Sensor surface for surface plasmon resonance assays

Abstract

The present invention relates to a method for production of an improved sensor surface for an SPR instrument, comprising forming a self assembled monolayer (SAM) on a surface and attaching ligands and protein resistant groups, preferably polyethylene glycol (PEG), directly to functional groups on said surface. The invention also relates to a sensor surface produced by these methods use thereof in SPR (surface plasmon resonance) assays or interactions.

Claims

1. A method for preparing a sensor surface for a surface plasmon resonance (SPR) instrument, comprising forming a self assembled monolayer (SAM) on a surface by reacting said surface with one or more thiol C10-C30 alkane reagent(s) having at least one type of functional groups, wherein one or more protein resistant compound(s) is/are coupled to a first fraction of said functional group(s), wherein the one or more protein resistant compound(s) comprise polyethylene glycol (PEG) or a derivative thereof; and wherein one or more capturing molecule(s) is/are coupled directly or via a linker which is not said protein resistant compound(s) to a second fraction of said functional group(s) on said surface, wherein no capturing molecule(s) is/are coupled to the protein resistant compound(s), wherein the second fraction of said functional group(s) comprises a portion of the at least one type of functional groups not coupled to the one or more protein resistant compound(s) after the coupling of the protein resistant compound(s) via the first fraction of the functional group(s), and wherein the first and second fraction of said functional group(s) are of the same or different type.

2. The method according to claim 1, wherein the surface is a metal plated surface.

3. The method according to claim 1, wherein the functional group of the one or more C10-C30 thiol alkane reagent(s) is one or more of a carboxyl, a hydroxyl, an amino, an aldehyde, an epoxy, a vinyl, a carbonyl or thiol group.

4. The method according to claim 1, wherein the C10-C30 thiol alkane reagent is MHA (mercapto hexadecanoic acid) or derivatives thereof.

5. The method according to claim 1, wherein the one or more protein resistant compound(s) have a molecular weight (mw)<20000.

6. The method according to claim 1, wherein the polyethylene glycol (PEG) has a mw of 100-20000.

7. The method according to claim 1, wherein the second fraction of the functional group(s) are activated with an activation reagent before coupling of the one or more capturing molecule(s) and an amount of the one or more capturing molecule(s) coupled to the second fraction of the functional group(s) is inversely proportional to a length of activation time; and wherein the activation reagent is selected from N-Ethyl-N′-(3-Dimethylaminopropyl) Carbodiimide/N-Hydroxy-succinimide (EDC/NHS), epichlorhydrine or bifunctional reagents.

8. A method for running an SPR assay using a sensor surface produced according to claim 1, comprising adding an analyte; interacting the analyte with the capturing molecule on the sensor surface and performing a kinetic assay and/or concentration assay.

9. A sensor surface produced according to claim 1, comprising a substrate with a self assembled monolayer (SAM) provided with functional group(s) available on said SAM, wherein the SAM is a thiol-alkane C10-C30, wherein a protein resistant compound is coupled to a first fraction of the functional group(s), wherein the protein resistant compound is polyethylene glycol (PEG) or a derivative thereof, and wherein a capturing molecule is coupled to a second fraction of the functional group(s) on the surface, and wherein no capturing molecule is coupled to the protein resistant compound, wherein the second fraction of said functional group(s) comprises a portion of said functional group(s) not coupled to the protein resistant compound after the coupling of the protein resistant compound via the first fraction of the functional group(s), and wherein the first and second fraction of said functional group(s) are of the same or different type.

10. The sensor surface according to claim 9, wherein the protein resistant compound and the capturing molecule are coupled to the same type of functional group.

11. The sensor surface according to claim 9, wherein the protein resistant compound and the capturing molecule are coupled to different types of functional groups.

12. The sensor surface according to claim 9, wherein the surface is a metal plated surface and the thiol-alkane C10-C30 is MHA-SAM.

13. The sensor surface according to claim 9, wherein the protein resistant compound is PEG mw 5000 in a concentration of 0.5-5 mM.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic view of the method steps in the production of a sensor surface of the invention;

(2) FIG. 2A is a schematic view with arrows to the functional groups where specific ligands are attached to the sensor surface in FIG. 1, and FIG. 2B shows that the PEG groups are not involved in ligand immobilization:

(3) FIG. 3 is a graph showing the results of activation time vs ligand level starting with a sensor surface of the invention; and

(4) FIG. 4 is a sensorgram from a kinetic characterization assay with immobilized ligand (30 s activation) on PEG surface synthesized according to FIG. 1/Experiment 3.

DETAILED DESCRIPTION OF THE INVENTION

(5) In a pre-study the inventors have found that self-assembled monolayers (SAMs) created from oligo(ethylene glycol) (OEG) derivatized alkanethiol compounds, or a mixture of these compounds with alkanethiol compounds without OEG, showed high non-specific binding. Poor result from cyclic voltametric analysis indicated unfavourable packing density and poor surface coverage and therefore high probability of non-wanted and non-specific binding to gold surface/disrupted SAM from samples. This result indicates that surfaces with SAM alkanethiol-OEG are unsuitable for high sensitivity applications.

(6) The present invention provides a novel sensor surface wherein the capturing molecule will be bound closely to the sensor surface and not bound to any matrix molecule to minimize baseline drifts. The capturing molecule is bound directly to an alkanethiol SAM layer (or through linker on SAM), not via PEG or OEG or any other hydrophilic or protein resistant part. The invention provides a method comprising creating a sensor surface with alkane-SAM to achieve isolating properties, wherein in a second step a hydrophilic matrix is introduced to achieve protein repellant properties while providing available functional groups for ligand immobilization on the SAM to achieve low baseline drifts.

(7) With thiol-alkanes, such as C.sub.10-C.sub.30 alkanes, reagents self-assembled monolayers (SAMs) can be created on gold surfaces. SAM C.sub.16 show high density SAM with good isolating properties which is needed to avoid non-specific binding from sample components to the gold surface and any disrupted SAM component on the sensor surface.

(8) A protein resistant compound forming matrix, such as a PEG matrix, is covalently immobilized through functional groups on the SAM layer. The matrix molecule is added solely for its protein repellant properties or other properties beneficial for the assay. Thus, both the ligand and protein resistant compound (matrix) are coupled directly to the SAM monolayer or via a linker. The linker may for example be epichlorohydrine The linker is a short molecule and not to a hydrophilic compound as in prior art.

(9) An advantage of the invention is that there is no ligand immobilization to the protein resistant compound (matrix). If identical ligands were to be immobilized on the matrix and SAM then the immobilized ligands would represent two different ligand populations, one population immobilized on the SAM layer and one population immobilized on the matrix. This would impact the assay results negatively. With the invention, all ligands are immobilized only on SAM functional groups since the PEG matrix or alternative matrices or mixture of matrices are immobilized in a separate step on SAM functional groups and do not have functional groups to be used for ligand immobilization. After deactivation (optional) remaining functional groups on the SAM monolayer are available for ligand immobilization.

(10) In the present invention, a preferred thiol-alkane reagent is MHA 16 (mercapto hexadecanoic acid). If the thiol-alkane reagent is MHA, a PEG molecule with a primary amine can be covalently attached to the carboxy moiety on the MHA SAM layer on the sensor surface. The PEG molecule with primary amine group can be covalently attached to a SAM layer with COOH (carboxy groups) through EDC/NHS chemistry. Sensor surfaces with amine functionalized PEG-molecules (such as 5 kDa) coupled to a carboxylated alkanethiol SAM show good matrix stability characteristics and proved to have high resistance towards non-specific binding from plasma samples and detergents.

(11) Protein resistant compounds (matrix) and ligands can also be immobilized through other functional groups on the SAM layer. Example of possible functional groups on the SAM layer for immobilization are: hydroxyl, amino; carboxyl, aldehyde, carbonyl, epoxy, vinyl and thiol.

(12) Example of possible functional group on the protein resistant compound (matrix) to be involved in immobilization on the SAM layer are: hydroxyl, amino; carboxyl, aldehyde, carbonyl, epoxy, vinyl and thiol. After immobilization of the protein resistant compound (matrix) to the SAM layer, any remaining functional groups on the protein resistant compound (matrix) must not be available, that is not used for ligand/capture immobilization, and/or de-activated if needed. Alternatively, they can be different from the functional group on SAM to be used for ligand immobilization.

(13) Immobilization of protein resistant compound (matrix) to the SAM layer occurs through spontaneous covalent coupling or through activation of functional groups on either the matrix or on the SAM layer, for example aldehyde on the SAM and hydrazine on the matrix. Deactivation is performed of non-reacted activated or reactive groups. Stabilization of matrix or linker might be included, for example the coupling between aldehyde and hydrazine is stabilized by NaCNBH.sub.4.

(14) Activation and de-activation procedures are known for a skilled person in the art and may be for example activation of SAM or matrix by introducing an active ester through EDC/NHS chemistry or introducing epoxide through epichlorhydrine. Alternatively, activation with established chemistry through bi-functional reagents, for example amine groups on the surface and NHS derivative on the bifunctional reagents and reactive disulphide from bi-functional reagents for ligand immobilization.

(15) Ligand Immobilization

(16) There are a number of different coupling chemistries to enable ligand immobilization to COOH groups on sensor surfaces (chip surface). The most common route is to use amine coupling, whereby carboxyl groups on the chip surface are used for covalent amide bonds with primary amine groups in proteins (ligands). However, this process does not occur spontaneously, the carboxyl groups need to be activated. Activation is performed with a mixture of N-Ethyl-N′-(3-Dimethylaminopropyl) Carbodiimide (EDC) and N-Hydroxy-succinimide (NHS). EDC reacts with the carboxyl group and forms a reactive intermediate which in turn reacts with NHS to form an active NHS ester. As ligand is passed over the sensor chip surface, the NHS-moiety (which is a good leaving group) reacts spontaneously with a primary amine group on the ligand and covalent bond between ligand and chip surface is formed. Most proteins contain several primary amines, and thus, immobilization can be achieved without seriously affecting the ligand's biological activity.

(17) To facilitate ligand immobilization, attractive electrostatic forces are employed in a process called pre-concentration. The ligand is dissolved in a coupling buffer with pH below the isoelectric point (pI) of the protein, but above the pI of the chip surface.

(18) Hereby,

(19) the ligand and chip surface obtain opposite net charges. Positively charged ligand molecules are electrostatically attracted to the negative surface and a high ligand concentration near the surface results in more efficient immobilization

(20) After ligand immobilization, excess NHS-activated carboxyl groups are deactivated with for example ethanolamine or NaOH that removes residual NHS esters so that no more protein can be immobilized to the surface during analyte injection. The process may also be performed with a positively charged surface and negatively charged ligands.

(21) The ligand level can be controlled through variation of ligand concentration, contact time for activation or ligand, composition and pH for ligand immobilization/coupling buffer or combination of this parameters. The present inventors have surprisingly found that with sensor surfaces with a MHA SAM layer according to the present invention, the ligand level is often increased with decreased activation time. This is inversed compared to available sensor surfaces with hydrogels such as CMDx. For the latter ligand level is increased with increased activation time (contact time).

(22) Experimental Section

(23) Experiment 1: Synthesis of Sensor Surface with MHA SAM and PEG

(24) For this Example reference is made to FIG. 1.

(25) Substrates comprising glass chips with gold surface were immersed in 80%/20% Ethanol/water with 1 mM MHA at 25° C. and incubated over-night. The gold chips were washed with following solutions for 5 minutes and in mentioned order; 100% Ethanol, 80% Ethanol, 50% Ethanol, 20% Ethanol and 100% water.

(26) The sensor chips were dried with nitrogen gas and immersed 0.144 M EDC and 0.050 M NHS in water and incubated for 30 minutes at 25° C. The sensor chips were washed 4 times with water and thereafter immersed in 0.252 M sodium phosphate buffer pH 8.5 with 1.79 mM O-(2-aminoethyl) polyethylene glycol mw 5000 and incubated for 30 minutes at 25° C. The PEG solution was discarded and remaining active esters were deactivated with 1 M Ethanolamine pH 8.5. Finally the sensor chips were washed four times with water. The chips were dried with nitrogen gas.

(27) Experiment 2: Immobilization of Ligand (Antibody) to Carboxyl Groups on SAM MHA Using Different Activation Times

(28) The chip from Experiment 1 was assembled with plastic carrier and hood. Thereafter, the chip was docked in Biacore 3000 according to the manufacturer's instruction in control software. The carboxyl groups on the SAM were activated in the instrument with a mixture of 200 mM EDC and 50 mM NHS in water for 30, 60, 90 and 180 seconds.

(29) 50 μg/ml Mouse IgG1 antibody in 10 mM sodium acetate pH 5 was injected over the activated chip surface for 7 minutes. The chip surface (not reacted succinimide esters) was deactivated with 1 M Ethanolamine pH 8.5 for 5 minutes. Immobilization with different activation times were performed in different flow cells. Running buffer HBS-EP (BR100188) (GE Healthcare).

(30) For this Example reference is made to the graph in FIG. 3 which shows that the immobilization level is inversely proportional against the activation time after 30 seconds.

(31) Experiment 3: Assay Using Sensor Surface of the Invention

(32) The chip from Experiment 1 was assembled with plastic carrier and hood. The chip was docked in a Biacore® 8K according to instruction in control software. The carboxyl groups on SAM were activated in the instrument with a mixture of 200 mM EDC and 50 mM NHS in water for 30 seconds.

(33) 30 μg/ml Mouse anti-β.sub.2μ in 10 mM sodium acetate pH 5 was injected over the activated chip surface for 7 minutes. The chip surface (not reacted succinimide esters) was deactivated with 1 M Ethanolamine pH 8.5 for 5 minutes. Different concentration of β.sub.2μ (1, 2, 4, 8, 16 nM) in running buffer were injected over the immobilized surface and a reference spot/surface without ligand. Running buffer was HBS-P+. β.sub.2μ binding kinetics to Mouse anti-β.sub.2μ was possible to determine. Fit to a one to one binding model was applied.

(34) For this Example reference is made to FIG. 4 which shows the reference subtracted sensorgram. Through the Biacore® 8K evaluation software the association constant (ka), dissociation constant (kd) and the affinity constant (KD) were calculated. The result was in accordance to previously established available result.