GB2387903A - Multiparameter analysis using tagged molecules - Google Patents

Multiparameter analysis using tagged molecules Download PDF

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Publication number
GB2387903A
GB2387903A GB0209319A GB0209319A GB2387903A GB 2387903 A GB2387903 A GB 2387903A GB 0209319 A GB0209319 A GB 0209319A GB 0209319 A GB0209319 A GB 0209319A GB 2387903 A GB2387903 A GB 2387903A
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United Kingdom
Prior art keywords
support
substrate
analyte
analyses
main surface
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GB0209319A
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GB0209319D0 (en
Inventor
Caroline Garey
Jodie Hadley
Christian Bunke
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Smartbead Technologies Ltd
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* SMARTBEAD TECHNOLOGIES LTD
SMARTBEAD TECHNOLOGIES LTD
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Priority to GB0209319A priority Critical patent/GB2387903A/en
Publication of GB0209319D0 publication Critical patent/GB0209319D0/en
Publication of GB2387903A publication Critical patent/GB2387903A/en
Application status is Withdrawn legal-status Critical

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex

Abstract

A system for multiparameter analysis comprising: <SL> <LI>(i) a substrate 10 including a main surface 12; <LI>(ii) at least one primary analyte bound to the main surface 12 of the substrate, the main surface being capable of accomodating a liquid 11 including at least one secondary analyte; and <LI>(iii) measuring apparatus for monitoring the surface 12. ```The system is distinguished in that: <LI>(iv) at least one support (microparticle or bead) is suspended in the liquid 11; <LI>(v) the support comprises an identification tag (e.g. a barcode, transponder, radio frequency identification chip (RFID)); <LI>(vi) at least one secondary analyte is attached to the support; and <LI>(vii) the measuring apparatus detects any reaction between the primary analyte and the secondary analyte based on the identification and final position of the support on the substrate 10. </SL>

Description

SYSTEM AND METHOD FOR MULTIPARAMETER ANALYSIS OF

ANALYTES

Field of the Invention

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

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

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 nucleotides thereat. Each site is effectively labelled by virtue of its spatial position on the surface of the integrated circuit. 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 probes on the microarray. This may tsult in low reliability of test results thereby obtained from the arrays. Such low reliability has, in turn, resulted in extensive quality control requirements during - 2

manufacture of the microarrays and spot arrays to ensure the quality of spotting.

Moreover, the reproducibility of sample hybridization on the microarrays has proved to be difficult to ensure during experiments leading to difficulty in attaining reliable results when reproducing experimental results.

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.

Other methods employed when undertaking parallel experiments in biochemical testing include, for example: 96-well plate ELISAs, gel-based analysis and dynamic allele - 3

J specific hybridization (DASH). All these methods of testing have limitations in the number of different parameters that can be analysed simultaneously. In 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 analyses.

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. As reactions 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 analyses 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.

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In further preferred embodiments of the invention, the analyses 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 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 traditional arrays with support assay technology.

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 I is a plan view and a side view of a single support (microparticle) comprising a sequential identification; Figure2 is a schematic sectional side view of a single support with analyses attached thereto;

Figures is a schematic diagram of a system for multiparameter analysis of analyses; Figure 4 is a schematic diagram showing the experiment reaction between supports, probes and a test sample, 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. 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 from virtually any insoluble or solid

material, for example one or more of polymers, silicates, glasses, fibres, metals or metal alloys. In 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. Each support 1 of this type has a largest dimension 3 of less than circa 250 1lm, more preferably less than 150 Em, and most preferably less than circa 100 lam in length. 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. Moreover, the support 1 has a thickness 5 which is preferably less than circa 3,um, and more preferably less than circa 1 m. The thickness of less than circa 1 lam has been shown to provide sufficient mechanical support strength for rendering the support I useable in harsh experimental conditions. A preferred embodiment of the invention concerns supports 1 having a length 3 of circa 100 m, a width 4 of circa 10 lam and a thickness 5 of circa 1 Em; such supports are capable of storing more than l OO,OOO different identification sequence bar codes 2. Experimental demonstrations of up to 100,000 different variants of the supports 1 for use in bioassays for analyte characterization experiments have been undertaken. Supports 1 of different lengths 3 in a range of 40 lam to 100,um, 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 - 7

1! manufacturing techniques used to manufacture and pattern a material layer to yield separate solid supports I with bar-coded identification 2.

A fabrication process for manufacturing a plurality of supports similar to the support I involves the following steps: (l) 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 analyses 8 are attached to a section 7 of the support 1. As mentioned the analyses 8 may be either probes or test samples depending on how experiments utilizing the supports l are designed and customised. Different types of analyses 8 may be attached to supports l 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 analyses 8, the attachment between analyses 8 and supports 1 is improved. Anodising the attachment surface 6 of the supports I is one way of providing such improved attachment enhancement. Aluminium - 8

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 8. Using an avidin-

biotin system is another approach for improving chemical binding between the supports I and their associated analyses 8. The support's 1 surface 6 may also be treated with a polymer material such as silane and/or biotin, to further enhance attachment properties.

The supports I preferably have silane baked onto their surfaces 6. Attaching linking molecules, for example avidin-biotin sandwich system, to the analyses 8 further enhances their chemical molecular attachment properties.

The enhanced attachment is preferably achieved through having covalent bonds between attachment surface 6 of the support 1 and the analyses 8. The covalent bonds prevent the analyses 8 from being dislodged from the supports 1 and causing disturbing background

noise during analysis. There is also a potential problem that loose analyses 8 could prevent the identification of reactions that have occurred. It is found to be important to wash the active supports l, said supports l having analyses 8 attached thereto, after attachment to remove any excess analyses 8 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.

In Figure 3, there is shown schematically a system indicated generally by 9 comprising a substrate (array) lO and a quantity of liquid l l 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 I 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. 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 loaded supports I placed on a substrate 10. 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 analyses 8 experiment many different types of analyses 8 may be used. For the life science industry the analyses 8 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 analyses can be, for example, dyes, preservatives, labelling chemicals (for example for tracking the movement of counterfeit products), radioactive labelling chemicals, and food. In Figure 4, an assay reaction 15 is depicted which takes place on the substrate 10 according to a first embodiment of the invention. The assay 15 consists of a liquid solution with suspended supports I and analyses 8. The analyses 8 are made up of probes 16, test samples 17, and signal emitting labels 18. Many different probes 16 are used for functioning as reaction molecules in the experiment to be performed, with each type of probe 16 being attached to a support 1 with a specific identification 2. The support I preferably with at lest one covalently bound probe 16 thereon is suspended in the liquid solution 11, which is then is placed on the main surface 12 ofthe substrate 10. The liquid - 10

solution I I may also be placed on the substrate 10 prior to the probe 16 loaded supports I being added. The test sample 17 is then added to the liquid solution 11 on the substrate 10 together with the signal emitting labels 18. One or more test sample 17 may also already be bound to the substrate 10 through, for example, a covalent bond prior to adding the supports I loaded with probes 16. This would allow the use of a prespotted 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 17. There are several detection methods that can be used as the signal emitting label 18. Examples of these signal emitting labels 18 are surface plasma resonance, 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 probe(s) 16 and one or more test sample(s) 17, they will mutually bind, preferably through a hydrogen bond, to generate a new unit 19.

To indicate such bonding between at least a probe 16 and at least a test sample 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 probe 16 and test sample 17. The signal emitting label 18 may be bound to either the support, the probe 16 or the test sample 17. As the analyses, i.e. probe 16 or test sample 17, 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.

An example of this embodiment could be the use of several supports 1 with appropriately attached probes 16 suspended in a liquid solution 11 placed in a well of a 96-well or 384-well ELISA plate 10 and a specific test sample 17 being added to 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. Each well in the ELISA plate may have multiple test samples 17 bound therein before the probe 16 loaded supports are added. Another example concerns the liquid solution 11 with probe 16 loaded supports I on a non-spotted slide 10 having added thereto a test sample 17. This - 11

example is capable of providing a very cost effective way of tailoring microarrays 10 as it potentially eliminates the need for the highly dense spotting of probes 16 onto the microarray 10. A further example of this embodiment relates to a substrate 10 with multiple test samples 17 predeposited thereon. This embodiment increases the number of parameters that can be analysed when adding the liquid solution 11 with suspended probe 16 loaded supports I to the test sample 17 spotted substrate 10.

In a second embodiment of the invention, specific test samples 17 are attached to individual supports I preferably through covalent bonds. These test samples 17 can, for example, be related to individuals in clinical trials or other research. Multiple test samples 17 can be tested against a specific probe 16 by placing the liquid solution 11 with suspended supports I on a substrate 10 with a probe 16 attached thereto. An example is employing a 96-well or 384-well ELISA plate to simultaneously test multiple test samples 17 attached to individual supports I in the same well against a specific probe 16. It is also possible to test against more than one probe 16 if several probes 16 were already attached to the substrate 10 prior to adding the liquid solution 11 with the supports 1 and test samples 17. The results of the reaction between test samples 17 and probes 16 will be based on the final position of the supports 1 together with their identification code 2. Another benefit that a system 20, illustrated in Figure 5, provides for all the embodiments of the invention is for tailoring the of experiments as the supports I with analyses 8 may easily be added to the substrate 10 eliminating the long turnaround time and expensive manufacture of conventional substrates 10. The system 20 gives an improved flexibility and customizability over conventional microarrays.

According to a third embodiment of the invention, it is possible to have a system 9 with primary analyses 16, 17 attached to a substrate 10, such as a slide or homebrew.

Secondary analyses 16, 17 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 - 12

secondary analyses 16, 17, 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, 17 as there will be a 3 dimensional interaction between the analyses as the support 1 is suspended in the liquid solution 11. The system 9 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 analyses 16, 17 may be e.g. either probes 16 or test samples 17 as outlined above.

Appropriate identification of supports 1, as mentioned above, refers to the importance of using a specific identification for a specific analyte 8, for example the probe 16 or the test sample 17. Such an arrangement also allows the use of predetermined identification codes 2 for certain analyses 8 but will also allow for matching of identification codes 2 and analyses 8 as desired when designing the experiment.

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

Table 1:

< ----- Probes ----------------- > Single Multiple A) Dipstick test B) Microarray, ELISA e g. glucose e.g. screening I test sample Single => monitoring of a against many oligos on a Test patient or home mcroarray pregnancy test Samples C) Screening tests D) Microarray+Beads, e.g. blood donor Lab-on-a-chip Multiple = > screening for HIV or Time points of the analysis newborn screening could also be added as an for cystic fibrosis additional parameter here.

When performing tests of multiple probes 16 against multiple test samples 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 - 13

pharmaceutical profiling where changes over time are important to record. It would also be possible to attach more than one type of probe 16 or test sample 17 to a support I 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 substrate 10 with loaded supports I suspended thereon in a liquid solution 11 will now be described with reference to Figures 5, 6a and fib. Laser, ultra violet (UV) 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 analyses 8. The test result of reacting analyses 8 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 analyses 8. The measuring unit has a detection unit 27 to detect the fluorescent reaction signal 19 form the interacted analyses 8 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 bar-codes 2 of the supports 1. It is preferable to have different type of signal for the support I 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.

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 - 14

example, statistical analysis such as mean calculation and standard deviation calculation can be executed for fluorescence 10 associated with each type of probe 16 and/or test sample 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 between the analyses 8 characteristics has occurred. 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 I 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 substrate 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 I read speed. A stepper-motor actuated base plate 40 supporting and bearing the substrate 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 I are tracked so that they are analysed once only.

The measuring unit's 25 reader unit 30 for image-processing is used to capture digital images of each field of the substrate 10 with a liquid solution 11 suspending supports I

with attached analyses 8 thereon. Digital images thereby obtained correspond to light - 15

transmitted through the substrate lo and past a base plate 40 and then through the supports I rendering the supports I in silhouette view; such silhouette images of the supports I 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 I 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 probe 16 and a test sample 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 analyses 8 on a support.

The intended uses of the system 20 may be in any process where experiments requiring the analysis of multiparameter analysis of analyses. The applications where several parameters are involved are for example in biochemical detection of one or more analyte - 16

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 analyses. 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 probes 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 analyses 8, would be based on the final position of the analyte 8 loaded supports 1 on the array 10 post reaction, as well as the identification code 2 of the support 1. In this situation the size of the supports l would preferably be of similar size to the surface area covered by each analyte deposited on the array.

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Claims (1)

1. A system for multiparameter analysis of analyses, 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, characterized 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, c h a r a c t e r i s e d 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, c h a r a c t e r i s e d 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 I or 2, c h a r a c t e r i s e d in that the primary analyte attached to the main surface of the substrate is a test sample and that at - 18
a t least one secondary analyte attached on a support with a specific identification in the liquid is a probe.
S. A system according to any one or more of the Claims I to 4, c h a r a c t e r i s e d in that analyses are fixedly arranged on the main surface of the solid substrate.
6. A system according to Claim 5, characterized 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 I to 4, c h a r a c t e r i s e d 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 l to 7, c h a r a c t e r i s e d 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 I to 8, c h a r a c t e r i s e d 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, c h a r a c t e r i s e d 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.
1 1. A system according to Claim 10, c h a r a c t e r i s e d in that the signalling label is a fluorescent label which is at least one of deactivated and displayed by interaction between the analyses when the system is in use.
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12. A method of multiparameter analysis of analyses, 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 substrate 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 analyses and one or more secondary analyses based on the identification and final position of the support on the substrate.
13. A system for multiparameter analysis of analyses substantially as hereinbefore described with reference to one or more of Figures I to 6b.
14. A method of multiparameter analysis of analyses substantially as hereinbefore described with reference to one or more of Figures I to 6b.
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GB0209319A 2002-04-24 2002-04-24 Multiparameter analysis using tagged molecules Withdrawn GB2387903A (en)

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GB0209319A GB2387903A (en) 2002-04-24 2002-04-24 Multiparameter analysis using tagged molecules
AU2003224297A AU2003224297A1 (en) 2002-04-24 2003-04-23 System and method for multiparameter analysis of analytes
PCT/GB2003/001725 WO2003091731A1 (en) 2002-04-24 2003-04-23 System and method for multiparameter analysis of analytes

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GB2387903A true GB2387903A (en) 2003-10-29

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