Appeal No. 2001-0940 Page 2
Application No. 08/467,712
Yomo et al. (Yomo), “Preparation and kinetic properties of 5-ethylphanazine-glucose-
dehydrogenase-NAD+ conjugate, a semisynthetic glucose oxidase,” Eur. J. Biochem.,
Vol. 200, pp. 759-766 (1991)
Claims 12-17 stand rejected under 35 U.S.C. § 103 as unpatentable over
Nacamulli and Yomo.
We reverse the rejection.
DISCUSSION
The present invention is directed to an electrochemiluminescence-based assay
for determining the concentration of a substrate of an oxidoreductase. As explained in
the specification, the oxidoreductase used in the assay “is not present in its natural
form, but [ ] has been chemically modified so that it has two unnatural appendages,”
converting the enzyme into a chemiluminescent biosensor. Specification, page 5. “One
appendage is a covalently attached [enzyme cofactor, e.g. NAD] . . . specifically
attached in a way that it can bind in the active site of the enzyme and function as a
redox reagent as part of the natural enzyme mechanism” and “[t]he second appendage
is an [electrochemiluminescent] label,” e.g., Ru(bpy)32+, also attached near the active
site of the enzyme. Id.
According to the specification, during the course of an assay, analyte (i.e., an
oxidoreductase substrate) is oxidized in the presence of the biosensor, which converts
the NAD+ containing appendage to NADH. The Ru(bpy)3 2+ and the NADH are oxidized
at the surface of an electrode, forming Ru(bpy)33+ and NADH +• (a radical). The NADH+•
spontaneously loses a hydrogen, forming NAD•. The NAD•, a strong reductant, reacts
with Ru(bpy)33+, a strong oxidant, to form the excited state of the label, Ru(bpy)32+*. The
label decays to the ground state through a normal fluorescence mechanism, emitting a
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