WO2007098755A1

WO2007098755A1 - A method for obtaining a microarray and an assay

Info

Publication number: WO2007098755A1
Application number: PCT/DK2006/000125
Authority: WO
Inventors: Hanne Kaas; Kristine Garde; Lars Christian Von Gersdorff
Original assignee: Toxispot A/S
Priority date: 2006-03-01
Filing date: 2006-03-01
Publication date: 2007-09-07

Abstract

A method for obtaining a substrate having a first binding partner immobilized in a region of a detection area of the substrate surface is pro- vided. The method comprises the steps of: (a) attaching a linking moiety to a detection area of the substrate surface, (b) spotting a solution of a first binding partner onto a region of the detection area provided linking moiety, and (c) treating the detection area with an agent forming a coupling between the linking moiety and the first binding partner. Also disclosed is an assay for detecting a binding partner in a fluid.

Description

A method for obtaining a microarray and an assay

Introduction

The present invention relates to a method for obtaining a sub- strate having a first binding partner immobilized in a region of a detection area of the substrate surface and an assay for detecting a binding partner in a fluid using the substrate. In a certain aspect of the invention an attachment technology applicable for a non-nucleic acid microarray is developed.

Prior art

A microarray generally requires a plurality of different probes to be attached in spatially separated regions on a substrate surface. Mi- croarrays of DNA probes were introduced commercially in the last dec- ade of the past century. The development has progressed rapidly since towards a miniaturisation of the spots to obtain a higher density.

In the early days of DNA microarrays membranes were used as support for attaching probes. Today, however, a rigid solid support is generally used. The rigid solid support may be glass, silicon, oxidised silicon, fused silica, poly(methyl-meta acrylate) (PMMA), poly(dimethylsiloxan)(PDMS), glas, etc.

There are three fundamental ways to fabricate a DNA micro array: contacting, non-contacting, and in situ synthesis of microarrays. Only the contacting and non-contacting technology is of interest as background of the present invention. According to the contacting and non-contacting technology pre-synthesised DNA is spotted onto a platform, usually a microscope slide. Microarray fabrication using contact printing is based on high definition pins that, upon contact with the microarray substrate deposit a small amount of probe solution. The pins are attached to a robotic arm that moves the pins between the different probe solutions, the glass slides where the microarray is created and a washing station. Non-contact printing is similar in terms of robotics but instead of pins, small dispensing systems are mounted on the robotic arm. The dispensing system can be based on inkjet, bubblejet or piezo actuation technology and can usually dispense in the range of 100 pL to 2 mL. Contact printing generally results in spot densities of 2000-4000 spots/cm² while non-contact printing can have slightly higher spot density.

Various techniques for immobilising the DNA to the solid support exist in the filed. Generally the solid support is modified for attach- ing the DNA strands. Unmodified DNA has been immobilised on solid supports treated with e.g. polylysine, amine, epoxy, diazonium ion, SU- 8, or agarose. DNA modified with amine groups has been immobilised on solid supports treated with aldehydes, epoxy, or isothiocyanate. DNA modified with thiol groups may be immobilised on solid supports modi- fied with gold, mercaptosilanes, maleimide, or iodoacetyl. DNA strands modified with a silane group may be immobilied on unmodified glass support.

It has also been disclosed to modify the substrate with a protein for capturing a DNA strand modified with a small molecule. Thus, avidin or streptavidine have been suggested for immobilising a DNA strand modified with a biotin moiety (Sosnowski RG, Tu E, Butler WF, O'Connell JP, Heller MJ: Rapid determination of single base mismatch mutations in DNA hybrids by direct electric field control, Proc Natl Acad Sci USA 1997;94: 1119-23 and Sabanayagam CR, Smith CL, Cantor CR. Oligonucleotide immobilization on micropatterned streptavidin surfaces. Nucleic Acids Res 2000;28:E33).

In protein microarrays, the protein to be immobilised may be expressed together with a tag or subsequently provided with a tag which may be recognised by an immobilised capturing entity. The tag may be a hexa histidine-tag which binds to solid support coated with NTA-agarose or the tag may be biotin which binds to a solid support coated with avidine or streptavidine. An immune array has been pre- parred in which biotinylated capture antibodies were immobilised onto an avidin-coated surface of a solid support (Rowe CA, Scruggs SB, FeId- stein MJ, Golden JP, Ligler FS: An array immunosensor for simultaneous detection of clinical analytes, Anal. Chem. 71(2), 433-439 (1999). It is suggested in MacBeath G and Schreiber, SL Printing Proteins as Microar- rays for High-Throughput Function Determination, Science, vol 289 (2000) to use slides that have been treated with an aldehyde-containing silane reagent. The aldehydes react readily with primary amines on the proteins to form a Schiff 's base linkage.

While the immobilisation technologies for attaching DNA or protein to a solid support is considered advanced a method applicable for a wider palette of compounds are not presently at hand for the skilled man.

Small molecule arrays has been suggested by Belleville, E., et al. "Quantitative microarray pesticidic analysis", J. Immunological Methods 286 (2004) 219-229, disclosing a quantitative, competitive microar- ray immunoassay, which permits quantification of the dichobenil degradation product 2,6-dichlorobenzamide (BAM) and the herbicide atrazine in a sample. The analytes were prepared for immobilization to a support through a complicated process in which the analyte initially was con- jugeted to ovalbumin, which subsequently was conjugated to a reagent comprising anthraquinone. The analyte-ovalbomin-anthraquinon reagent was immobilised by spotting a solution comprising the reagent and subsequently exposing the reagent to UV light.

The present invention aims at providing a method applicable for immobilisation of a binding partner to a substrate without the need for prior preceding chemical modification. In a certain aspect of the invention, the binding partner to be immobilised can be of a versatile chemical nature, including nucleic acid, protein, and small molecules.

Summary of the invention

The present invention relates to a obtaining a substrate having a first binding partner immobilized in a region of a detection area of the substrate surface, comprising the steps of:

(a) attaching a linking moiety to a detection area of the sub- strate surface,

(b) spotting a solution of a first binding partner onto a region of the detection area provided linking moiety, and

(c) treating the detection area with an agent forming a coupling between the linking moiety and the first binding partner.

In step (a) of the method a detection area of the substrate surface is covered with a linking moiety. Preferably, the linking moiety is evenly distributed on the surface. The surface may or may not be modified for enhancing the compatibility to the linking moiety. In step (b) a solution of a first binding partner is spotted onto a region of the detection area provided with the linking moiety. The spot does usually not interfere with other spots, which may appear in the detection area and the spot is generally surrounded by the layer of linking moieties distancing the spot from other optionally appearing spots. In step (c) the de- tecting area harbouring the spot is treated with an agent forming a coupling between the linking moiety and the first binding partner. Suitably, the coupling is a covalent linkage for an attachment reliable under various conditions to be obtained.

In the present description and claims the term "first binding partner" refers to a molecule being immobilised. The first binding partner may also be referred to herein as probe. The term "second binding partner" refers to the molecule being captured by the first binding partner. The second binding partner may also be referred to herein as target. The term "substrate" denotes the solid support onto which the binding partner is immobilised. The term "platform" is usually used herein to denote the assembly of substrate and immobilised binding partner.

The substrate may be any conventionally material and may have any convenient design. To be able to fit into a standard scanning apparatus the dimension of the substrate usually resembles that of a microscope slide (25mm x 76mm). Suitable materials for the substrate include glass, poly(methyl-meta acrylate) (PMMA), and poly(dimethylsiloxan)(PDMS). However, any rigid, impervious transparent or non-transparent material may be used. In an aspect of the invention the detection area is pre-treated with an agent providing a chemical group capable of reacting with a chemical group of the linking moiety. Various methods are available for the skilled person for surface modification of a substrate, including pro- viding the substrate with a chemical group or compound selected from polylysϊne, amine, epoxy, diazonium ion, SU-8, agarose, gold, mercap- tosilanes, maleimide, iodoacetyl, aldehyde, isothiocyanate, thiol, silane, aldehyde, keton or NO_x. For references, see review by Dufva, M in Bio- molecular Engineering, Volume 22, Issues 5-6, December 2005, Pages 173-184. Preferably, the chemical group provided on the substrate is selected from the group consisting of aldehyde, isothiocyanate, and epoxy.

In a certain embodiment the chemical group attached to the substrate by pre-treatment is an aldehyde group. The aldehyde groups may be provided by a number of chemical reactions. In a preferred aspect of the invention, the agent providing the chemical group on the substrate is a polysaccharide reacted with 1O₄ ^", thereby forming aldehyde groups. An example of a method of providing aldehyde groups involve heat treatment of an aqueous agarose solution with NaIO₄, and subsequent spreading on the detection area of a substrate. Specific embodiments are disclosed in Dufva M, et al. : "Characterisation of an inexpensive, non-toxic, and highly sensitive microarray substrate", BioTech- niques 37:286-296 (August 2000) and Afanassiev V et al. : "Preparation of DNA and protein micro arrays on glass slides coated with an agarose film", Nucleic Acid Research, 2000, Vol. 28, No. 12.

The linking moiety preferably comprises a chemical group capable of reacting with the chemical group of the substrate, thereby forming a linkage between the substrate surface and the linking moiety. The linkage is preferably a covalent bond, but may be established due to hydrophobic/hydrophilic interactions or hydrogen-bonds. The chemical group of the linking moiety is preferable selected so as to provide for a chemical reaction to take place. Examples of chemical groups of the linking moiety include: amine, epoxy, diazonium ion, mercaptosilane, maleimide, iodoacetyl, aldehyde, isothiocyanate, thiol, silane, aldehyde, keton and NO_x. In the event the detection area is pre-treated with an agent providing an aldehyde, the chemical group of the linking moiety is preferably a nucleophilic group. In a certain aspect of the invention the linking moiety comprises an amine group. The linking moiety may be selected from a wide group of compounds. However, an important feature of the linking moiety is the ability, apart from binding to the substrate, also to be able to bind to a first part of a binding partner. Therefore, preferred linking moieties are bi- functional molecules having the ability to react at one part of the mole- cule with a chemical group of the detection area to provide for attachment of the linking moiety to the substrate and to react at another part of the molecule with the first part of a binding partner. In a preferred embodiment the linking moiety is peptide or amino acid.

When the linking moiety is selected as an amino acid, said amino acid suitably has a side chain comprising an amine group. In an embodiment of the invention the amino acid is arginine.

A peptide is generally preferred as the linking moiety because a variety of functional groups are available for reaction with the first binding partner. The functional groups include hydroxyl groups from tyro- sine, serine, and threonine; thiol groups from cysteine; amine groups from lysine, arginine, asparagines, glutamine, and histidine; and car- boxylic acids from aspartate and glutamate. The functional groups may be treated with a suitable reagent to provide for a coupling to be formed to the first binding partner. Suitable linking moieties include bovine se- rum albumin (BSA) or ovalbumin (OA).

The linking moiety may be provided on the detection area in a zone beneath the spot or a group of spots. However, the full benefit of the invention is obtained when the linking moiety is attached to the entire surface of the detection area. When a standard microscope slide is used as substrate, the entire surface of the substrate can be covered with a layer of the linking moiety. Suitably, the linking moiety is applied the surface as an aqueous solution. The solvent is allowed to evaporate and the dry or moist surface is used for spotting.

The first binding partner is a member of a binding pair in addi- tion comprising a second binding partner. The first and the second binding partner have a certain affinity for each other. The affinity may be relatively high, i.e. in or above the micromolar range. In a preferred aspect of the invention the affinity is 10^"4M or higher. The affinity of the binding partners towards each others is usually specific, i.e. the tendency to cross-bind to other substance is low.

Suitable examples of binding pairs include antigen-antibody interaction, compound-aptamer interaction, antibody-antibody interactions, protein-small molecule interactions, enzyme-substrate interac- tions, nucleic acid-nucleic acid interactions, nucleic acid-protein interactions etc. When a first binding partner is selected as an antibody the assay is generally referred to as an immunoassay or antibody microarray and when the first binding partner is a protein the assay is referred to as a protein assay or protein microarray. If the first binding partner is a nucleic acid the assay may be referred to a DNA microarray.

In a certain aspect of the invention, the method provides a platform for performing a competitive assay, in which the first binding partner is immobilised on the substrate. The first binding partner may be selected from a variety of sources including bacterial toxins, fungal toxins, algal toxins, shellfish toxins, fish toxins, enzymes occurring in the synthesis pathway of toxins, markers for the occurrence of pathogenic micro organisms, geosmins, plant protection products, pharmaceutical products and degradation products of the above mentioned group of compounds. A certain group of binding partners of interest is algal toxins, such as cyanotoxins, especially hepatoxins and neurotoxins. Specific examples of hepatoxins include microcystein (MC) like microcystins-LR (MC- LR) and microcystin-RR (MC-RR), nodularin, and cylindrospermopsin. Specific examples of neurotoxins include anatoxins and saxitoxins. Other algal toxins of interest include paralytic shellfish toxins/poisoning (PST, PSP, saxitoxins and gonyautoxins), diarrheic shellfish toxins/poisoning (DST, DSP, ocadeic acid, yessotoxin, etc.), amnesic shellfish toxins/poisoning (AST, ASP, domoic acid), ciguatera fish toxins/poisoning (CFT, CFP, ciguatera etc.), neurotoxins shellfish tox- ins/poisoning (NST, NSP, brevetoxin, etc.), and endotoxins (lipopolysac- charides, LPS).

The chemical nature of the binding partners may vary considerable. In a certain embodiment the first binding partner is a polypeptide, such as a microcystin, or an alkaloid.

The solution of the first binding partner to be used for spotting is preferably an aqueous solution. An organic solvent may be selected in special circumstances, e.g. in the event the binding partner to be immobilised is not sufficient soluble in water. The spotting buffer, i.e. the solution in which the binding partner is dissolved or dispersed, may comprise salts, polymers, and further additives. As an example, the spotting buffer may contain a certain amount of formaldehyde, betaine, or DMSO for reducing the evaporating time or to avoid or inhibit degradation of the first binding partner. Generally, it is desired to add to the spotting solution such additives which will provide for a sufficient high viscosity that reduces mass transport by capillary actions and provide for sufficient low evaporation for allowing the binding partner to react with the linking moiety. An adjusted evaporation rate is also important for spot homogeneity and morphology. Various commercial spotting buffers are suitable. An example of such buffers is Genetix's amine spotting buffer.

The spotting solution is generally applied in a small quantity in the sub-μl range. In a preferred embodiment, the solution of the first binding partner is spotted in an amount of lOnl or less. Suitably, each spot is applied in an amount of about InI or less. A deposited amount of spotting solution of InI corresponds to a spot diameter of around lOOμm.

The substrate usually comprises more than a single spot. Generally, a plurality of different first binding partners are spotted in corre- sponding discrete regions of the detection area. Such design is generally referred to as a microarray.

Manufacture of a microarray according to the invention may be effected using contact printing or non-contact printing. Contact printing implies that a hollow pin is allowed to soak a minor amount of spotting solution. Subsequently, the spotting solution is deposited by the pen on the detection area. Non-contact spotting makes use a small dispenser instead of the pen. The dispensing system can be based on inkjet, bub- blejet or piezo actuation technology. The binding partners attached to the substrate in discrete regions may be of the same or a related chemical nature. As an example two different proteins are regarded to have a related chemical nature. According to a certain embodiment of the invention, at least two of the plurality of first binding partners are chemically unrelated. An example of chemically unrelated compounds is a protein and a small molecule not exclusively comprising naturally occurring amino acids

After the first binding partner has been spotted onto the substrate, it is treated with an agent forming a coupling between the linking moiety and the first binding partner. The agent may e.g. be of a chemi- cal nature or radiation. Coupling agents of a chemical nature are well- known in the art and include mineral acids, oxidation agents, and reducing agents. When radiation is used as a coupling agent, the radiation may be ultra violet light, gamma radiation, alpha-radiation, or beta- radiation. In a suitable embodiment, the agent used for treating the spotted detection area is ultra violet (UV) light. Suitably, the formed coupling is a covalent bond.

After the first binding partner has been coupled to the linking moiety, the platform should be handled in a suitable fashion to avoid degeneration. Depending on the specific design of the microarray, the platform may used immediately upon manufacture or it may be stored under dry or moist conditions at ambient or cold temperatures.

The invention also relates an assay for detecting a binding partner in a fluid comprising the steps of contacting the detection area of the substrate with a sample suspected of comprising a free first bind- ing partner and a second binding partner capably of binding to the free and immobilised first binding partner, removing excess non-reacted second binding partner and free first binding partner, and analysing the region comprising the immobilised first binding partner to detect whether the second binding partner has reacted with the immobilised first binding partner.

In one aspect of the invention the second binding partner is an antibody or a fragment thereof having retained activity, said antibody or fragment being directed to the first binding partner. This assay design is commonly referred to as competitive immunoassay because the free and the bound binding partner, i.e. antigen, compete for binding with the antibody. Thus, a large amount of free antigen in solution will result in a low binding to the immobilised antigen and conversely low antigen concentration in solution results in a high tendency. In an alternative design, which is also included in the present scope of protection, the antibody is immobilised and the sample is spiked with a labelled antigen that competes with the unlabelled antigen suspected in being contained in the sample. The second binding partner may also be an aptamer or a protein-displaying phage having affinity for the first binding partner.

To detect the binding or non-binding of the second binding partner, the second binding partner may be directly labelled. Suitable labels include fluorescence labels like Cy-3 or Cy-5. Generally, however, it is suitable to incubate with a secondary antibody capable of binding to the second binding partner before analysing. Preferably, the secondary antibody comprises a detectable label or an enzyme capable of producing a detectable label.

The detectable label of the second label may be a fluorescence label or a radio active label. Preferred fluorescence labels include Cy-3 and Cy-5.

Alternatively, reactions between the first and the second binding partner may be measured using scanning electron microscopy or by using colorimetric detection of nanoparticles, such as nanogold particles, similar to the method disclosed in Han A. et al "Detection of analyte binding to microarrays using gold nanoparticle label and a desktop scanner" Lab Chip, 2003, 3, 329-332.

A sample comprising a first binding partner may be quantified with respect to the first bind partner. Generally, the concentration of first binding partner in a sample is calculated by comparing the meas- ured signal of a label in a region comprising an immobilised first binding partner with a standard curve prepared for know concentrations of the first binding partner.

Basically, the sample to be analysed by the present assay can stem from any source. In a certain aspect of the invention, the sample is a water sample and the first binding partner it is suspected of containing is selected from the group consisting of bacterial toxins, fungal toxins, algal toxins, shellfish toxins, fish toxins, enzymes occurring in the synthesis pathway of toxins, markers for the occurrence of patho- genie micro organisms, geosmins, plant protection products and pharmaceutical products, and degradation products of the above mentioned group of compounds.

Samples may comprise a variety of compounds to be analysed by the assay of the invention. Therefore a fast parallel method of de- tecting a multitude of compounds is provided. In the event the substrate comprises a plurality of different first binding partners immobilised on the substrate a corresponding number of parallel measurements of these compounds may be conducted. Thus, in an aspect of the invention multiplex quantification of a plurality of first binding partners are performed simultaneously.

Description of the drawings

Fig. 1 discloses a schematic representation of the immobilisation method, Fig. 2 shows a typical slide and array layout

Fig. 3 schematically shows the slide layout used in example 1 Fig. 4 shows the principle of the competitive assay Fig. 5 Shows a diagram of the results of example 2. Fig. 6 shows a standard curve obtained in example 2 Examples

Example 1

Manufacture of the microarray chip targeting microcystin (MIA Bio-Chip)

Manufacture of activated chip surface

An 1% activated agarose solution was prepared by dissolving 1.5 g of UltraPure Electrophoresis grade Agarose (Ivitrogen, UK) and 0.32 g NaIO₄ (Fluka Chemie GmbH, Switzerland) in 150 ml MiIIiQ water (Millipore, Bedford, MA, USA) (sufficient for preparing 100 - 120 slides). The agarose was dissolved in the MiIIiQ water by heating the solution in a microwave owen until the agarose was completely dissolved before the NaIO₄. For preparation of microarray substrates, 76 x 26 mm unmodified microscope glass slides (Superfrost, Menzel-Glaser, Germany) were coated with a thin 1% activated agarose film. 1 ml of the hot 1% agarose solution was distributed evenly onto each slide and left to solidify 30 minutes at room temperature. After solidification of the agarose, the slides were immersed in MiIIiQ water for three hours at room temperature and afterwards dried overnight at room temperature. The acti- vated slides were stored until use.

Microarray Spotting, and Antigen Immobilization

Using a QArray microarray printer (Genetix, UK), 8 identical arrays were printed. Each array consisted of different spots: microcystin- LR (MC-LR) and negative controls. The spots were printed onto the ac- tived slides in a cooled chamber (temperature below 12 ⁰C). As a control also EasySpot (U-vision Biotech Inc., Taiwan) microarray substrates were applied. Immobilization of microcystin to the slide surface was ensured by pre-incubating the slide surface in 0.1% BSA for 15 minutes at medium agitation prior to printing, and exposing the printed arrays to UV light after spotting (UV light at a wavelength of 254 nm in a Strata- linker 2400 (Stratagene, USA) for 4 minutes). The final microcystin concentration in the printing solution was 0.5 mg/ml, obtained by diluting a stock solution with Genetix amine spotting buffer (Genetix, UK). Spot volumes were approximately 1 nl/spot delivered by CMP3 Chipmaker spotting pins (TeleChem International, USA). See fig. 2 for array and spotting layout. Two negative controls were spotted: A negative control for the spotting procedure (MiIIiQ water) and a negative control for un- specific binding of the antibodies (Avidin). The spot density was ~563 spots/cm².

Surface Inactivation /Blocking

Three in-house manufactured activated agarose (AG) slides and three commercial EasySpot (ES) slides were used in the experiment.

Following immobilization, the microarray substrate surface not involved in immobilizing the printed spots were inactivated for preventing unspecific background. Thus, the microarray substrates were blocked by incubating the slides for 10 minutes in TBST/0.1% BSA, fol- lowed by a 2 minute rinse in MiIIiQ water. The slides were dried by spinning in a Mini Centrifuge (National Labnet co., NJ, USA) for 30 seconds.

The 8 individual arrays were separated by applying a Blue Film layer with at a structure cut using a CCVIaser, see Figure 3. The dimensions are (height x width) 80 x 26 mm, designed to match a standard microscope slide with a 5 mm slip at the bottom for swift displacement of the film before scanning. 10 wells are cut in the film, but only well 1 - 6 are used in this experimental setup.

The processed microarray substrates were stored in dry conditions at room temperature until use.

Quantification of Microcystin (MC)

See Figure 4 for a schematic overview of the competitive MIA BioChip process. In a first step, a water sample was incubated with the primary detection molecule (mAb against MC) on the BioChip. The mAb was the anti-microcystin antibody MC10E7 obtained from Alexis Bio- chemicals. The primary mAb was bound in part to MC in the water sample, and to the MC-LR on the BioChip. After incubation, primary mAb not bound to MC-LR on the BioChip is washed away. In a second step, the BioChip was incubated with a secondary detection molecule (pAb-Cy3) which binds to the mAb bound on the BioChip. After incubation, secondary pAb-Cy3 not bound on the BioChip was washed away. The BioChip was then subjected to scanning and quantification.

Generation of standard curves using the MIA BioChip

Solutions with known microcystin derivate MC-LR concentrations were prepared for generation of a standard curve. In this setup 6 MC-LR concentrations were used.

Table 1

Before exposed to the microarray, the standard solutions were mixed with primary antibody in a 1 :1 reaction mix prepared by mixing 10 μl of standard with 10 μl of primary monoclonal antibody (mAb-LR) (MC10E7, Alexis Biochemicals), diluted 5,000-fold (200 ng/ml) in TBS incubation buffer, supplemented with concentrations of each 0.05% (w/v %) BSA Fraction V and (v/v %) Tween 20. Each standard solution (20 μl) was then exposed to an individual array for 60 minutes. The mi- croarrays were incubated uncovered in a humid chamber at room temperature.

Following incubation, the microarrays were washed for 10 minutes in TBS buffer supplemented with 0.1 % (v/w %) Tween 20, rinsed with MiIIiQ water for 2 minutes and spin-dried.

The microarrays were then incubated with the secondary anti- body, a Cy3 labelled polyclonal anti-mouse antibody (pAb-Cy3) (Sigma-

Aldrich, Germany). Incubation time was 25 minutes using 20 μl for each array of a 5,000-fold (200 ng/ml) dilution of the secondary antibodies in TBS buffer, supplemented with each 0.05% (w/v %) BSA Fraction V and (v/v %) Tween20. The microarrays were incubated uncovered in a humid chamber at room temperature.

Following incubation, the microarrays were washed for 10 min- utes in TBS buffer supplemented with 0.1 % (v/w %) Tween 20, rinsed with MiIIiQ water for 2 minutes and spin-dried.

Data acquisition and analysis

Fluorescent pAb-Cy3 emissions were acquired with a ScanArray LITE confocal laser scanner (Perkin Elmer, MA, USA) and quantified us- ing the freeware software ScanAlyze 2.50

(http://rana.lbl.gov/EisenSoftware.htm). The signal intensity of each spot was quantified along with the background fluorescence from each slide. The background value was then subtracted from the signal intensity values These data were then used to generate standard curves in a semi-log graph, and the standard error of mean (SEM) was calculated for each point in standard curve. To quantify the MC-concentration in a sample, the standard curve was modelled by using the 4-parameter- logistic method by Rodbard according to the equation:

Wherein

V₂: Fluorescence signal bi: Maximum fluorescence signal (upper asymptote) b₄: Minimum fluorescence (lower asymptote)

V₁: Concentration of analyte b₃: IC50 of signal b₂: Transition between bl and b4. (reference: Rodbard, D. Statistical Quality-Control and Routine

Data-Processing for Radioimmunoassays and Immunoradiometric As- says. Clinical Chemistry 20, 1255-1270 (1974)). The model is then used to quantify the MC-concentration in the sample.

Results

In general the signal intensity from the ES slides were accept- able, however some background fluorescence were noticed. The signal intensity from the AG slides was a bit lower than the ES slides, but still acceptable. The background fluorescence from the AG slides was considerably higher than from the ES slides. The higher background level may be explained by the scanning method, as the focus of the laser scanner is very sensitive to the height of the agarose film. To evaluate if the fluorescence background observed on the AG slides is caused mainly by the agarose film, a blank unspotted and unprocessed slide was scanned under the same conditions as the spotted processed AG slides. As this gave fluorescence background similar to that observed in the spotted processed AG slides, this seems not to be the case. Other scanners, as a CCD-based scanner (e.g. ArrayWoRx) may reduce the background signal since they are less sensitive to the height of the agarose film.

Figure 5 shows the results of the microcystin quantification on ES and AG based microarrays, respectively. It is observed that the signal intensity from the ES slides generally is higher than the AG slides. This may be explained by a higher background observed in the AG slides, as the background signal is subtracted from the signal intensity values. The chip-to-chip variation is low for the individual substrates (ES 1, ES 2 and ES 3, respectively AG 4, AG 5 and AG 6). Hence, the method is capable of producing standard curves applicable for quantification of unknown microcystin concentrations.

Example 2

In this experiment the MIA BioChip is used to quantify the microcystin concentration in an extract made from a natural sample col- lected at a Polish water work.

Experimental setup

The setup of the experiment was identical to Example 1 with the following modifications: The glass carrier was coated with agarose and the arrays were separated by applying hydrophobic borders using a PAP-pen instead of the structure cut in blue film. The sample volume applied in each array was increased to 30 μl, with the reaction mix ratio still being 1 : 1. Only 5 standard solutions were applied.

Table 2

MC-LR standards and sample dilutions used

Array no. AOl - A05 were used for generation of a standard curve, while A06 were used to estimate microcystin concentration in Sl and A07 and A08 were used for a double estimation of S2.

Results

The standard curve from the experiment was used to quantify the microcystin-concentration in the water sample. Figure 6 shows the resulting standard curve generated from BioChip AG. The standard curve is acceptable and close to the shape of an ideal standard curve. The results of the analyses of the extract samples are indicated. The concentration in Sl and S2 are estimated to 7.7 and 0.64 ug/L, respec- tively, i.e. the signal of the samples differed by approximately a factor 10. This agrees with the differences in dilution (1 :10.000 contras 1: 100.000).

The estimated concentrations were compared with correspond- ing results obtained by HPLC analyses of the same sample. This comparison is not straight forward as although primary targeting MC-LR, the applied antibody does also detect other microcystins with similar structure; which other derivates is not obvious. In the comparison the HPLC values is calculated by summing the concentrations of derivates that to our best knowledge can be expected to be included in the immunoassay.

Table 3 gives the concentrations estimated using the two methods. The results cooperates the applicability of the described assay (using agarose coated slides and fluorescence detection) with samples ob- tained from natural sources. The results of MIA BioChip were comparable to the HPLC analyses.

Table 3

A comparison between microcystin concentrations of the extract and stan- dard curve, using HPLC and MIA BioChip analyses, respectively.

Claims

P A T E N T C L A I M S

1. A method for obtaining a substrate having a first binding partner immobilized in a region of a detection area of the substrate surface, comprising the steps of: (a) attaching a linking moiety to a detection area of the substrate surface,

2. The method according to claim 1, wherein the detection area is pre-treated with an agent providing a chemical group capable of reacting with a chemical group of the linking moiety.

3. The method according to claim 2, wherein a linkage between the substrate surface and the linking moiety is formed by reaction of the chemical groups of the substrate and the linking moiety, respectively.

4. The method according to claim 3, wherein the chemical group provided on the substrate is selected from the group consisting of aldehyde, isothiocyanate, and epoxy.

5. The method according to any of the claims 2 to 4, wherein the agent providing the chemical group on the substrate is a polysaccharide reacted with 1O₄ ^", thereby forming aldehyde groups.

6. The method according to claim 1, wherein the linking moiety comprises an amine group.

7. The method according or claim 1, wherein the linking moiety is peptide or amino acid.

8. The method according to claim 7, wherein the amino acid is arginine.

9. The method according to claim 6 or 7, wherein the linking moiety is bovine serum albumin (BSA) or ovalbumin (OA).

10. The method according to any of the claims 1 to 9, wherein the linking moiety is attached to the entire surface of the detection area.

11. The method according to claim 1, wherein the substrate material is selected from the group consisting of glass, poly(methyl- meta acrylate) (PMMA), and poly(dimethylsiloxan)(PDMS).

12. The method according to any of the claims 1 to 11, wherein the first binding partner is selected from the group consisting of bacterial toxins, fungal toxins, algal toxins, shellfish toxins, fish toxins, en- zymes occurring in the synthesis pathway of toxins, markers for the occurrence of pathogenic micro organisms, geosmins, plant protection products and pharmaceutical products, and degradation products of the above mentioned group of compounds.

13. The method according to claim 1, wherein the first binding partner is a polypeptide or an alkaloid.

14. The method according to any of the claims 1 to 14, wherein the solution of the first binding partner is spotted in an amount of lOnl or less.

15. The method according to any of the preceding claims, wherein a plurality of different first binding partners are spotted in corresponding discrete regions of the detection area.

16. The method according to claim 15, wherein at least two of the plurality of first binding partners are chemically unrelated.

17. The method according to any of the claims 1 to 16, wherein the agent used for treating the spotted detection area is ultra violet

(UV) light.

18. An assay for detecting a binding partner in a fluid comprising the steps of: contacting the detection area of the substrate obtained accord- ing to any of the claims 1 to 17 with a sample suspected of comprising a free first binding partner and a second binding partner capably of binding to the free and immobilised first binding partner, removing excess non-reacted second binding partner and free first binding partner, and analysing the region comprising the immobilised first binding partner to detect whether the second binding partner has reacted with the immobilised first binding partner.

19. The assay according to claim 18, wherein the second binding partner is an antibody or a fragment thereof having retained activ- ity, said antibody or fragment being directed to the first binding partner.

20. The assay according to claim 18, wherein the second binding partner is an aptamer or a protein-displaying phage having affinity for the first binding partner.

21. The assay according claim 18, comprising the further step of incubating with a secondary antibody capable of binding to the second binding partner before analysing.

22. The assay according to claim 21, wherein the secondary antibody comprises a detectable label or an enzyme capable of producing a detectable label.

23. The assay according to any of the claims 18 to 22, wherein the concentration of first binding partner in a sample is calculated by comparing the measured signal of a label in a region comprising an immobilised first binding partner with a standard curve prepared for know concentrations of the first binding partner.

24. The assay according to any of the claims 18 to 23, wherein the sample is a water sample and the first binding partner it is suspected of containing is selected from the group consisting of bacterial toxins, fungal toxins, algal toxins, shellfish toxins, fish toxins, enzymes occurring in the synthesis pathway of toxins, markers for the occurrence of pathogenic micro organisms, geosmins, plant protection products and pharmaceutical products, and degradation products of the above mentioned group of compounds.

25. The assay according to any of the claims 18 to 24, wherein multiplex quantification of a plurality of first binding partners are performed simultaneously.

26. Use of a substrate obtained according to any of the claims 1 to 17 for detecting a first binding partner in a fluid.

27. Use of a substrate obtained according to any of the claims 1 to 17 for multiplex quantification of a plurality of first binding partners in a fluid.