Enhanced molecular recognition signal in allosteric biosensing by proper substrate selection.

Area of Science:

• Biochemistry
• Biosensor Technology
• Immunodiagnostics

Background:

• Enzyme-based biosensors offer rapid molecular diagnostics by comparing reaction products with and without an effector.
• Beta-galactosidase biosensors are explored for detecting human immunodeficiency virus (HIV) antibodies.

Purpose of the Study:

• To evaluate the performance of five beta-galactosidase substrates in an HIV antibody detection assay.
• To determine how substrate choice impacts the dynamic range and sensitivity of the biosensor.

Main Methods:

• Tested five beta-galactosidase substrates for sensor activation by anti-HIV antibodies.
• Analyzed the dynamic range, signal/background ratios, and detection limits for each substrate.
• Assessed linear sensing responses using immune human sera.

Main Results:

• Substrate selection significantly influences the dynamic range and sensitivity of beta-galactosidase biosensors.
• Ortho-nitrophenyl beta-D-galactopyranoside detected anti-gp41 antibody at <0.024 ng/µL.
• Phenol red beta-D-galactopyranoside showed the most dynamic response (12-fold signal/background) with a detection limit of ~0.071 ng/µL.

Conclusions:

• Chromogenic substrates are advantageous over chemiluminescent, amperometric, or fluorescent signals for beta-galactosidase biosensing.
• The findings support the potential of chromogenic substrates for automated, miniaturized, and high-throughput diagnostic applications.