A Surface Plasmon Resonance Sensor for Substance P Using Gold-Modified Calmodulin and Melittin

Abstract

A surface plasmon resonance (SPR) biosensor for the quantification of a neuropeptide substance P (SP) is described based on an inhibition assay using Au colloid-modified calmodulin (Au-CaM) and a target peptide melittin immobilized on carboxymethylated dextran. The modification of CaM with streptavidin Au colloids was achieved in a sample solution by the amine coupling method. The SPR signal sharply increased, corresponding to the formation of a Ca2+-Au-CaM-melittin complex on the sensor surface, and approached a steady state within 5 min. When SP was added to a sample solution, the SPR signal was decreased, due to the formation of a Ca2+-Au-CaM-SP complex in the sample solution. The modification of CaM with streptavidin Au colloids was effective for enhancing the SPR signal for SP. A decrease in the SPR signal was observed for SP in the concentration range from 0.10 to 5.0 microM, whose lower limit was ten-times superior to that (1.0 microM) with unmodified CaM. The response was highly selective to SP and the selectivity was in the order of SP >> neurokinin A > neurokinin B > neurotransmitters (glycine, GABA, L-glutamate, acetylcholine, norepinephrine, 5HT) - substance P fragment (1 - 7). The potential use of the present sensor for the quantification of SP in mouse brain extracts is demonstrated.