited at the spatial position sites, while maintaining the quality of the experiment results. There are also challenges with high background noise and expensive manufacturing procedures for these microarrays.

Several companies manufacture microarrays for the use in fields such as cancer research, genotyping, neurobiology, toxicology and many others. As the number of probes tested on each microarray has increased in recent years to hundreds or even several thousand, the demand for associated manufacturing equipment miniaturization and specialized materials handling has rendered the fabrication of such microarrays increasingly complex and costly. For example, when analysing genes, each contemporary microarray is arranged to allow parallel analysis of up to 12000 probes in the form of gene fragments. The characteristics of the probes being monitored on such microarrays must often also be known and isolated beforehand; such prior knowledge makes it a complicated and costly process to manufacture specific microarrays to customer requirements for each different type of organism, species or specific tests to be studied.

A further disadvantage of spotted microarrays is the variable quality of the spotted or synthesised probes on the microarray. This may result in low reliability of test results thereby obtained from the arrays. Such low reliability has, in turn, resulted in extensive quality control requirements during manufacture of the microarrays and spot arrays to ensure the quality of spotting. Moreover, the reproducibility of sample hybridisation on the microarrays has proved to be difficult to ensure during experiments leading to difficulty in attaining reliable results when reproducing experimental results. There are several different reporter systems, such as fluorescent, photoabsorptive, luminescent or radioactive labels used to indicate that a reaction has occurred between a probe (query molecule) and a test sample on a microarray. These reporter systems give poor signals depending on the quality of the query molecule. When using fluorescent reporters the signal deteriorates following exposure and when using more than one die to multiplex multiple analytes in a test sample care must be taken to choose wavelengths which do not interfere with each other.

Further disadvantages associated with microarrays are low flexibility, poor customisation properties, long manufacturing turnaround times, high cost of reagents and poor sensitivity. In attempting to solve the poor data quality and sensitivity of the interaction between the probes and test sample on microarrays, it is common practice to increase the number of identical probes on an array to hence increase the exposure to the probes (reactants) on the array. Other techniques used to improve the reaction kinetics and therefore the quality of results obtained from microarrays include for example, improvements to surface-to-volume ratios of microarrays. This can include the use of channels and porous materials as described in Akzo Nobel's published international PCT application no. WO 99/02266. Another method of increasing the surface area for the sample attachment is described in the United States patent no. US 6,133,436. It describes the attachment of an oligonucleotide to a solid array support via a bead to improve the surface area of attachment. Conventionally, the position of each experiment in a microtitre plate or in a tube was used to label experiments. Such an approach is very labour intensive and hence limits the usefulness as the number of required tests increase. It further has the disadvantage of requiring substantially large quantities of reagents, probes and test samples. Often scientists who are looking for specific tests set up their own arrays, so called "home brews", by placing their experiments on a membrane or slide. The numbers of tests that can be performed on these home brews are very limited and also have the drawbacks described above. The reading of these "homebrews" is time and labour intensive with respect to the number of data points read. These solutions use the same reporter systems as described previously.

Another approach is to perform simultaneous testing, so called multiplexing, which uses several coded arrays in one vessel rather than a microarray. This can include the use of programmable matrices with memories as described in IRORI's published international PCT application no. WO 96/36436. A recording device stores the information of what molecules and biological materials are linked to the matrix material of each programmable matrice. These matrices can be in solution in one vessel or each linked to a well of a microtitreplate. Several matrices can also be arranged in an array taking the form of a microarray. Other particle array solutions from Virtual Arrays Inc and University of Hertfordshire are described in the published international PCT applications no. WO 00/63419 and WO 00/01475 respectively. These particle based arrays allow greater customisation of the probes (query molecules), which probes are attached to coded particle arrays and tested against a test sample, than the positional based microarray solutions. This solution has problems with cross reactivity of probes (query molecules) when performing many multiplexed assays, each attached to uniquely coded arrays, in one vessel. The reporter systems described previously are used to detect the interaction between probes (query molecules) and the test sample.

Other methods employed when undertaking parallel experiments in biochemical testing include, for example: 96-well plate ELISAs, gel-based analysis and dynamic allele- specific hybridisation (DASH). All these methods of testing have limitations in the number of different parameters that can be analysed simultaneously, h most cases, these limitations result in multiple sets of experiments, each comparing one specific parameter against another, run in sequence or parallel.

Summary of the Invention

A first object of the invention is to provide an improved system for the detection of multiparameter characteristics of analytes.

A second object of the invention is to improve the parallel testing throughput of currently used two-dimensional arrays systems.

According to a first aspect of the invention, there is provided a system as defined in the accompanying Claim 1.

The system is of advantage in that it is capable of addressing at least one of the aforementioned objects of the invention.

The system is beneficial in that it is flexible and can be used to complement and/or improve existing array technology. The nature of at least one primary analyte is known by virtue of its position of binding on a solid substrate and the nature of at least one secondary analyte is determined by its support's identification means. This provides the information of binding characteristics of primary and secondary analytes by analysing their interaction. As reactions of secondary analytes in the system are tagged by individual identifiable supports, the throughput previously achieved using arrays is efficiently improved. Such improvement also allows the use of adapted conventional reading means rather than requiring new more advanced readers used for reading arrays with greater spotting density. As multiple analytes are present in the system, an improvement in the number of parameters that can be analysed simultaneously is achieved. In a preferred embodiment of the invention, the identification means is a barcode allowing for easy identification using well-established standards and methods.

In further preferred embodiments of the invention, the analytes comprises a probe and/or a test sample, which are bound to identifiable supports. The identification of the reaction can hence be established by the final position of the support on the substrate and the identification code of the support.

According to an especially preferred embodiment of the invention, the probes are spotted or synthesised onto the main surface of the array allowing the use for commonly used microarrays, which allows easy adaptation of the new system which combines the established microarrays with particle based support assay technology. Further it is preferable that the support with identification means is smaller than the area of spotted or synthesised probe on the microarray surface.

In another preferred embodiment at least two primary analytes and at least two secondary analytes are used in an experiment to truly benefit from the multiparameter analysis of the solution.

According to a second aspect of the invention, there is provided a method as defined in the accompanying Claim 12.

The method is of advantage in that it is capable of addressing at least one of the aforementioned objects of the invention.

It will be appreciated that features of the invention can be combined in any combination without departing from the scope of the invention. Description of the Drawings

Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings wherein:

Figure 1 is a plan view and a side view of a single support (microparticle) comprising a sequential identification;

Figure 2 is a schematic sectional side view of a single support with analytes attached thereto;

Figure 3 is a schematic diagram of a system for multiparameter analysis of analytes;

Figure 4a, 4b are schematic top views diagrams showing the experiment reaction between a substrate with attached primary analytes and secondary analytes bound to supports with identification means before and after wash;

Figure 5 is a schematic diagram illustrating a planar-based reader for interrogating the system; and

Figures 6a, 6b are schematic top views of a planar substrate illustrating examples of the measuring path taken by the planar-based reader of Figure 5.

Description of Embodiments of the Invention

In Figure 1, there is shown an illustration of a preferred embodiment of a support 1 for use in a system according to the invention where the supports 1 are placed on a substrate 10 to allow multiplexed detection of binding activity of analytes attached thereto. There is shown a single support 1; such a support 1 will also be referred to as a microparticle or "bead" in the following description. The support 1 can be fabricated f om virtually any insoluble or solid material, for example one or more of polymers, silicates, glasses, fibres, metals or metal alloys, fn the preferred embodiment of the invention, the support 1 is fabricated from a metal, such as gold, silver, copper, nickel, zinc or most preferably aluminium. It would also be possible for the support 1 to be partially or totally coated in either of the above-mentioned materials. The support 1 incorporates an identification 2 also referred to as a tag in the following description. Examples of identification means 2 may be based on sequential identification, varied shape and size of the supports, transponders (for example Radio Frequency Identification Chips, RFIDs) attached to the support 1, or different colours of the supports. Preferably, the identification 2 is a sequential identification which can be in the shape of at least one (or any combination thereof) of grooves, notches, depressions, protrusions, projections, and most preferably holes. The identification 2 being part of the support 1 is advantageous in that there is no need to label each support 1 after manufacture. The sequential identification 2 is suitably a transmission optical barcode. An associated sequential identification code is thereby recorded in the support 1 as a series of holes using coding schemes similar to those found on conventional bar code systems, for example as employed for labelling merchandise in commercial retailing outlets. Such a code allows the use of existing reader technology to determine the identification 2 of the supports 1, thereby decreasing the initial investment when adopting technology according to the invention.

The support 1 can be of many different types of shape, but has preferably a substantially planar form with at least a principal surface 6 as illustrated in Figure 1, which allows the supports 1 to settle flat with its main surface on a main surface of the substrate 10. Each support 1 of a preferred type has a largest dimension 3 of less than circa 250 μm, preferably less than circa 150 μm, more preferably less than circa 100 μm, and most preferably less than circa 50 μm in length. The substrate 10 and support 1 have primary analytes 17 (also referred to as probes or query molecules hereinafter) and secondary analytes 16 (also referred to as test samples hereinafter) attached thereto. Preferably the largest dimension of the supports 1 is smaller than the largest dimension of the area spotted with primary analyte 17 on the substrate 10. The rniniaturisation of the supports 1 decreases the use of reagents and secondary analyte 16 in experiments. With the support 1 smaller than the size of the spot of analyte on the microarray there is also a decreased risk in the supports 1 being associated with more than one spot of analyte when unbound supports 1 have been washed away. The support 1 has suitably a width 4 to length 3 ratio in a range of circa 1:2 to circa 1:20, although a ratio range of circa 1:5 to circa 1:15 is especially preferred. This allows good flow properties of the supports 1 during liquid handling and dispensing. It also allows good utilisation of the support's 1 surface area for the identification means 2. Moreover, the support 1 has a thickness 5 which is preferably less than circa 3 μm, and more preferably less than circa 1 μm. The thickness of less than circa 1 μm has been shown to provide sufficient mechanical support strength for rendering the support 1 useable in harsh experimental conditions. A preferred embodiment of the invention concerns supports 1 having a length 3 of circa 100 μm, a width 4 of circa 10 μm and a thickness 5 of circa 1 μm; such supports are capable of storing more than 100,000 different identification sequence bar codes 2. Experimental demonstrations of up to 32 bit binary coding schemes theoretically allowing several billion different variants of the supports' 1 codes for use in bioassays for analyte characterization experiments have been undertaken. The identification means 2 is preferably a barcode made up of sequentially arranged holes, which include error checking and directional orientation features to prevent incorrect identification of the support 1. Also end markers 8 of the support's 1 coding system may be used to indicate the intactness or reading direction of the support 1. Supports 1 of different maximum lengths 3 in a range of circa 40 μm to 100 μm, carrying between two and five decimal digits of data in the sequential identification 2, have been fabricated for use in different experiments for the detection of analyte characteristics.

Around ten million such supports 1, namely microparticles, can be fabricated on a single 6-inch diameter semiconductor-type wafer, for example a silicon wafer, using contemporary established manufacturing techniques. Advantageously the shape of the support 1 is such that it optimises the number of supports 1 manufactured per wafer and also substantially optimises the number of identification codes possible on the supports 1. Conventional photolithography and dry etching processes are examples of such manufacturing techniques used to manufacture and pattern a material layer to yield separate solid supports 1 with bar-coded identification 2. The photolithography also allows all types of planar shapes of supports to be manufactured in shapes such as elliptical, circular, rectangular, square, multi-angled and many more.

A fabrication process for manufacturing a plurality of supports similar to the support 1 involves the following steps:

(1) depositing a soluble release layer onto a planar wafer;

(2) depositing a layer of support material onto the release layer remote from the wafer;

(3) defining support features, including the sequential identification 2, in the support material layer by way of photolithographic processes and etching processes;

(4) removing the release layer using an appropriate solvent to yield the supports released from the planar wafer; and

(5) optionally treating the support material to improve its attachment properties.

Many methods of chemically treating or physically altering the support material may be used for the optional step (5) to facilitate the attachment of an analyte, such as a test sample and or a probe used in multiparameter experimental analysis, to the support 1. The treatment of the supports 1 can be performed after the release from the wafer as described above or alternatively prior to the release from the wafers or earlier in the manufacturing process steps. Alternatively, the treatment of the support material layer, step (5), could be omitted.

Figure 2 shows how secondary analytes 16 are attached to a section 7 of the support 1. As mentioned in the following examples the secondary analytes 16 are referred to as test samples, but it will be apparent to the person skilled in the art that the test samples may instead be attached to the substrate 10 with the primary analytes (probes/query molecules) 17 being attached to the supports 1 depending on how experiments are designed and customised. However, different types of secondary analytes 16 may be attached to supports 1 fabricated by steps (1) to (5) above either before or after executing photolithographic operations or releasing the supports 1 from the planar wafer. By modifying the surface 6 of the supports 1 or the secondary analytes 16, the attachment between secondary analytes 16 and supports 1 is improved. This allows good attachment of each type of secondary analyte 16 to supports 1 with a specific identification code used for multiplexed experiments. Anodising the attachment surface 6 of the supports 1 is one way of providing such improved attachment enhancement. Aluminium is a preferred material for the supports 1, and there are known methods of growing porous surfaces through aluminium anodisation to improve the attachment properties thereof. Likewise, processes for sealing such porous surfaces are also known. The Applicant has exploited such knowledge to develop a relatively simple process for growing an absorbing surface having accurately controlled porosity and depth. Such porous surfaces 6 are capable of achieving a mechanical binding to preferred analyte 16, 17. Using an avidin-biotin system is another approach for improving chemical binding between the supports 1 and their associated analytes 16, 17. The support's 1 surface 6 may also be heated with a polymer material such as silane and/or biotin, to further enhance attachment properties. The supports 1 preferably have silane baked onto their surfaces 6. Attaching linking molecules, for example avidin-biotin sandwich system, to the analytes 16, 17 further enhances their chemical molecular attachment properties.

The enhanced attachment is preferably achieved through having electrostatic or more preferably covalent bonds between attachment surface 6 of the support 1 and the secondary analytes 16, so called fixedly attaching the analytes 16 to the supports 1. The covalent bonds prevent the analytes 16 from being dislodged from the supports 1 and causing disturbing background noise during analysis. There is also a potential problem that loose analytes 16 could prevent the identification of reactions that have occurred. It is found to be important to wash the active supports 1, said supports 1 having analytes 16 attached thereto, after attachment to remove any excess analytes 16 that could otherwise increase the noise in the experiment during analysis. Discrimination of the tests is thereby enhanced through a better signal-to-noise ratio.

hi Figure 3, there is shown schematically a system indicated generally by 9 comprising the substrate (array) 10 and a quantity of liquid 11 including supports 1. The substrate 10, which herein after also is referred to as an array or microarray, has two substantially planar main surfaces 12 and can be of any desired shape, but is most preferably square or rectangular. The substrate 10 may also be made of a variety of materials, such as glass, metal, plastics materials, wafers, membranes or any other material contemporarily used for fabricating microarrays. Most preferably, the substrate 10 is fabricated from a material, for example glass (microscope slide) or plastics material (for example an acrylate), which is light transmissive. This would allow a support 1 with a transmissive bar-code identification 2 to be used with the substrate 10. The substrate's 10 top main surface 12 is planar or may be divided into sections by partitioning features, for example wells or boundaries, to prevent cross contamination between sections. The main surface 12 of the substrate 10 has preferably a surface area in a range of 0.25 cm² to 50 cm², more preferably in a range of circa 1 cm² to 25 cm² and most preferably in a range of circa 2 cm² to 6 cm². On the planar main surface 12 of the substrate 10 primary analytes 17 are fixedly attached by spotting or synthesis. The primary analytes 17 may also be electrostatically or more preferably covalently attached to the substrate 10 as discussed for the supports 1. The primary analytes 17 may then be identified through their position on the substrate 10. The liquid 11, which is placed on the substrate 10, is appropriately a liquid solution and is normally an aqueous solution. The system 9 can be considered to be an assay comprising the liquid solution 11 with secondary analytes 16 loaded supports 1 placed on a substrate 10 with position loaded primary analytes 17. The system 9 is of considerable advantage in that it is capable of providing the benefits of using two dimensional substrates 10 with established reader technology, multiplexing as well as the advantages of the contemporary assays with higher throughput, and good sensitivity and reaction kinetics.

When performing a multiparameter analysis of analytes 16, 17 experiments many different types of analytes 16, 17 may be used. For the life science industry the analytes 16, 17 may be antibodies, antigens, proteins, enzyme substrate, carbohydrates, peptides, nucleic acids, peptide nucleic acids, cell lines, chemical components, oligonucleotides, serum components, drugs or any derivatives or fragments thereof. For other industries, the analytes can be, for example, dyes, preservatives, labelling chemicals (for example for tracking the movement of counterfeit products), radioactive labelling chemicals, and food.

h Figure 4a & 4b, an assay reaction experiment is depicted which takes place on the substrate 10 according to a first embodiment of the invention.

Figure 4a shows the analyte loaded substrate 10 with analyte loaded supports 1 before reading and figure 4b shows the same when non reacting supports 1 have been washed away ready for reading. The assay reactions which takes place on the substrate 10 consists of a liquid solution with suspended supports 1 and analytes 16, 17. The analytes are made up of test samples (secondary analytes) 16, probes (primary analytes) 17, and signal emitting labels 18. Many different test samples 16 are used for functioning as reaction molecules in the experiment to be performed, with each type of test samples 16 being attached to a support 1 with a specific identification 2. The support 1 preferably with at lest one covalently bound test samples 16 thereon and more preferably coated with test samples 16 is suspended in the liquid solution 11, which is then is placed on the main surface 12 of the substrate 10. The liquid solution 11 may also be placed on the substrate 10 prior to the test sample 16 loaded supports 1 being added. One or more probe 17 is preferably bound to the substrate 10 through, for example, a covalent bond prior to adding the supports 1 loaded with test samples 16. This would allow the use of a pre- spotted microarray as the substrate 10 in the system 9. The signal emitting labels 18 may be added to the liquid solution 11 before or after adding the test sample 16 loaded supports 1. There are several detection methods that can be used as the signal emitting label 18. Examples of these signal emitting labels 18 are radioactivity or preferably fluorescence. All these signal emitting labels 18 are used for quantitative evaluation purposes. If there is a match between one or more supports 1 with test samples 16 and one or more probes 17, they will mutually bind, preferably through a hydrogen bond, to generate a new unit 19. To indicate such bonding between at least a test sample 16 and at least a probe 17, the signal emitting label 18 emits a fluorescent signal when optically interrogated. The signal could be in the form of the activating or deactivating the fluorescent label 18 when in interaction with the bonded test sample 16 and probe 17. The signal emitting label 18 is normally suspended in the liquid solution 11, but may also be bound to either the support, the test sample 16 or the probe 17. As the analytes, i.e. probe 17 or test sample 16, are bound to each other through a hydrogen bond it allows the bond to be broken separating the substrate with a primary analyte from the support with the secondary analyte, which allows the reuse of the substrate 10. Again it will be apparent to the person skilled in the art that the probes 17 and test samples 16 can be interchanged and used either as primary or secondary analytes depending on the required experiment.

Prior to reading the substrate 10 it is important to wash the excess of unbound supports 1 with test samples 16 and any excess of signal emitting labels 18 away from the main surface 12 of the substrate 10. The reading of the identification means 2 of the supports 1 with test samples 16 and their final positions having bound to appropriate probes 17 on the substrate 10 indicates the reactions that have occurred. The supports' 1 may be the same size as the area of spotted probes 17 on the substrate 10. It is however more preferable for the support 1 to cover less than circa 75%, preferably less than circa 50%, more preferably less than circa 25% and most preferably less than circa 10% of each probe's 17 spotted area on the substrate 10. By the supports 1 being much smaller than the area of spotted probe 17 on the substrate 10, several supports 1 may bind to the spot at any one time. The signal emitting label (reporter system) 18 which has bound to the probes 17 and test samples 16 will then be emitting its signal e.g. fluorescent signal which gives the quantitative measurement. The advantage of using the test sample 16 loaded supports 1 to indicate reaction is that it elhninates the need for several types of reporters. The differently coded supports 1 can be used to indicate the interaction between different probes 17 and test samples 16 while a single fluorescent reporter is used to indicate the quantitative level of the interaction. Traditionally when using fluorescent reporter systems to detect multiple interactions on a microarray it becomes difficult to perform multiplexing experiments of many probes 17 against more than 2 different types of test samples 16 as the different fluorescent reporter labels 18 used have problems with spectral overlap and poor results. It would also be possible in an alternative embodiment to perform quantitative measurements using only the test sample 16 coated supports 1 as the only reporter system when the number of supports 1 attaching to each spot of primary analyte 17 on the substrate corresponds to the amount of test sample 16 being analysed in the experiment.

An example of this embodiment could be the use of several supports 1 with appropriately attached test samples 16 suspended in a liquid solution 11 placed in a well of a 96-well or 384-well ELISA plate 10 with specific probes 17 attached to the bottom of each well. This dramatically improves the throughput of contemporary methods of batching ELISA plates, which pause until a sufficient number of plates are ready for analysis, while still allowing the use of conventional ELISA plate readers. Another example of this embodiment related to the use of a pre-spotted microarray as the substrate 10, while drastically increasing the number of probes 17 analysed through the use of several supports 1 with identification means 2 coated in test samples 16. A further example of this embodiment relates to a microscope slide with multiple probes 17 pre-deposited thereon like a "home brew microarray". This embodiment increases the number of parameters that can be analysed when adding the liquid solution 11 with suspended test sample 16 loaded supports 1 to the probe 17 spotted slide 10.

In a second embodiment of the invention, specific secondary analytes 16 are attached to individual supports 1 preferably through covalent bonds. These secondary analytes 16 can, for example, be test samples related to individuals in clinical trials or other research. Multiple secondary analytes 16 on uniquely coded supports 1 can be tested against specific primary analytes (probes) 17 by placing the liquid solution 11 with the suspended supports 1 on a substrate 10 with primary analytes 17 attached thereto. The results of the reaction between probes 17 and test samples 16 will be based on the final position of the supports 1 together with their identification code 2. Using the supports '1 as the only reporter system eliminates the use of any currently reporter system, such as radioactivity or preferably fluorescence to indicate that reaction has occurred. This gives a very good qualitative result irrespectively of the quality of the spotted or synthesised primary analyte on the substrate 10. It also gives the advantage as discussed previously in that a large number of test samples (secondary analytes) 16 attached to supports 1 may be tested against many different probes (primary analytes) 17 attached to the substrate 10.

A system 20, illustrated in Figure 5, provides further benefits for all the embodiments of the invention as it allows tailoring of experiments as the supports 1 with test samples 16 may easily be added to the substrate 10 with probes 17 eliminating the long turnaround time needed if using many different substrates 10 for testing against each test sample 16. The system 20 gives an improved flexibility and customisability over conventional microarrays, and increases the throughput allowing multiparameter analysis. Again it is preferable if the supports 1 are smaller than the deposited primary analyte on the subsfrate 10 to avoid unambiguous analysis and reading. It is also possible to have the test sample as a primary analyte and the probe as a secondary analyte in this embodiment of the invention.

According to a third embodiment of the invention, it is possible to have a system 9 with primary analytes 17 attached to a substrate 10, such as a slide, microarray or homebrew. Secondary analytes 16 attached to the identifiable supports 1 and suspended in a liquid solution placed on the substrate 10 as in the first and second embodiments above. By adding a tertiary analyte to the liquid solution another parameter of analysis can be added to the system. This can be useful if it is difficult to attach a certain analyte to the substrate 10 or support 1. The tertiary analyte may then interact with either the primary or secondary analytes 17, 16, which interaction is shown through the use of a signal emitting label 18, such as a change in colour or fluorescence. The tertiary analyte will have very good sensitivity and reaction kinetics with the secondary analyte 16 as there will be a 3 dimensional interaction between the analytes as the support 1 is suspended in the liquid solution 11. The system then uses the advantages of the existing technologies of 2 dimensional microarrays and 3 dimensional solution based arrays. As in the other embodiments the different analytes may be e.g. either probes 17 or test samples 16 as outlined above. This embodiment of the invention is comparable to a competitive ELISA experiment with quantitative measurements where there is competition between the different analytes when interacting. O 03/09173

Appropriate identification of supports 1, as mentioned above, refers to the importance of using a specific identification for a specific analyte, for example the secondary analyte (test sample) 16 or the primary analyte (probe) 17 if used on the support 1 instead of the substrate 10. Such an arrangement also allows the use of predeteπnined identification codes 2 for certain analytes 16, 17 but will also allow for matching of identification codes 2 and analytes 16, 17 as desired when designing the experiment.

The different embodiments of the system are summarised in table 1.

Table 1: < Probes >

Single Multiple

Single =>

Test Samples

Multiple = >

When performing tests of multiple test samples 16 against multiple probes 17, as described in panel D of Table 1 above, it can also be beneficial to analyse the experiments at different time points. This temporal analysis is potentially useful in pharmaceutical profiling where changes over time are important to record. It would also be possible to attach more than one type of test sample 16 or probe 17 to a support 1 allowing detection of several different signal indicators 18, for example differently fluorescing signals, from the same support 1. This could be useful when performing genetic analysis e.g. using primary extension of single nucleotide polymorphism (SNP) analysis.

A reading means used for reading the subsfrate 10 with loaded supports 1 suspended thereon in a liquid solution 11 will now be described with reference to Figures 5, 6a and 6b. When the liquid solution 11 with secondary analyte 16 loaded supports 1 have been placed on the primary analyte 17 loaded substrate 10 movement of the supports 1 over the substrate's 10 main surface can be done using established laboratory equipment such as rockers, agitators, shakers or belly dancer. This is to assure that the secondary analytes 16 of the supports 1 are given the opportunity to interact with all the primary analytes 17 on the substrate 10. When the reaction time has been sufficient allowing full interaction between the analytes 16, 17 the substrate 10 is washed removing all the secondary analyte bound supports 1 and signal indicators 18 before the substrate 10 is read and analysed.

Laser, ultra violet (UN) or light emitting diode (LED) reader equipment currently used for the analysis of for example microarrays may also be employed with the aforementioned system for analysing multiple parameters of analytes 16, 17. The test result of reacting analytes 16, 17 is measured as a yes/no binary result or by the degree of fluorescence 10 emitted from the signal emitting label 18. The system 20 consists of a reader, as shown in figure 5. The reader includes a measuring unit indicated by 25 which measures the identification 2 of the supports 1 tagged to analytes 16. The measuring unit has a detection unit 27 to detect the fluorescent reaction signal 19 form the interacted analytes 16 and a reader unit 30 to read the identification code 2 of the supports 1. The detection unit 27 has a fluorescence microscope when detecting the fluorescent signal 19 indicating reaction. The reader unit 30 has a barcode reader to read the transmissive barcodes 2 of the supports 1. If required to have multiple reporter systems it is preferable to have different type of signal for the support 1 identification 2 and the reaction detection 19, as there then is a limited risk of the signals being mixed up or being overlapping (spectral overlap). This allows for greater multiplexing (multiple simultaneous reactions) possibilities. The best solution for qualitative analysis is to use the support 1 with identification 2 as the only reporter system eliminating all the need for any radioactive or fluorescent reporters needing mercury lamps or similar to be able to detect the reporter signal. The support 1 may also be detected on the subsfrate 10 in reflective or transmissive light using a bright field light source. Once a sufficient number of supports 1 have been read, a processing unit 28 of the measuring unit 25 calculates the results of the tests associated with the supports 1. This sufficient number is preferably between 10 and 100 copies of each type of supports 1; this number is preferably to enable statistical analysis to be performed on test results. For example, statistical analysis such as mean calculation and standard deviation calculation can be executed for fluorescence 10 associated with each type of test sample 16 and/or probe 17 present. A processing unit 28 is also included for controlling the detector and reader units 27, 30 so that the each individual support 1 is only analysed once.

Normally, all the supports 1 on the substrate 10 are analysed to verify the total quality of the experiment. In cases where there could be an interest in saving time and/or processing capacity, the software of the processing unit 28 can preferably be configured to analyse only the supports 1 that give off a signal 19, for example through a fluorescent signal label 18, indicating that an interaction hetween the analytes 16, 17 characteristics has occurred. This could also be used to avoid detection of supports 1 with secondary analytes 16 which have not reacted with the primary analytes 17 on the substrate 10 but not been washed off prior to analysis. The analysis of the loaded substrate 10 using the measuring unit 25 is a very cost effective, easy to perform and suitable way to multiply the analysing capacity for low to medium sample numbers in the range of, for example, single figures to a few thousand supports 1 on each substrate 10.

Preferred paths 50 for systematically interrogating the substrate 10 are shown in Figure 6a and 6b. Figure 6a is a depiction of a meander-type interrogation regime, whereas Figure 6b is a depiction of a spiral-type interrogation regime. There are of course many other possible paths 50 apparent to one skilled in the art, for example moving the substrate 10 in an opposite direction to the path 50, moving the subsfrate in a meandering diagonal path, or covering the whole substrate in one substantially linear path across its surface. However, the regimes of Figures 6a, 6b are efficient for achieving an enhanced support 1 read speed. A stepper-motor actuated base plate 40 supporting and bearing the subsfrate 10 may be used to move the substrate 10 around while the measuring unit 25 is held stationary. The most preferable method of analysis would, however, be to move the measuring unit 25 while the substrate is held stationary. The positions of supports 1 are tracked so that they are analysed once only. It would also be preferable to scan the substrate 10 in one movement from substantially side to side while detecting the supports

1 with identification codes 2, plus any additional reporter signal 18 if used.

The measuring unit's 25 reader unit 30 for image-processing is used to capture digital images of each field of the subsfrate 10 with a liquid solution 11 suspending supports 1 with attached analytes 16 thereon. Digital images thereby obtained correspond to light transmitted through the substrate 10 and past a base plate 40 and then through the supports 1 rendering the supports 1 in silhouette view; such silhouette images of the supports 1 are analysed by the reader unit 30 in combination with a processing unit 28. The sequential identification 2, for example a bar-code, associated with each support 1 is hence identified from its transmitted light profile by the reader unit 30. The signal emitting unit 29 generates a fluorescent signal, which signal makes the labels 18 on supports 1 fluoresce indicating a positive reaction 19 between a test sample 16 and a probe 17. A detector unit 27 detects the magnitude of fluorescence 19 from activated supports 1 to identify the degree of reaction. The fluorescent signal 19 integrated over activated supports' 1 surface 6 is recorded in association with the identification bar-code

2 to construct data sets susceptible to statistical analysis.

The processing unit 28 is connected to the light source 45, the signal unit 29, the reader unit 30, and the detector unit 27 and to a display 46. Moreover, the processing unit 28 comprises a control system for controlling the light source 45 and the signal unit 29. The light silhouette and fluorescent signals 19 from the supports 1 pass via an optical assembly 41, for example an assembly comprising one or more lenses and or one or more mirrors, towards the detector unit 27 and reader unit 30. A mirror 42 is used to divide the optical signals into two paths and optical filters 43, 44 are used to filter out unwanted optical signals based on their wavelength. Alternatively, the light source 45 and signal unit 29 can be turned on and off at intervals, for example mutually alternately. Signals are received from the reader unit 30 and detector unit 27, which are processed and corresponding statistical analysis results presented on a display 46. Similar numbers of each type of supports 1 are required to give optimal statistical analysis of experiments. Such statistical analysis is well known in the art. A similar approach using filters and optical lenses may be used if multiple fluorescent signals 18 are to be detected from reactions with multiple types of analytes 16 on a support 1. When the test sample 16 loaded supports are used as the only reporter system the reading is simplified as no e.g. fluorescence or radioactivity needs to be measured speeding up the analysis and giving good quality results.

The intended uses of the system 20 may be in any process where experiments requiring the analysis of multiparameter analysis of analytes. The applications where several parameters are involved are for example in biochemical detection of one or more analyte characteristics including gene expression, SNPs analysis, nucleic acid testing, antibody or protein analysis, lead target identification and drug targeting. There will be many other applications for this system for alternative industries requiring multiparameter analysis of analytes.

It will be appreciated that modifications can be made to embodiments of the invention described in the foregoing without departing from the scope of the invention as defined by the appended claims. For example, when a conventional spotted microarray 10 with test samples 16 attached directly to the array's 10 surface 12 is used as the array (substrate) 10 in the system 20, the identification of the multiparameter reaction between analytes 16, 17, would be based on the final position of the analyte 16 loaded supports 1 on the array 10 post reaction, as well as the identification code 2 of the support 1. The primary analytes 17 and secondary analytes 16 which can be either test samples or probes can be interchanged so that there is a multiparameter analysis experiment possible irrespectively which analyte is attached to the coded support 1 or the position based coded substrate 10. In the situation of e.g. performing qualitative analysis the size of the supports 1 should be less than the half of the smallest distance between to spots of deposited analyte on the substrate 10 to avoid overlapping supports 1 attached to different spots.

Claims

1. A system for multiparameter analysis of analytes, the system comprising:

(1) a solid substrate including a main surface extending substantially in a two dimensional plane;

(2) at least one primary analyte bound to the main surface of the substrate, the main surface being capable in use of accommodating a liquid including at least one secondary analyte; and

(3) measuring means arranged in visual communication with the main surface of the solid substrate for monitoring the surface,

characterised in that:

(4) at least one support suspended in use in the liquid;

(5) the support comprises identification means for identification thereof;

(6) at least one secondary analyte is attached to the support; and

(7) the measuring means is arranged to detect any post-reaction interaction between one or more primary analyte and one or more secondary analyte based on the identification means and final position of the support on the substrate.

2. A system according to Claim 1, characterised in that identification means comprises a barcode enabling identification of the support independently of its spatial position with respect to the substrate.

3. A system according to Claim 1 or 2, characterised in that the primary analyte attached to the main surface of the substrate is a probe and that at least one secondary analyte attached on a support with a specific identification in the liquid is a test sample.

4. A system according to Claim 1 or 2, characterised in that the primary analyte attached to the main surface of the substrate is a test sample and that at least one secondary analyte attached on a support with a specific identification in the liquid is a probe.

5. A system according to any one or more of the Claims 1 to 4, characterised in that analytes are fixedly arranged on the main surface of the solid substrate.

6. A system according to Claim 5, characterised in that the solid substrate is one or more of: a spotted microarray, and an ELISA assay including wells with associated interrogation molecules therein.

7. A system according to any one of Claims 1 to 4, characterised in that the solid substrate is divided into sections by barriers operable to discourage the movement of the liquid between the sections.

8. A system according to any one or more of the Claims 1 to 7, characterised in that the solid substrate has at least one well for the containment of the liquid.

9. A system according to any one or more of the Claim 1 to 8, characterised in that the liquid solution comprises at least one or more tertiary analyte operable to interact with the primary and/or secondary analyte.

10. A system according to any one of the of the preceding claims, characterised in that the system, post reaction, in use is capable of arranging for a signalling label to be in contact with the primary analyte that is in contact with the secondary analyte and a support to indicate interaction there between.

11. A system according to Claim 10, characterised in that the signalling label is a fluorescent label which is at least one of deactivated and displayed by interaction between the analytes when the system is in use.

12. A method of multiparameter analysis of analytes, the method including the steps of:

(1) providing a solid substrate including a main surface extending substantially in a two dimensional plane;

(2) binding at least primary analyte to the main surface of the substrate, the main surface being capable in use of accommodating a liquid including at least one secondary analyte; and *

(3) providing measuring means arranged in visual communication with the main surface of the solid subsfrate for monitoring the surface,

c h a r a c t e r i s e d in that the method further comprises the steps of:

(4) attaching at least one secondary analyte to at least one support;

(5) including identification means on the support for identification thereof;

(6) suspending the support in the liquid; and

(7) arranging for the measuring means to detect post-reaction any interaction between one or more primary analytes and one or more secondary analytes based on the identification and final position of the support on the subsfrate.

13. A system for multiparameter analysis of analytes substantially as hereinbefore described with reference to one or more of Figures 1 to 6b.

14. A method of multiparameter analysis of analytes substantially as hereinbefore described with reference to one or more of Figures 1 to 6b.