Optical biosensor referencing method
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.
1. A method for measuring a reference bulk refractive index response and a binding response in a single 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 single sensing region wherein a second exit port at a second end of the single sensing region is open and a first exit port at the first end of the single sensing region is closed such that the first sample is caused to flow from the first end of the single sensing region to the second end of the single 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, and wherein the second sample comprises a deactivating agent sufficient to render the first portion unable to immobilize the ligand;
modifying the flow rate of the second sample in a manner sufficient to cause the second sample to contact a first portion of the single sensing region thereby defining a second portion of the single sensing region that is not contacted by the second sample;
terminating the injection of the second sample;
injecting a third sample comprising the 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 single sensing region are exposed to the third sample, wherein the ligand is only immobilized to the second portion of the single sensing region due to deactivation of the first portion;
terminating the injection of the third sample;
injecting a fourth sample through the first input port wherein the second exit port is open and the first exit port is closed such that the fourth sample is caused to flow over the first and second portions of the single sensing region, wherein the fourth sample comprises an analyte;
interrogating the first portion of the single sensing region using low surface plasmon resonance from a single range of increasing reflectance angles to generate a first surface plasmon resonance dip;
interrogating the second portion of the single sensing region using high surface plasmon resonance from a single range of increasing reflectance angles to generate a second surface plasmon resonance dip;
wherein the steps of interrogating the first portion of the single sensing region and interrogating the second portion of the single sensing region creates said first surface plasmon resonance dip and said second surface plasmon resonance dip in said single sensing region;
measuring a first response at the first portion of the single sensing region and a second response at the second portion of the single sensing region as the fourth sample flows over the sensing region; and
subtracting the first response from the second response to determine a binding response attributed to the interaction of the analyte with the ligand.
2. The method of claim 1 , wherein the activating agent is a mixture of ethyl (dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide.
3. The method of claim 1 , wherein the deactivating agent is selected from the group consisting of sodium hydroxide and ethanolamine.
4. The method of claim 1 , wherein the flow cell contains additional sensing regions and the method is repeated for each additional sensing region.
5. The method of claim 1 , wherein the ligand is of a low molecular weight.
6. 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.
7. The method of claim 1 , wherein the fourth sample further comprises an additive in an amount sufficient to shift the resonance to elicit a high mass surface plasmon resonance dip at the second portion while maintaining a low mass surface plasmon resonance dip at the first portion.
8. The method of claim 7 , wherein the additive is selected from the group consisting of sucrose and glycerol.
9. 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.
10. The method of claim 9 , wherein the ligand is of a low molecular weight and the non-interacting agent is ovalbumin.