Amine-Reactive Biosensor
Described are methods, articles of manufacture, and kits for coupling an analyte-binding molecule to the surface of a biosensor through formation of amide bonds. One amide bond is formed between a first carboxyl group on a polymer and a first reflecting surface comprising an aminoalkyl moiety. A second amide bond is formed between a second carboxyl group on the polymer and an amine group on an analyte-binding molecule to be coupled. The present invention thus provides for covalent attachment of the analyte-binding molecule to the biosensor, thus providing advantages over non-covalent attachment methods of the prior art. These advantages include the ability to couple without using bio tin (which, in some instances can alter functional properties of a molecule) the ability to improve the fidelity with which a binding or dissociation reaction that takes place on the surface of the biosensor represents a solution phase reaction, and the ability to regenerate the sensor by stripping ligands from the covalently bound analyte-binding molecule.
1 . A method for derivatizing an optical assembly, comprising:
providing said optical assembly, said assembly comprising an optical element adapted for coupling to a light source via a fiber, said optical element comprising a transparent material, a first reflective surface comprising an aminoalkyl moiety, and a second reflecting surface separated from said first surface by a distance, d;
a polymer comprising plurality of carboxyl groups; and
an analyte-binding molecule comprising an amine group;
activating said plurality of carboxyl groups by exposing said polymer to a water-soluble carbodiimide and an N-hydroxysuccinimide;
exposing said plurality of activated carboxyl groups to a reaction mixture comprising said analyte-binding molecule to form a first amide bond between said polymer and said aminoalkyl moiety and a second amide bond between said polymer and said analyte-binding molecule; and
quenching said reaction mixture, thereby derivatizing said optical assembly.
2 . The method of claim 1 , wherein said polymer is a polypeptide.
3 . The method of claim 2 , wherein said polypeptide is bovine serum albumin.
4 . The method of claim 1 , wherein said analyte-binding molecule is a polypeptide.
5 . The method of claim 1 , wherein said analyte-binding molecule is a nucleic acid.
6 . The method of claim 1 , wherein said distance, d, is at least 50 nm.
7 . The method of claim 1 , wherein said water-soluble carbodiimide is EDC.
8 . The method of claim 1 , further comprising exposing the optical element to a solution comprising a sugar and subsequently drying the optical element.
9 . The method of claim 8 , wherein said sugar is sucrose.
10 . A method for regenerating an optical assembly derivatized according to the method of 1 , comprising exposing the derivatized assembly to a chaotrope, wherein said exposure is sufficient to remove a non-covalently bound material from said optical element.
11 . The method of claim 10 , wherein said chaotrope is selected from the group consisting of an acid, a base, a salt, a detergent, urea, and a guanidinium ion.
12 . The method of claim 11 , wherein the detergent is sodium dodecyl sulfate.
13 . An optical assembly according to the method of claim 1 .
14 . An optical assembly according to the method of claim 8 .
15 . A kit for derivatizing an optical assembly with a layer of analyte-binding molecules comprising an amine group, comprising at least three of a polymer comprising a plurality of carboxyl groups; a water-soluble carbodiimide; an N-hydroxysuccinimide; a quencher; instructions for use; and packaging.
16 . The kit of claim 15 , wherein said water-soluble carbodiimide is EDC.
17 . The kit of claim 15 , wherein said polymer is a polypeptide.
18 . The kit of claim 15 , further comprising a sugar.
19 . The kit of claim 15 , further comprising a chaotrope.
20 . The kit of claim 15 , further comprising an optical assembly, said assembly comprising an optical element adapted for coupling to a light source via a fiber, said optical element comprising: a transparent material; a first reflecting surface comprising an aminoalkyl glass; and a second reflecting surface separated from said first reflecting surface by a distance, d.