Enhanced molecular recognition signal in allosteric biosensing by proper substrate selection

Abstract

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.

Screening HIV‐1 antigenic peptides as receptors for antibodies and CD4 in allosteric nanosensors

“…All of them were inserted between amino acids 795 and 796 of the bacterial beta-galactosidase by means of recombinant DNA technology, and the resulting enzymes were produced in E. coli and purified by affinity chromatography as described elsewhere [Ferraz et al, 2006b]. After production, all the proteins were well recognized by anti-beta-galactosidase antibodies in Western blots, showing sufficient proteolytic stability (data not shown).…”

“…The biosensing assay was specifically adapted to the new proteins as done previously for NF795gpC with a sera pool from 36 HIV-1-infected individuals [Ferraz et al, 2006b] (Table 2) were selected for the analysis of individual sera samples.…”

“…After running and transfer, bands were identified with a sera pool [Ferraz et al, 2006b] from 36 HIV-1 infected patients (1/300), and visualized with goat anti-human IgG antibody peroxidase conjugated (1/2000) from Pierce. Beta-galactosidase immunoreactivity was determined similarly as described [Ferrer-Miralles et al, 2001].…”

“…In particular, the beta-galactosidase variant NF795gpC displays the immunodominant epitope P1 from the envelope glycoprotein gp41 of HIV-1, between residues 795-796 [Ferrer-Miralles et al, 2001]. By protein engineering [Cazorla et al, 2002], adjusting the reaction conditions [Ferraz et al, 2004] and selecting a convenient beta-galactosidase substrate [Ferraz et al, 2006b], the analytical signal (that is, the amount of substrate processed per time unit) has been enhanced up to more than 10 fold than that observed in absence of effector (the background signal). This has permitted the use of this enzyme for high-throughput functional screening of human sera [Ferraz et al, 2006a] and to explore the functional modification of the humoral response promoted by antiretroviral therapy [Ferraz et al, 2008].…”

Fast electrochemical detection of anti-HIV antibodies: Coupling allosteric enzymes and disk microelectrode arrays

“…These custom-made enzymes, when used as biosensing tools, showed specificities and sensitivities comparable to conventional antibody detection immunoassays [22]. Solving the structural rationale for the sensing mechanics [41][42][43][44][45][46] has permitted to optimize the performance of the engineered proteins by dramatically shortening the reaction time [47] and reaching signal:background ratios higher than 10 [48,49]. The improved versions of the allosteric sensing reactions permit the use of these molecular sensors in high-throughput sera analysis [50].…”