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 beta-galactosidase substrates during the activation of a beta-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 senso-enzymatic reaction. While ortho-nitrophenyl beta-D-galactopyranoside allows the detection of a model anti-gp41 monoclonal antibody below 0.024 ng/microL, phenol red beta-D-galactopyranoside offers the most dynamic response with signal/background ratios higher than 12-fold and a detection limit around 0.071 ng/microL. 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 beta-galactosidase-based biosensing to high-throughput applications.
Institut de Biotecnologia i de Biomedicina and Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain.
Rosa María
RM
Ferraz
Anna
A
Arís
Antonio
A
Villaverde
D000494:Allosteric Regulation; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015374:Biosensing Techniques; D002863:Chromogenic Compounds; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D004926:Escherichia coli / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D015483:HIV Antibodies / Q000032:analysis; D015700:HIV Envelope Protein gp41 / Q000032:analysis; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D006868:Hydrolysis; D008163:Luminescent Measurements; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D015202:Protein Engineering; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013997:Time Factors; D001616:beta-Galactosidase / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D002863:Chromogenic Compounds; D000939:Epitopes; D015483:HIV Antibodies; D015700:HIV Envelope Protein gp41; D001616:beta-Galactosidase