Enhanced molecular recognition signal in allosteric biosensing by proper substrate selection

Among protein biosensors, those based on enzymatic responses to specific analytes offer convenient instruments for fast and ultra-fast molecular diagnosis, through the comparative analysis of the product formed in presence and in absence of the effector. We have explored here the performance of five β-galactosidase substrates during the activation of a β-galactosidase sensor by antibodies against the human immunodeficiency virus (HIV). Interestingly, the employed substrate determines the dynamic range of the allosteric signal and significantly influences the sensitivity of the sensoenzymatic reaction. While ortho-nitrophenyl β-D-galactopyranoside allows the detection of a model anti-gp41 monoclonal antibody below 0.024 ng/μL, phenol red β-D-galactopyranoside offers the most dynamic response with signal/background ratios higher than 12-fold and a detection limit around 0.071 ng/μL. The hydrolysis of both chromogenic substrates generates linear sensing responses to immune human sera and parallel time-course topologies of the allosteric reaction. Therefore, the obtained results stress the potential of chromogenic substrates versus those rendering quimioluminescent, amperometric, or fluorescent signals, for the further automatization, miniaturization, or adaptation of β-galactosidase-based biosensing to high-throughput applications. © 2006 Wiley Periodicals, Inc.