Catalytic precipitation and subsequent
electrochemical oxidation
or reduction of a redox-active precipitate has been widely used in
electrochemical biosensors. However, such biosensors often do not
allow for low detection limits due to a low rate of precipitation,
nonspecific precipitation, loose binding of the precipitate to the
electrode surface, and insulating behavior of the precipitate within
a normal potential window. Here, we report an ultrasensitive electrochemical
immunosensor for parathyroid hormone (PTH) detection based on DT-diaphorase
(DT-D)-catalyzed formation of an organic precipitate and electrochemical
oxidation of the precipitate. In the present study we found that DT-D
can be used as a catalytic label in precipitation-based affinity biosensors
because DT-D catalyzes fast reduction of 3-(4,-5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) to MTT-formazan precipitate; the MTT reduction does
not occur in the absence of DT-D; and a high electrochemical signal
is obtained at low potentials during electrodissolution of MTT-formazan
precipitate. The immunosensor is fabricated using a silane copolymer-modified
ITO electrode surface that is suitable for both efficient and strong
adsorption of MTT-formazan precipitate. When the enzymatic MTT-formazan
precipitation and subsequent MTT-formazan electrodissolution is applied
to a sandwich-type immunosensor, PTH can be detected over a wide range
of concentrations with a very low detection limit (∼1 pg/mL)
in artificial serum. The measured concentrations of PTH in clinical
serum samples showed high similarity with those obtained using a commercial
instrument.