Ion channels are ionophores that play an essential role in the movement of ions across cell membranes. Membranes are lipid bilayers and they build a hydrophobic, low dielectric barrier to hydrophilic and charged molecules. Charged molecules or atoms cannot penetrate this barrier. Ion channels provide a high conducting, hydrophilic pathway across the hydrophobic interior of the membrane. A well-known example of an ion channel molecule (or ionophore) is the polypeptide gramicidin, a naturally occurring antibiotic known to insert in biological membranes and to form transient dimers that facilitate the flux of ions through biological membranes.
A biosensor is a device for detecting a biologically active analyte. One type of biosensor is an immunosensing device based on an electrical detection of open ion channels. An example of this type of biosensor is the ICS Biosensor disclosed by Australian Membrane and Biotechnology Research Institute (where ICS is ion channel switch) (WO 98/55853). In the presence of an applied potential, ions flow between a reservoir and an external compartment when the ion channels are open. Recognition molecules specific for a desired analyte (typically fragmented antibodies) are linked to mobile gramicidin monomers in the outer leaflet of the bilayer. When an analyte is present and binds to the recognition molecule, the mobile gramicidin becomes crosslinked, preventing the formation of a conductive dimeric state with the tethered gramicidin channels in the inner half of the membrane. This crosslinking closes off open channels, which results in a reduction on the impedance current.
The current technology for the attachment of recognition moieties to gramicidin relies on a non-covalent complexation or association between biotin and streptavidin. Both gramicidin and the recognition molecule are chemically derivatized to contain a terminal biotin moiety. Addition of streptavidin produces a non-covalent mediated linkage between gramicidin and the recognition moiety by forming a ternary complex with streptavidin. (U.S. Pat. Nos. 5,874,316; 5,443,995; 5,753,093; 5,741,409; and 5,874,316).
The association between biotin and streptavidin is subject to a number of limitations that reduce the effectiveness of the biosensor. For example, the biotin and streptavidin system are subject to dissociation characterized by the kinetic rate constant, koff. This limitation is accentuated due to the low concentration of components in the biosensor such that the association (kon) of the biotinylated species to streptavidin is disfavored. This dissociation contributes to a relatively short stability of the biosensor (days instead of months) when stored in hydrated form and decreases the overall sensor performance.
Furthermore, the addition of streptavidin to the biotinylated gramicidins and transmembrane lipids results in an aggregation or “streptavidin gating” that reduces the admittance. In some cases, admittance may be reduced by a factor of approximately 2-20. It would be desirable to avoid the signal and sensitivity loss associated with streptavidin gating.
General biosensor and membrane technology and particularly ion-channel switch biosensors are described in U.S. Pat. Nos. 5,443,955; 5,741,409, and 5,741,712; the contents of which are incorporated herein by reference.
WO 02/079394 discloses a method of producing a glycoprotein by reacting a protein with a glycosylated methanethiosulfonate reagent under conditions effective to produce a glycoprotein. The glycoprotein has altered functional characteristics when compared with the protein.
There is a need for an improved method of associating ionophores to recognition molecules. Additionally, there is a need for biosensors that are more stable and have better performance.