Nonseparation binding/immunoassays using polycation-sensitive membrane electrode detection

Two novel homogeneous potentiometric immunoassay formats suitable for the d etection of small molecules are described based on polycation-sensitive mem brane electrode detection. One approach involves the use of synthetic polyc ationic-analyte conjugates (polycation labeled analyte) and a limited amoun t of binding protein sites (antibodies, etc.) to modulate the electrochemic al response toward the conjugate. Upon conjugate binding to large binding p roteins/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 incr ease in EMF response is observed in proportion to the concentration of anal yte present. With a 15 min reaction incubation period and 3 min electrochem ical response time, the assay is demonstrated to function effectively for m easurement 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/im munoassay 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 solu tion. 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 smal ler fragments by the enzyme. This new electrochemical homogeneous enzyme bi nding/immunoassay method is shown to respond to the analyte at > 10 nM leve ls, again without need for washing or separation steps.