Nonseparation Binding/Immunoassays Using Polycation-Sensitive Membrane Electrode Detection

Abstract

Two novel homogeneous potentiometric immunoassay formats suitable for the detection of small molecules are described based on polycation-sensitive membrane electrode detection. One approach involves the use of synthetic polycationic-analyte conjugates (polycation labeled analyte) and a limited amount of binding protein sites (antibodies, etc.) to modulate the electrochemical response toward the conjugate. Upon conjugate binding to large binding proteins/antibodies in solution, the polycation label cannot be efficiently extracted into the transduction membrane of the polycation-sensitive device, greatly diminishing the observed EMF response. In the presence of analyte molecules, competitive binding frees up more of the conjugate, and an increase in EMF response is observed in proportion to the concentration of analyte present. With a 15 min reaction incubation period and 3 min electrochemical response time, the assay is demonstrated to function effectively for measurement of theophylline (model analyte) at sub-micromolar levels without any discrete washing or separation steps. The second approach is based on the use of trypsin as an enzymatic label in a homogeneous enzyme binding/immunoassay scheme. Trypsin-biotin conjugates (biotin used as model analyte) are shown to be inhibited nearly 100 % by avidin, but catalytic activity is regained as free biotin is present at increasing levels in the sample solution. The activity of trypsin can be conveniently monitored via polycation-sensitive membrane electrode detection by following the decrease in the EMF response to the polycationic protein protamine, as it is cleaved into smaller fragments by the enzyme. This new electrochemical homogeneous enzyme binding/immunoassay method is shown to respond to the analyte at ≥10 nM levels, again without need for washing or separation steps.