We developed a dye‐binding method for albumin in urine based on bis (3′,3″‐diiodo‐4′4″‐dihydroxy‐5′5″‐dinitrophenyl)‐3,4,5,6‐tetrabromosulfonphthalein (DIDNTB), a dye that has a higher chemical sensitivity and specificity for albumin when compared to two other commonly used dyes. We prepared urine dipsticks with DIDNTB and certain other compounds to prevent "nonspecific" binding to the dipstick matrix. The detection limit for albumin with DIDNTB as the dye is about 10 mg/L. The extent of dye binding to proteins and other compounds was studied using ultracentrifugation and a selectively permeable membrane that permitted the passage of free but not bound dye; we believe this method is superior to photometric titration. The affinity of the dyes for albumin was found to be pH dependent with stronger binding at pH 1.8 than at pH 7.0. At pH 1.8, DIDNTB had a ca.10‐fold greater binding coefficient to albumin when compared to the widely used dyes, tetrabromophenol blue (CI 4430‐25‐5) or bromophenol blue (CI 115‐39‐9). We developed a system that minimized nonspecific binding by the dye through the use of polymethyl vinyl ethers and bis‐(heptapropylene glycol) carbonate. DIDNTB showed a greater chemical specificity for albumin when compared to most other proteins. The new albumin dipsticks are resistant to many potential interferences at substantial concentrations, making the dipsticks suitable to screen for albuminuria. J. Clin. Lab. Anal. 13:180–187, 1999.© 1999 Wiley‐Liss, Inc.

Amplification of the electrochemical impedance and capacitance signals in a biosensor is demonstrated for the model fluorescein/anti-fluorescein system. Following immobilization of fluorescein onto Au through formation of a self-assembled monolayer, goat anti-fluorescein conjugated with 10-nm Au nanoparticles is introduced into the system. This results in an increase in the capacitance of approximately 400 nF/cm(2), whereas no change can be observed for goat anti-fluorescein without the Au nanoparticle conjugate. An even greater sensitivity is obtained by introduction of a redox probe, [Fe(CN)6]3-/4-, whereby the charge-transfer resistance (R(ct)) is reduced to approximately 25% of its original value. This allows construction of high-sensitivity electrochemical impedance biosensors at a single low frequency, where the signal is sensitive to the interfacial R(ct). This change in the electrochemical impedance signal upon binding to goat anti-fluorescein conjugated with Au nanoparticles can be attributed to the much higher electrochemical activity of Au surfaces relative to the underlying organic layer.

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