Optical biosensor referencing method

Abstract

A referencing method for an optical biosensor system using a single sensing region is provided. The method involves limiting the ligand immobilized in a single sensing region to only a portion thereof. In one embodiment, this is accomplished by selectively deactivating a portion of the sensing surface to prevent immobilization of ligand to that portion. As a result, a reference response can be recorded in the same sensing region as a molecular interaction response. Thus, the bulk refractive index can be accurately accounted for in measuring the kinetics of a molecular interaction.

What is claimed is: 
     
         1 . A method for providing a reference bulk refractive index response and a binding response in a sensing region defined along a channel in a flow cell of a biosensor system comprising the steps of:
 injecting a first sample through a first input port at a first end of the sensing region wherein a second exit port at a second end of the sensing region is open and a first exit port at the first end of the sensing region is closed such that the first sample is caused to flow from the first end of the sensing region to the second end of the sensing region, wherein the first sample comprises an activating agent sufficient to permit immobilization of a ligand;   terminating the injection of the first sample;   injecting a second sample at a flow rate through the first input port, wherein the first exit port is opened and the second exit port is closed;   modifying the flow rate of the second sample in a manner sufficient to cause the second sample to contact a first portion of the sensing region thereby defining a second portion of the sensing region that is not contacted by the second sample, wherein the second sample comprises a deactivating agent sufficient to render the first portion unable to immobilize the ligand;   terminating the injection of the second sample;   injecting a third sample comprising a ligand through the first input port with the second exit port open and the first exit port closed such that first and second portions of the sensing region are exposed to the third sample, wherein the ligand is only immobilized to the second portion of the sensing region due to deactivation of the first portion; and   terminating the injection of the third sample.   
     
     
         2 . The method of  claim 1 , wherein the first portion encompasses an area of the sensing region that is interrogated by low surface plasmon resonance angles and the second portion encompasses an area of the sensing region that is interrogated by high surface plasmon resonance angles. 
     
     
         3 . The method of  claim 1 , wherein the ligand is immobilized to the second portion at a density sufficient to generate a high mass surface plasmon resonance dip. 
     
     
         4 . The method of  claim 1 , wherein the third sample further comprises a non-interacting agent that co-immobilizes with the ligand at the second portion.